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Encounters with oxygen: Aerobic physiology and HO production of Lactobacillus
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Hertzberger, R.Y.
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Hertzberger, R. Y. (2014). Encounters with oxygen: Aerobic physiology and HO production of Lactobacillus
johnsonii
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Download date: 18 Jun 2017
Encounters with oxygen Aerobic physiology and H2O2 production of Lactobacillus johnsonii
Uitnodiging
Voor het bijwonen van de
openbare verdediging van
mijn proefschrift
Encounters with oxygen
Aerobic physiology and
H2O2 production of
Lactobacillus johnsonii
op vrijdag 4 juli 2014 om
13:00 in de Aula van de
Universiteit van Amsterdam,
Singel 411, Amsterdam
Rosanne Y. Hertzberger
[email protected]
Encounters with oxygen
|
Rosanne Y. Hertzberger
Aerobic physiology and
H2O2 production of
Lactobacillus johnsonii
Rosanne Y. Hertzberger
Paranimfen
Johan van Beilen
[email protected]
Sarah Chorus
[email protected]
Encounters with oxygen
Aerobic physiology and H2O2 production of
Lactobacillus johnsonii
Rosanne Y. Hertzberger
2014
The work presented in this thesis was carried out in the frame of a PhD-project of
NIZO Food Research, funded by Nestlé Research Centre and carried out at the
Molecular Microbial Physiology lab, Swammerdam Institute for Life Sciences, University
of Amsterdam. The project was part of the Kluyver entre for Genomics of Industrial
Fermentation.
Chapter 2 and 4 of this thesis have been published:
Hertzberger RY, Arents J, Dekker HL, Pridmore RD, Gysler C, Kleerebezem
M, Teixeira de Mattos MJ. 2014. H2O2 production in species of the Lactobacillus
acidophilus group, a central role for a novel NADH dependent flavin reductase. Appl.
Environ. Microbiol.
Hertzberger RY, Pridmore RD, Gysler C, Kleerebezem M, Teixeira de Mattos MJ.
2013. Oxygen relieves the CO2 and acetate dependency of Lactobacillus johnsonii NCC
533. PLoS One. 8:e57235.
Cover
Lactobacillus johnsonii colonies after 9 hours of growth in a 20%
O2 and 5% CO2 atmosphere on AnoporeTM inorganic membranes
on MRS agar. The colonies were stained with SYTO9 and propidium
iodide and photographed through a green filter (showing only live/
SYTO9 stained cells) with a cooled charge-coupled device camera
mounted on an Olympus BX-60 fluorescence microscope.
Cover design MidasMentink.nl
and layout
Printing
Ipskamp drukkers
Encounters with oxygen
Aerobic physiology and H2O2 production of
Lactobacillus johnsonii
academisch proefschrift
ter verkrijging van de graad van doctor
aan de Universiteit van Amsterdam
op gezag van de Rector Magnificus
prof. dr. D.C. van den Boom
ten overstaan van een door het college voor promoties
ingestelde commissie,
in het openbaar te verdedigen in de
Aula der Universiteit op 4 Juli 2014, te 13:00 uur
door
Rosanne Yente Hertzberger
geboren te Rotterdam
Promotiecommissie
Promotores
Prof. dr. M.J. Teixeira de Mattos
Prof. dr. M. Kleerebezem
Overige Leden
Prof. dr. J.T. Pronk
Prof. dr. O.P. Kuipers
Prof. dr. J. Hugenholtz
Prof. dr. R. Kort
Dr. F. Branco dos Santos
Dr. E.J. Smid
Faculteit der Natuurwetenschappen, Wiskunde en Informatica
“Science is the only news; alle else is hearsay and gossip.”
Lynn Margulis, condensed from Stewart Brand - Mind, Life and Universe
Table of contents
Chapter 1:
General introduction. Living on the edge: Reactive Oxygen
10
Species production and scavenging in Lactic Acid Bacteria.
Chapter 2:
Hydrogen peroxide production in species of the Lactobacillus
38
acidophilus group, a central role for a novel NADH dependent
flavin reductase.
Chapter 3:
Transcriptome response in Lactobacillus johnsonii identifies
68
an oxygen induced NADH oxidase that contributes to H2O2
production.
Chapter 4:
Oxygen relieves the CO2 and acetate dependency of
92
Lactobacillus johnsonii NCC 533
Chapter 5:
Genome-wide transcriptome response to CO2 depletion in
114
Lactobacillus johnsonii
Chapter 6:
General discussion and outlook.
138
References
151
Summary (for a scientific audience)
170
Samenvatting (voor breed publiek)
174
List of Abbreviations
177
Dankwoord
178
About the author
180
Chapter 1
General introduction
Living on the edge: Reactive Oxygen Species
production and scavenging in Lactic Acid
Bacteria.

Abstract
Lactic acid bacteria (LAB) are generally classified as aerotolerant anaerobes. They grow
relatively well in the presence of oxygen but cannot use oxygen as a terminal electron
acceptor due to an incomplete electron transfer chain. Oxidases, such as cytochrome
oxidase, NADH oxidase, lactate oxidase and pyruvate oxidase play a central role in
the aerobic lifestyle of lactic acid bacteria, either protecting against oxidative stress
(cytochrome oxidase, H2O-forming NADH oxidase) or aggravating oxidative stress
(pyruvate oxidase, lactate oxidase, H2O2-forming NADH oxidase). LAB employ several
ROS-scavenging enzymes and molecules to provide resistance against oxidative stress.
Here we give an overview of the literature that describes the role of oxidases and ROSscavenging enzymes in aerobic metabolism and oxidative stress resistance of LAB.
10
Introduction
“There’s something new here—in the middle of the drop they are lively,
going every which way.” Gently, precisely, a little aimlessly, he moved the
specimen so that the edge of the drop was under his lens... “But here at
1
the edge they’re not moving, they’re lying round stiff as pokers.” It was so
with every specimen he looked at. “Air kills them,” he cried, and was sure
he had made a great discovery.
– Paul de Kruijf, Microbe Hunters, 1926 –
This is Paul de Kruif’s interpretation of what happened when Louis Pasteur first peered
through his microscope at a specimen of rancid butter in 1860 (1). Previously, Antonie
van Leeuwenhoek in the 17th century and Lazarro Spalanzani in the 18th century had
already noted that some of these swarming animalcules live happily in the absence of
air. Now Pasteur could add a new category to the list of lifestyles in the microbe world
that were dictated by oxygen: air could also be toxic (2).
By now, we know that oxygen is not only toxic to these butyric acid producing clostridia
that Pasteur was looking at, but that all forms of life that encounter oxygen need some
strategy to deal with its hazardous byproducts. The risk is embedded in the chemical
makeup of the oxygen molecule. The ground state of the oxygen molecule is the triplet
state, which means that the single electron transfer to oxygen is thermodynamically
highly favorable, but quantum-mechanically (spin) forbidden. As a consequence it is
a relatively inert gas: the building blocks of the cell -lipids, amino acids, nucleotidesare only weak univalent electron donors so their autoxidation is a sluggish reaction.
However, oxygen rapidly oxidizes other cellular compounds that are strong univalent
electron donors, such as flavins and quinones (3). The products that are formed by these
reaction are much stronger univalent electron acceptors than triplet oxygen and are
known as reactive oxygen species (ROS, Figure 1.1). They can cause extensive damage
through different pathways. Metal centers are especially vulnerable to superoxide. The
enzymes that commonly rely on catalytically active [4Fe-4S]-clusters, such as fumarase
and aconitase, are quickly inactivated by superoxide (4, 5). The release of free iron
metals from damaged centers accelerates further ROS formation: hydrogen peroxide
can interact with free ferric ions producing the highly reactive hydroxyl radical. If this
so-called Fenton-reaction occurs in the proximity of DNA, it may cause DNA lesions and
subsequent cell death. An excellent review on the specific damage that ROS may cause
was written by James Imlay (6).
11

flavins, quinones
NOX, LOX, POX, flavins
COX, NOX
was written by James Imlay (6).
·O2-­‐
e-­‐
H2O2
SOD
O2
e-­‐
e-­‐
·OH
e-­‐
Fenton reac@on H2O
catalase, peroxidases
Figure 1.1; Generation and scavenging of reactive oxygen species. NOX: NADH oxidase, POX: pyruvate oxidase,
LOX: lactate oxidase, COX: cytochrome oxidase, SOD: superoxide dismutase. Adjusted from (6)
It is therefore not surprising that, on a planet with an atmosphere that consists for
more than 20% of oxygen, there is an urgent need for many organisms to invest in
mechanisms that eradicate reactive oxygen species from the cells (7). Even species that
are dependent on a continuous supply of oxygen for aerobic respiration, require enzymes
that continuously scavenge the toxic byproducts. The anti-oxidative arsenal consists of a
variety of proteins specialized at this task, including catalase, and superoxide dismutase
(SOD) that react with hydrogen peroxide and superoxide radicals, respectively (8).
The discoverers of superoxide dismutase, McCord and Fridovich (9, 10) hypothesized
that ROS scavengers were the facilitators of an aerobic lifestyle, since their occurrence
seemed to correlate with the aerotolerance of bacteria (11). Since then, it was shown
that several bacteria that express these proteins indeed rely on their presence for
aerotolerance (7, 12, 13). Yet, the physiology of other bacteria indicate that the proposed
rule does not apply universally: obligate anaerobes such as the butyric acid producing
clostridia that Pasteur had observed, express a variety of ROS-scavengers, including a
functional superoxide dismutase (14, 15). Moreover – as McCord and Fridovich already
observed for Lactobacillus plantarum - several lactic acid bacteria are aerotolerant
but are devoid of a functional SOD or catalase. Such bacterial species challenge our
understanding as to how micro-organisms may deal with the consequences of a life in
the presence of oxygen.
12
1. Lactic acid bacteria
LAB are a phylogenetically diverse group of Gram-positive, non-sporulating, rod- or
coccoïd shaped bacteria that have a fermentative metabolism with lactic acid as a
major end product. The intimate relationship between LAB and humans is related to
1
the prominent role of these bacteria in food fermentations. They contribute to the taste,
texture and shelf life of a wide variety of fermented food products including yoghurt,
cheese, butter milk, sour cream, pickles, olives, sauerkraut, sourdough and meat.
Besides their presence in these food products, LAB are associated with the mucosal
surfaces of the human body. They are amongst the very first microbes that colonize
newborns: the lactobacilli that inhabit the vaginal cavity of the mother are microbial
pioneers in the neonatal gut (16). Further bacterial transmission from the mother to the
infant during breastfeeding also mostly consists of LAB (17, 18). LAB, and in particular
streptococci, constitute a considerable part of the upper oro-gastrointestinal tract
microbiota (19, 20).
In the gastro-intestinal tract, specific LAB species and strains have been proposed to
exert a positive effect on the health of the host. Administration of adequate amounts
of lactobacilli and bifidobacteria in products designated as probiotics can reduce the
severity of antibiotic-associated diarrhea (21, 22), infectious diarrhea (23) and Clostridium
difficile –related diarrhea (24). Probiotics can be effective in the prophylactic treatment
or necrotizing enterocolitis in preterm infants (25) and they have been proposed to be
effective in reducing the risk of atopic dermatitis development in children (26).
Several characteristics of LAB were identified that may contribute to these probiotic
effects. Firstly, probiotic LAB generally display substantial acid and bile resistance
that enables them to survive the hostile environment of the stomach and the upper
digestive tract (27). Secondly, they express proteins that allow competitively adherence
to intestinal mucus (28), and thirdly, they can strengthen the mucosal barrier and/or
immune function (29, 30).
2. LAB and oxygen
In terms of their classification with respect to oxygen, LAB are generally referred
to as aerotolerant anaerobes. This classification reflects two commonly conserved
characteristics in LAB: (i) due to the incompleteness of their electron transport
chain, they are unable to use oxygen as a terminal electron acceptor (hence the
term “anaerobes”), while (ii) oxygen is also not particularly damaging either, which is
13

illustrated by the relatively good growth of many LAB in aerobic environments (hence
the term “aerotolerant”). This tolerance does not mean that LAB are unresponsive to
environmental oxygen levels. Oxygen can have a profound effect on growth, metabolism
and viability. The presence of oxygen can dictate which pathways are used for ATP
generation and which fermentation metabolites are produced (31, 32). The enzymes
involved in pyruvate dissipation are particularly influenced by exposure to oxygen (see
Figure 1.2). For example, pyruvate formate lyase (PFL) is highly sensitive to oxygen
(33) and as a consequence, formate production is completely abolished upon oxygen
exposure. The pyruvate oxidase (POX) pathway depends on molecular oxygen as a
substrate (34) and facilitates acetate production with additional generation of ATP from
central metabolism. Furthermore, oxygen allows the utilization of lactate through the
lactate oxidase (LOX) pathway and the regeneration of oxidized reducing equivalents
catalyzed by NADH oxidase (NOX) (Figure 1.2)
glucose ADP NAD+ ATP NADH pyruvate pfl formate NAD+ pdh NADH acetyl-­‐CoA .
NADH NAD+ ldh lactate lox H2O2 O2 O 2 pox O2
+C
2
H 2O
pat NADH adh NAD+ acetyl-­‐P ack ADP ATP ethanol acetate O2 H2O2 / H2O NADH nox NAD+ Figure 1.2; Overview of pyruvate metabolism in LAB (for simplicity the pathways for acetoin/2,3-butanediol were
omitted). LDH: lactate dehydrogenase, LOX: Lactate oxidase POX: pyruvate oxidase, PFL: pyruvate formate lyase,
PDH: pyruvate dehydrogenase, PAT: phosphate acetyltransferase, ACK: acetate kinase, NOX: NADH oxidase)
Besides the additional metabolic reorientation that oxygen may elicit, the generation of
hydrogen peroxide (H2O2) and superoxide species in aerobic metabolism, for instance
by NOX or POX activity, is a major source of intrinsic oxidative stress in LAB. Especially
14
in obligate homofermentative lactobacilli H2O2 can freely accumulate and may cause
growth stagnation and cell death (35-38). At the same time, oxygen can also provide
a benefit in terms of additional ATP generation through the POX-pathway, involving
activity of POX and acetate kinase (ACK, see Figure 1.2). Accordingly, the degree of
1
aerotolerance encountered in individual LAB species and strains shows considerable
variation. Some species may lean more towards “aerotolerant”, such as species in the
Leuconostoc genus that can reach approximately 2-fold higher cell densities in aerobic
environments as compared to anaerobic environments (39). Other species may lean
more towards “anaerobes”, such as Lactobacillus delbrueckii subsp bulgaricus that
reaches approximately 2-fold reduced cell densities in aerobic conditions (37). Results
presented in this thesis demonstrate that Lactobacillus johnsonii also belongs to this
latter group.
In the environments where LAB are regularly found, they will frequently encounter
molecular oxygen and its detrimental derivatives. The epithelial and mucosal linings of
the mouth, vagina and intestine are considered to contain considerable levels of oxygen
(40-45). Similarly, LAB used in the preparation of fermented food products are exposed
to oxygen during processing, shelf life and consumption (35, 46, 47). This regular
oxygen exposure has a considerable impact on their overall metabolism and physiology
(48), on their interactions with other bacteria (49) and on their interactions with the
host organism (40, 50).
In this literature review we explore the aerobic physiology of LAB and in particular their
aerotolerance. We consider
two aspects that are central
Definitions used in this thesis:
for the ability of cells to retain
ROS Hydrogen peroxide, superoxide, hydroxyl radical.
viable and to proliferate in the
For simplicity, we restrain ourselves to these three
presence of oxygen. Firstly, we
compounds (Figure 1.1).
will discuss aerobic metabolism
Oxidative stress Cellular damage (or the risk of cellular
in LAB, which is presumably
damage) caused by ROS.
mediated by the activities of
Oxidative stress resistance: The ability to grow
four
cytochrome
and/or retain viability in the presence of ROS-generating
oxidase, POX, LOX and NOX.
compounds (e.g. paraquat and plumbagin) or hydrogen
Secondly,
peroxide.
oxidases:
we
will
discuss
expression of ROS-scavenging
Aerotolerance: The ability to grow and/or retain
enzymes and other common
viability in the presence of oxygen.
anti-oxidative strategies that
15

confer resistance against oxidative stress to LAB. Taken together, these aspects provide
insight into the different consequences of oxygen exposure for different LAB species.
3. Cytochrome oxidase and respiration
A conserved characteristic amongst LAB is their inability to constitute a functional electron
transfer chain that uses molecular oxygen as a terminal electron acceptor. Nevertheless,
the requirements for a rudimentary electron transfer chain are present in several LAB,
such as NADH dehydrogenase that accepts electrons from reducing equivalents (51)
and cytochrome oxidase to catalyze the final electron transfer to oxygen (52). However,
with a few exceptions (53, 54) most LAB are unable to synthesize quinones, that
are essential for electron shuttling between NADH dehydrogenase and cytochrome
oxidase. Furthermore, all LAB lack the essential genes to constitute a complete synthesis
pathway for heme (55), an essential and functional prosthetic group of cytochromes
and cytochrome oxidases. Therefore, LAB cannot assemble cytochromes and cannot
produce functional cytochrome oxidases (52). In several species a respiratory phenotype
can be induced by supplementation of hemin, indicated by increased biomass levels,
cytochrome synthesis, altered metabolite levels and proton pumping (56-58).
Several studies on the respiratory phenotype report a dramatic increase in survival in
respiration-permissive environments. The viability loss that is witnessed in certain LAB
during aerobic stationary phase can be prevented by the addition of hemin, as was
shown in L. lactis (59, 60), L. plantarum and S. agalactiae (57). Moreover, L. plantarum
displayed a considerably higher H2O2-tolerance in the presence of hemin (61).
Besides the production of additional ATP, hemin addition may also contribute to aerobic
robustness through reducing ROS production. The respiratory chain is an important
source of reactive oxygen species. Reduced quinone species can spontaneously react
with oxygen, resulting in superoxide and H2O2 formation. Enterococcus faecalis is an
exception amongst LAB since it is can synthesize demethylmenaquinones. Univalent
oxidation of these quinones was shown to be the cause of extracellular superoxide
production (62, 63). Activity of cytochrome oxidase through the addition of hemin
abolished this superoxide generation. Such an effect of cytochrome oxidase activity on
ROS-production was previously observed in Escherichia coli where a flux through the
electron transfer chain (by restoring the function of a cytochrome oxidase) could reduce
H2O2 production derived from autoxidation of the flavin cofactor of fumarate reductase
(64). In L. lactis, aerobic respiration also abolished ROS-production (53), although here
16
the major source of ROS was found to be a H2O forming NADH oxidase (48). We consider
however that lowering the NADH/NAD+ balance in L. lactis by shuttling electrons from
NADH in an electron transfer chain, would also lower the flux through the H2O forming
NADH oxidase and would therefore contribute to lowering superoxide-formation.
1
These studies show that in several LAB, aerobic respiration can increase yield and
robustness, and oxidative stress tolerance. Oxidative stress is reduced by diminshing
autoxidations of respiratory intermediates and by generating additional ATP. In addition
menaquinone and/or hemin has an impact on a variety of physiological aspects,
including metabolite profile, pH and redox-state. All these aspects may play a role in
the increased oxidative stress tolerance.
4. NADH oxidase
One of the consequences of lacking an intact electron transfer chain is that additional
redox constraints arise. Glucose to lactate fermentation is redox-neutral: the NADH that
is produced through the glycolytic production of pyruvate, is regenerated by the lactate
dehydrogenase (LDH) reaction in an equimolar stoichiometry. Pyruvate conversion to
acetic acid (through one of the three pathways: PDH, PFL, POX) requires the oxidation
of NADH through a different pathway, either by the production of reduced metabolites
such as ethanol or acetoin or through the use of molecular oxygen to regenerate NAD+
for example through the activity of NOX (Figure 1.2; (65-67).
A second complication in the correct identification of NOX is that other flavoproteins
that do not belong to the NOXs may catalyze NADH oxidation as a side activity.
Fumarate reductase of Escherichia coli (64, 68) and dihydroorotate dehydrogenase
of B. bifidum (69) are examples of proteins with a solvent-exposed flavin moiety that
easily autoxidizes, leading to superoxide or H2O2 production. Furthermore, free flavins
that are reduced by NADH flavin reductases may spontaneously react with molecular
oxygen producing considerable amounts of ROS (6). Reduced flavins can provide an
important source of H2O2 in lactobacilli, which is illustrated by the identification of a
flavin reductase as the main contributor to H2O2 production by L. johnsonii (chapter 2 of
this thesis). Two different NOX enzymes are expressed by LAB: one that catalyzes a four
electron transfer producing mainly water and one that catalyzes a two electron transfer
producing mainly H2O2. It is not trivial to distinguish between these two forms since LAB
may also express an NADH peroxidase (NPR), which in combination with the activity of
H2O2-producing NOX results in the same overall reaction as the one catalyzed by the
17

H2O-producing NOX (32, 70) (Table 1.1). In some cases, such as in the eukaryotic DUOX
enzyme (71) and bacterial alkyl hydroperoxide reductase (72), the two reactions could
even be attributed to a single enzyme. DUOX H2O2 producing and scavenging (NOX
and NPR activity) can even be attributed to a single enzyme. Nox-1, a 55 kDa protein
requiring exogenous flavin for activity, in Streptococcus mutans displayed a high level
of homology with the earlier identified ahpF gene in Salmonella typhimurium (73) that
is part of an alkylhydroperoxide reductase (AHPR, encoded by ahpC and ahpF, see also
below at 6b). This Nox-1 catalyzes H2O2 producing NADH oxidation but together with
the upstream located ahpC gene, it forms an AHPR, which can reduce H2O2 as well as
alkyl organic peroxides using NADH (74, 75). A similar feature and DUOX enzymes in
eukaryotes (71)
Table 1.1; Enzymatic reactions involving reactive oxygen species generally found in LAB.
Cytochrome bd oxidase (Q = menaquinone or ubiquinone)
Pyruvate oxidase (cofactors, TPP, Mg2+, FAD)
Lactate oxidase
NADH oxidase (H2O2-forming)
NADH oxidase (water-forming)
NADH flavin reductase (uses either FAD, FMN or riboflavin as substrate)
Catalase (with either hemin or manganese in the active site)
Thiol-based peroxidase. R could be substituted for TRX, GT or AHP.
18
NADH peroxidase
1
Superoxide dismutase
Fenton reaction
Streptococcus mutans also encodes a 50 kDa enzyme Nox-2 that catalyzes the transfer
of four electrons to oxygen, producing water. This Nox-2 appears to be essential for
regenerating NAD+, since a nox-2 deletion derivative of S. mutans displays hampered
growth on mannitol, which is a substrate that generates additional NADH when it
is catabolized to pyruvate. Moreover, this deletion mutant also redirected its carbon
metabolism towards a higher production of lactate, which is more reduced than the
acetate and CO2 combined (76). In other LAB, these type of metabolic effects of NOX
are also observed. In L. sanfranciscensis (65, 77) and in L. lactis (66, 77), NOX was
shown to exert direct control on the end-product of fermentation. L. lactis shifted
from homolactic to mixed acid fermentation upon controlled overexpression of a H2Oforming NOX when growing in the presence of oxygen (66, 74, 77).
These examples illustrate that redox balance (NADH/NAD+ ratios) plays a pivotal role in
the control of the pyruvate dissipating flux. Lactic acid bacteria may be forced towards
lactate formation to sustain redox balance under anaerobic conditions, but can shift
towards mixed acid fermentation under aerobic conditions through the alternative
electron sink provided by the NOX enzyme activity, with the concomitant benefit of
the additional ATP generated through the acetate formation pathway. This metabolic
control of redox balance has been exploited in LAB, and in particular in L. lactis to
redirect its metabolic flux towards higher production of flavour components like acetoin,
diacetyl and acetaldehyde (66, 78)
19

5. Aerobic lactate utilization: pyruvate and lactate oxidase
POX plays a prominent role in the metabolism of several well-studied LAB. It catalyzes
the oxidative decarboxylation of pyruvate in the presence of inorganic phosphate,
releasing acetyl-phosphate, carbon dioxide and H2O2. The enzyme is a homotetramere
that contains tightly bound flavin adenine nucleotide (FAD) and uses thiamine
pyrophosphate (TPP) and a divalent metal ion as cofactors (79, 80).
POX is part of the aerobic lactate utilization pathway. Upon glucose exhaustion in
the environment as a consequence of aerobic growth of L. plantarum of L. lactis, the
accumulated lactate can be oxidized by the combined activity of an NAD-dependent LDH
and POX (81, 82). The resulting acetyl-phosphate can support ATP production through
acetate kinase. Deletion of two (poxB and poxF) of this apparently five-fold redundant
function in L. plantarum WCFS1 completely abolished aerobic lactate utilization and
acetate production in stationary phase, implying that POX (and not PFL or PDH) is the
main acetate-producing reaction in this species under these condions (83).
A similar aerobic lactate utilization pathway is found in S. pneumoniae, although in
this organism the first step is catalyzed by LOX instead of LDH. S. pneumoniae (then
referred to as Pneumococcus) Expression of the lox (LOX) and pox (POX) genes in several
LAB is repressed in the presence of glucose through CCP-A mediated catabolite control
(84-87). This does not appear to be the case in S. pneumoniae where the transcription
of these genes (88) nor the reaction they catalyze (lactate utilization, acetate and H2O2
production) were influenced by environmental glucose levels (89).
Combined LOX and POX activity results in the accumulation of up to 1 mM of H2O2
during the stationary phase of growth in S. pneumoniae (90) and S. pyogenes (86).
Notably, deletion of either the pox or lox gene completely abolished H2O2 production
and prevented the dramatic viability loss during the aerobic stationary phase of growth
in several LAB (83, 86, 87, 89-91).
An exception with respect to the role of POX and LOX in aerobic metabolism is
encountered in the species belonging to the L. acidophilus group. The pox-gene is
present in these species and an active protein could be purified from L. delbrueckii
subsp bulgaricus (34). Furthermore, as we show in this thesis, L. johnsonii can produce
sufficient acetate and CO2 to satisfy its growth requirements, in a POX and oxygen
dependent manner (chapter 4). However, L. johnsonii and delbrueckii subsp bulgaricus
are strictly homofermentative and thus do not produce acetic acid under aerobic
conditions, which allows only a limited flux through the POX-pathway.
20
6. ROS scavenging enzymes
Many of the above-mentioned enzymes, such as POX, LOX, NFR and NOX, catalyze the
transfer of two electrons to oxygen, resulting in H2O2 formation. Consequently, aerobic
growth of some LAB is accompanied by the accumulation of substantial amounts (>1
1
mM) of H2O2. In streptococci this H2O2 is predominantly produced by POX and LOX
(85, 86, 89, 92). In other bacteria, such as the species belonging to the L. acidophilusgroup, H2O2 is produced in a reaction involving NADH (37, 93, 94). In this thesis we
report the identification of the enzyme involved in H2O2 production in L. johnsonii as
an NFR and identified a NOX enzyme to contribute to H2O2 production after prolonged
aeration (see chapter 2 and 3). Besides the direct involvement of these enzymes, H2O2
may also be generated in spontaneous oxidations of cellular components. Superoxide
radicals that are generated during autoxidation of dimethylmenaquinone in E. faecalis
(62) and NADH oxidase activity of L. lactis (48) can spontaneously be dismuted to form
H2O2. Even in aerobic, respiring bacteria such as E. coli, the deletion of the main H2O2
scavenging enzymes catalase and AHPR leads to H2O2 accumulation, indicating that this
is a universal characteristic of bacterial growth in oxygenated environments. (64, 95).
Besides the ability to use oxygen for ATP generation, e.g. in aerobic respiration or aerobic
lactate utilization, the ability to scavenge the toxic byproducts of oxygen reactions (ROS)
is an important factor which contributes to aerotolerance of cells. LAB employ a diverse
range of mechanisms to protect against the oxygen radicals that are generated during
aerobic metabolism. Below we discuss the enzymes that were shown to contribute to
aerotolerance and oxidative stress resistance in LAB by scavenging ROS. An overview of
the distribution of genes encoding these ROS-scavenging enzymes in LAB is presented
in Table 1.2 and S1.1).
21
22
1 Hemin-dependent catalase (HemCat) from E. faecalis V583 (96)
2 Mn-catalase (MnCat), L. plantarum ATCC 14431 (188)
3 Thioredoxin reductase (TRXR) L. plantarum WCFS1 (113).
4 Thiol peroxidase (TPX) from S. pneumoniae D39 (116)
5 Glutathione reductase (GSHR) L. sanfranciscensis DSM20451 (122)
6 Alkyl hydroperoxide reductase (AHPR), S. mutans NCIB 11723 (74)
7 NADH peroxidase (NPR) from Enterococcus faecalis V583 (117).
8 Manganese-superoxide dismutase (MnSOD) L. lactis MG1363 (48)
-
-
1
-
E. coli K-12
-
S. mutans UA159
-
-
-
S. thermophilus LMG 18311
-
-
-
-
S. pneumoniae D39
-
-
S. pyogenes HSC5
-
-
1
E. faecalis V583
-
-
-
MnCat2
(accession nr.
P60355)
-
L. sanfranciscensis TMW
1.1304
L. delbrueckii subsp
bulgaricus ATCC 11842
L. johnsonii NCC 533
-
L. rhamnosus GG
-
1
L. casei BL23
L. plantarum WCFS1
L. lactis subsp. cremoris
MG1363
HemCat1
ef_1597
2
2
2
2
5
3
3
4
3
2
2
3
2
TrxR3
lp_0761
1
1
-
1
1
1
1
-
1
1
1
1
1
Tpx4
SPD_1464
4
3
1
1
4
3
3
2
3
4
3
4
GshR5
(accession nr.
A1YAC0)
5
1
-
-
-
1
-
-
1
1
1
1
1
AhpC6
(accession nr.
O66265)
-
1
1
1
1
2
2
3
2
5
4
5
5
10
NPR7,
ef_1211
2
-
-
-
1
1
1
1
1
1
-
1
-
MnSOD8
llmg_0429
Table 1.2; Distribution of genes encoding antioxidative/ROS-scavenging mechanisms in LAB genomes as found by BlastP (cut-off values: minimal query coverage 60%,
maximal e-value 10-10, minimal identitical residues: 20%). The number of genes that fall within the selection criteria are between brackets. For gene annotations and ID see
Supplementary table S1.1.

a. Catalase
LAB are generally referred to as being catalase-negative and indeed, catalase encoding
genes are sparsely encountered in LAB genomes. Nevertheless, two different types of
catalases are found in LAB. A hemin-dependent catalase is present in the genomes of
1
certain strains of L. plantarum, L. sakei, L. casei and E. faecalis (96, 97). This gene can
produce a functional H2O2 scavenging enzyme when cells reside in hemin-containing
environments, thus providing H2O2 tolerance under specific conditions (98-100). An
alternative catalase that is not depending on hemin supplementation was identified in
L. plantarum strain ATCC 14431 (101, 102) and appeared to employ manganese ions
in its reactive center (103). The gene encoding MnCAT (accession nr. P60355) is very
rare in LAB, and only encountered in a few Pediococcus, Enterococcus and Lactobacillus
strains. A L. plantarum strain in which this MnCAT was absent produced H2O2 during
aerobic growth indicating that this enzyme is the main H2O2 scavenging activity for
these species in hemin-depleted conditions (100).
b. Thiol-based peroxidase
Whereas catalases are quite scarce in LAB, genes for a second type of H2O2-scavenging
enzyme, with an activity that revolves around a catalytically active cysteine residue, are
abundantly present on LAB genomes. The nomenclature in literature is quite diverse and
elsewhere this group may be referred to as peroxiredoxins, cysteine-based peroxidases,
thiol-specific antioxidants or NPRs. Here we will refer to them as thiol-based peroxidases,
following Mishra et al (7). We discuss them as a group, since they show significant
similarity, both in gene sequence and in reaction mechanism (104).
Thiol-based peroxidases catalyze the reduction of H2O2 to water, or organic hydroperoxides
to their corresponding alcohol, using NADH or NADPH as the electron donor. Different
types of thiol-based peroxidases have been detected in LAB: glutathione /glutathione
reductase (GSH/GSHR), thioredoxin /thioredoxin reductase (TRX/TRXR) and alkyl
hydroperoxide /alkyl hydroperoxide reductase (AHP/AHPR encoded by ahpC and ahpF,
respectively). The reaction mechanism consist of two steps: the smaller polypeptide
(either TRX, GSH or AHP) has an active site with two (seleno-)cysteine residues, or
in the case of GSH two molecules that each contain a single cysteine residue. One
of the cysteine residues reacts with H2O2, leading to the formation of a sulfenic acid
that subsequently reacts with the secondary cysteine residue forming a disulfide bond.
A dedicated flavoprotein reductase (either TRXR, GSHR or AHPR) can subsequently
reduce the disulfide bond. The active site of these reductases also contains cysteine
23

residues and employs NADH or NADPH as an electron donor (7, 105, 106). In the
case of thiol peroxidase (TPX), the disulfide bond is reduced by a TRX, which itself
is reduced by TRXR. Besides their role in oxidative stress tolerance, these peroxidases
protect against a wide variety of environmental stresses that are associated with the
formation of reactive oxygen species, such as temperature and low-pH stress (107, 108).
This type of NADH related peroxidase activity was shown to be complementary to
catalase activity in E. coli (109). However, an important difference is that thiol-based
peroxidases require input of electrons from reducing equivalents, whereas catalase
does not need a co-substrate besides H2O2. Moreover, thiol-based peroxidases are
active at lower ROS concentrations as compared to catalase (7, 74). Below, we discuss
the prevalence of four types of thiol-based peroxidases in LAB, (i) thioredoxin/thiol
peroxidase, (ii) glutathione reductase, (iii) alkyl hydroperoxide reductase and (iv) NADH
peroxidase (Table 1.2 and Table S1.1).
Thioredoxin reductase / Thiol peroxidase
The genes encoding TRX (trxA) and its corresponding TRXR (trxB) are ubiquitously present
in LAB (Table 1.2). TRX is a short polypeptide (103-106 residues) with a conserved
cysteine rich catalytic site (CXXC). It is present in all forms of life and in several species it
is indispensable. In bacteria, it has been associated with a variety of processes, including
gene regulation and signal transduction. TRX can also function as an electron acceptor
or hydrogen donor in redox reactions and can contribute to ROS scavenging (110-112).
The catalytic cysteine residues of TRX react either directly with H2O2 or indirectly with an
oxidized thiol from a so-called peroxiredoxin such as TPX. Oxidized TRX can be reduced
by a dedicated TRXR, which has a corresponding CXXC conserved catalytic site.
LAB generally have multiple copies of both the TRX encoding trxA gene and the TRXR
encoding trxB gene. TRXR is a member of a larger family of pyridine nucleotide-disulfide
oxidoreductases that also include GSHR, AHPR and DLD (catalyzing the second reaction
in the pyruvate dehydrogenase complex). There are similarities between the members
of this group, which complicates correct annotation. This is possibly the reason why not
all copies of genes annotated as TRXRs have the CXXC-motif which is deemed essential
for its activity (113) (Table S1.1).
In several LAB, the TRX/TRXR systems were shown to contribute to oxidative stress
resistance and aerotolerance. Mutation analysis of the TRXR encoding trxB gene in L.
casei subsp. shirota, in L. plantarum and in L. lactis showed that the TRX/TRXR system
24
is essential for tolerance against disulfide (diamide)- and H2O2-stress (113-115). TRX
expression was associated with the induction and repression of a variety of stress
proteins in L. plantarum (113) and L. lactis (115). The L. lactis trxB1 gene that contains
the canonical CXXC-motif and is predicted to encode a TRXR was not reported to play
1
a role in aerotolerance (115). Nevertheless, the L. lactis genome contains a second TRX
homolog, annotated as an AHP (33% identity) and contains the CXXC motif, which
may show TRXR activity when the primary TRXR was disrupted in L. lactis.
Notably, a TPX that functions in conjunction with the TRX system has been shown to
display NADPH peroxidase activity in S. pneumoniae (116), and to contribute to oxidative
stress tolerance in E. faecalis (117). Thereby the TRX, TPX system acts as a direct NPR
that is involved in aerotolerance. Moreover, this system has also been shown to play a
role in gene regulation related to the oxidative stress response in S. pneumoniae (116).
Glutathione reductase
Almost all LAB lack the enzymatic machinery for GSH synthesis: the GSH synthetase
encoding gene from E. coli has no full-length homologs in the Streptococcus,
Lactobacillus and Lactococcus genera. However, many LAB can import exogenous GSH
and maintain high intracellular levels (108, 118). Furthermore, many species encode
the corresponding GSHR, to regenerate reduced GSH. In L. lactis intracellular GSH was
estimated to increase to 60 mM when GSH-rich ingredients such as yeast extract were
present in the medium. GSH supplementation was correlated with H2O2 tolerance (119)
and provided cross-protection against acid stress (120). Similarly, in several LAB the
uptake and recycling of GSH is correlated with oxidative stress resistance (118, 121123).
Alkyl hydroperoxide reductase
In E. coli, the AHPR was observed to be the “first line of defense” against H2O2. The
system, encoded by ahpC and ahpF, scavenges H2O2 with high affinity and low-level
saturation (Km of 5 µM). It can fully complement catalase deficiency in aerobically
growing E. coli (109). As mentioned before, AHPR belongs to the same protein family
as TRXR, GSHR and DLD and the corresponding genes are ubiquitously present on LAB
genomes. However, copies of the corresponding AHP-encoding ahpC gene are not as
prevalent in LAB, indicating that this system is only functional in a subset of LAB (Table
1.2).
25

AHPR was first identified in S. mutans as a H2O2-producing NOX (76, 124). However,
the direct reaction with oxygen appeared to be a side-activity. In vivo its main electron
acceptor was identified as AHP, whose cysteine residues can be oxidized by H2O2,
indicating its function in ROS scavenging (74, 76, 105). Such a role was established in E.
faecalis, where deletion derivatives of ahpCF displayed reduced growth and viability in
the presence of exogenous H2O2 compared to the wild type (117). However, this role of
AHPR is apparently not universally valid, since ahpCF deletion derivatives of S. mutans
were not affected in terms of their sensitivity towards H2O2 as compared to the wild
type strains (125). Besides these apparently contradictory studies, the role of AHPR in
ROS tolerance in LAB has not been studied extensively, and its in vivo function remains
to be further elucidated.
NADH peroxidase
NPR is unique in the class of thiol-based peroxidases since both the reaction between
thiol and H2O2 as well as the NADH recycling of the oxidized thiol is catalyzed by a single
enzyme. The NPR of E. faecalis, encoded by the npr-gene, is a flavoenzyme with a single
active cysteine residue Cys42 (126, 127) that is essential for oxidative stress resistance in
this bacterium (117).
The NPR shows a remarkable redundancy in LAB. L. plantarum even has 10 genes that
show resemblance with high significance (BlastP cut-off values: maximum e-value 10-10,
minimum identical residues 20%, minimum query coverage 60%). 6 of these 10 genes
are annotated as NOXs and 5 have the conserved active site cysteine residue. One of these
NPR homologs (noxV, accession number F9UUC2), displayed H2O-forming NOX activity
but its capacity to convert H2O2 was not investigated (128). The high-level of sequence
homology between NOX and NPR may suggest that this group of flavoproteins could
display substrate promiscuity and thereby play a role in NADH-dependent conversion of
both oxygen and H2O2, and possibly also other electron acceptors.
c. Superoxide dismutase
Superoxide radicals in cells are formed during the autoxidation of cellular components
that are strong univalent electron donors, such as quinones and flavins. The one electron
transfer reaction between oxygen and flavin leads to the formation of a so-called
flavinsemiquinone and a superoxide molecule. The reactivity of the flavinsemiquinone
with triplet oxygen and the reactivity of fully reduced flavin with superoxide leads to
26
autocatalytic superoxide formation through redox cycling (129, 130). The reactivity of
the superoxide radical towards iron-sulfur clusters is an important factor in oxidative
stress damage. Damage to iron-sulfur clusters leads to the inactivation of proteins with
such clusters. The release of iron from damaged iron-sulfur clusters accelerates the
1
generation of ROS through the Fenton reaction (68, 131)(see Figure 1.1).
Although two superoxide molecules can spontaneously dismutate to form H2O2, the
catalysis of this reaction by SOD is in many species an important means to prevent
oxidative stress. The presence of a functional SOD in several LAB species is wellestablished (132-137). Interestingly, the main source of superoxide generation in L.
lactis was shown to be a supposedly water forming NOX, where micromolar amounts
of H2O2 appeared to be exclusively produced via the combined activities of NOX and
SOD (48).
Whereas SOD in other organisms either uses iron, manganese or copper ions in its
catalytic site, only the manganese-form was encountered in LAB. Expression of SOD
was shown to contribute to aerotolerance (132, 134) and to H2O2-stress tolerance (125,
138), indicating that superoxide is a central oxygen intermediate contributing to cellular
damage during oxygen exposure.
7. Physiological adaptations to protect against oxidative
stress
Despite the presence of several homologs of thioredoxin reductases, NPRs and
glutathione reductases, no NADH-related H2O2 scavenging activity can be detected in
the cell extract of the well-studied lactic acid bacteria L. lactis (48) and L. plantarum
(139, 140). Analogously, cell extracts of L. plantarum lack SOD activity but the cells
show remarkable resistance towards hyperbaric oxygen levels (141). These results
indicate that apart from expression of enzymes dedicated to ROS scavenging, lactic acid
bacteria employ physiological adaptations to protect against toxic oxygen derivatives.
Here, we discuss how LAB utilize metal homeostasis, accumulation of pyruvate and
thiol metabolism as a means to reduce ROS-induced damage.
a. Intracellular manganese accumulation
Apart from its role as a cofactor of catalase and SOD, manganese is accumulated to
high intracellular levels in L. plantarum (>20 mM) and functions as a superoxide sink.
LAB that show such high intracellular manganese levels (such as L. plantarum and L.
27

casei) are generally more resistant against superoxide-generating compounds such as
plumbagin as compared to LAB that do not accumulate high levels of manganese (such
as L. lactis and L. acidophilus) (141-143).
There are several clues as to how such high intracellular levels of manganese may
contribute to superoxide quenching. Bicarbonate-complexed manganese can contribute
to H2O2 scavenging, which could indirectly also reduce superoxide toxicity (144). The
authors of the original paper describing the phenomenon in L. plantarum suggested
that pyrophosphate-complexed manganese could directly react with superoxide in vivo
(141). Conversely, manganese accumulation in E. coli, which occurs in mutants lacking
the genes for catalase and peroxidase, was effective in protecting against oxidative
stress by replacing the reactive Fe2+ ions in metalloproteins and thereby reducing Fenton
chemistry (145, 146).
Attempts were made to identify the responsible manganese transporters in different
LAB. The product of the mnt-gene was identified as a functional Mn2+ uptake system
in S. oligofermentans (147) and L. plantarum strain ATCC 14917 (148, 149) but not
in strain WCFS1 (150). A recent study reported that in S. oligofermentans, manganese
import was regulated upon increasing H2O2 levels and mntA expression substantially
contributed to oxidative stress tolerance (147). However, the mechanism remains poorly
understood and apart from these studies, no new research has appeared creating a link
between regulation of manganese homeostasis and oxidative stress in LAB.
b. Regulation of intracellular iron levels
Compared to regulation of intracellular manganese levels, controlling intracellular
iron may be an even more fundamental physiological adaptation that underlies the
aerotolerant nature of LAB. Where other micro-organisms compete with each other
to obtain sufficient bioavailable iron sources, LAB seem to have sidestepped this rivalry.
Although there are several exceptions (in S. pneumoniae (91) and in heme supplemented
growth conditions (151)), the growth requirements of several Lactobacillus species
for iron are nearly zero and the intracellular iron levels are very low (152-154). As
was mentioned before, such intracellular unbound iron can engage in ROS formation
through the Fenton reaction which accelerates the damage to cellular components.
These low intracellular iron levels may be a direct consequence of the relatively low
numbers of iron sulfur binding gene products in LAB compared to other bacteria such
as E. coli. Moreover, several metalloproteins in LAB, such as catalase and SOD, were
28
shown to function with manganese ions instead of ferrous ions (see for instance our
previous discussion of catalase and SOD in LAB). Through this reduced iron-dependency
of protein function, lactic acid bacteria may avoid the need to maintain high intracellular
iron pools, and may thereby be less sensitive to the toxicity of H2O2.
1
Besides the lack of iron-sulfur clusters, LAB also lack copies of the genes encoding
bacterioferritin and ferritin, which are the major bacterial iron-storage proteins. The
third iron binding protein DPS, which is associated with preventing oxidative stress (155,
156), is present in many LAB and showed to sequester iron and prevent H2O2-induced
cell death in streptococci (75, 157, 158). We consider that even with low dependency
on iron for protein function, oxidative stress in certain LAB is still correlated with levels
of intracellular unbound iron.
c. Pyruvate accumulation
The metabolic intermediate pyruvate is an important branching point in the metabolism
of LAB. Besides its role as central intermediate, pyruvate can be an effective H2O2
scavenger since it reacts non-enzymatically with H2O2, producing CO2 and acetate. The
scavenging effect of pyruvate is clearly illustrated by the observation that exogenously
provided pyruvate can protect against H2O2-killing in different types of cells (159-161).
L. lactis is reported to accumulate substantial amounts of pyruvate (>90 mM) when
grown at low growth rates (48, 162). This excretion of pyruvate was found to effectively
reduce extracellular H2O2 levels and accompanying oxidative stress (48). It is not clear
whether this results from enhanced production rates or from reduced dissipation rates
due to stress-induced reduced function of metabolic enzymes (163).
d. Cystine metabolism
Cystine is a dimer of two cysteine molecules. The supplementation and uptake of cystine
by a cystine-binding protein encoded by cyuC was shown to prevent H2O2 accumulation
in L. reuteri BR11 (164). The intracellular conversion of cystine into smaller thiols such as
H2S is catalyzed by cystathionine-γ-lyase. The gene encoding cystathionine-γ-lyase (cgl)
is located in the same operon as cyuC, which also encodes a predicted cystine ABCtransporter (122, 165-167). The aerotolerance and oxidative stress resistance that this
operon confers to L. reuteri BR11 indicates that such thiol metabolism may be involved
in alternative pathways of oxidative stress resistance in LAB.
29

8. ROS production by LAB
Of the abovementioned enzymes and physiological adaptations that are correlated to
oxidative stress, only a few (SOD, catalase and AHPR) were shown to contribute to ROS
scavenging and prevent the accumulation of the most stable ROS species H2O2. An
analysis of the prevalence of these proteins in LAB leads to the conclusion that these
proteins are absent in LAB like S. pneumoniae and L. johnsonii. These species only encode
proteins such as TRXR, TPX and GSHR (Table 1.2), for which expression was found to be
associated with oxidative stress resistance but in vivo ROS-scavenging functionality was
not proven. An indication that these proteins are not, or only moderately involved in in
vivo ROS scavenging is provided by the relatively high-level of H2O2 production during
aerobic growth of LAB encoding these proteins.
Hydrogen peroxide production by species of the L. acidophilus group and some
streptococci presents an intriguing dilemma since the accumulating H2O2 leads to
growth stagnation and cell death. One would assume that this creates a substantial
evolutionary pressure for the acquisition and expression of genes encoding ROSscavenging proteins. The expression of either catalase, SOD or AHPR appears as an
almost zero-cost option which would allow these species to counteract the damaging
side-effects of the presence of oxygen. Nevertheless, despite their lack of ROS scavenging
capacities, that are considered essential for aerotolerance in other bacteria, these H2O2
producing species show remarkable aerotolerance, with an initial aerobic growth rate
that is comparable to the anaerobic growth rate and growth impairment only occurring
when endogenously produced H2O2 reaches millimolar levels.
9. Concluding remarks
In this literature review we have provided an overview of the different protein expression
and physiological adaptations LAB use to deal with oxygenated environments. We
find three overarching themes that play a role in aerotolerance and oxidative stress
resistance of LAB.
1
The extent in which LAB can profit from the presence of oxygen.
Hemin supplementation induces a respiratory phenotype in several LAB, which
results in higher biomass levels and lower oxidative stress. Acetate production
through the POX pathway can result in additional ATP generation. Furthermore,
acetate and CO2 production through the POX pathway could relieve growth
dependencies of some LAB and thereby expand the environmental niches that
30
can be colonized by these species (chapter 4 this thesis).
2 The extent in which a cell generates toxic derivatives during oxygen exposure.
Several oxidases such as NOX, POX, LOX and NFR generate ROS which cause
1
oxidative stress. Aerobic lactate utilization in many LAB (through LDH/LOX
and POX, facilitated by NAD+ recycling by NOX) can cause cell death due to
concomitant H2O2 production.
3 The extent in which a cell is resistant against the toxic derivatives of oxygen (ROS).
LAB express several ROS scavengers, such as thiol-based peroxidases, catalase
and SOD which are effective in protecting against oxidative stress. Furthermore,
regulation of metal homeostasis, such as intracellular accumulation of
manganese, and decreasing iron dependency of enzyme functions, leads to a
cellular physiology that is more oxidative stress-tolerant. The absence of ROS
scavengers are correlated with a considerably lower aerobic biomass levels.
We conclude that these cellular mechanisms underlie the general anaerobic aerotolerant
nature of LAB. The diversity in phenotypes with respect to oxygen that is encountered
within this group of bacteria, can in part be explained by the expression of oxidases
and oxygen-dependent pathways, by the presence or absence of ROS scavengers and
by physiological adaptations.
10. Lactobacillus johnsonii
Lactobacillus johnsonii NCC 533 (previously referred to as Lactobacillus acidophilus La1)
is a gram-positive, rod-shaped, non-sporulating, bacteriocin and exopolysaccharideproducing lactic acid bacterium, with a low G-C content (34.6%), belonging to the
Lactobacillus genus of the Firmicutes phylum. Within the genomically diverse Lactobacillus
genus, L. johnsonii is assigned to the L. acidophilus group on the basis of similarities in
DNA and rRNA sequence (168). This group of bacteria has received extensive attention
due to their proposed probiotic properties (169-171), their occurrence in the microbiota
of the gastro-intestinal tract as well as those encountered in the oral and vaginal
cavities in humans (94). Furthermore, species such as L. delbruecki subsp. bulgaricus, L.
kefirofaciens and L. helveticus are prominently present in different starter cultures for
the production of yoghurt, cheese and kefir. Importantly, to date, none of the species
of the L. acidophilus group has been recognized as a potential pathogen in mammals.
There has been a great effort in the last two decades to improve taxonomical classification
31

of this group, especially in order to refine the analysis of the enormous amount of human
intestine microbiota data that have appeared over the past decade and to support the
characterization of novel candidate probiotic strains. Comparative genomics studies
revealed a high similarity at DNA, rRNA, protein, as well as metabolic level between the
species of the L. acidophilus group (168, 172-175). For many of the species belonging
to this group of lactobacilli there are now multiple genome sequences available and
the group is continuously expanding due to the identification and sequencing of novel
strains and species, which are often isolated from the GI-tract of mammals.
L. johnsonii was the second species of the Lactobacillus-genus for which the complete
genome sequence was published (176). The sequence indicates that this species has
been subject to a process of reductive evolution, displaying a remarkable loss of genes
(177, 178). L. johnsonii lacks the genes for the biosynthesis of numerous compounds,
including secondary metabolites, amino acids, vitamins, and fatty acids. This is reflected
in its fastidious growth requirements and its lack of metabolic versatility.
L. johnsonii has been of particular interest to the food industry for its health-supporting
properties. It is commercially used as the probiotic ingredient in the LC-1 fermenteddairy products marketed by Nestlé. In this application context, several studies have
specifically looked at features related to adherence to the gut epithelium (171), its
activity against enterovirulent pathogens in vitro and in vivo (38, 179, 180) and its
immunomodulatory properties (181-183). In addition, broader understanding of the
physiology and metabolism of L. johnsonii has been of relevance (184-186) to improve
its survival and robustness in the harsh conditions encountered in industry and in the
upper-gastrointestinal tract. These metabolic characteristics, such as its fastidious
growth requirements, may also impact the interactions of L. johnsonii with its host
organism.
11. Outline of this thesis
L. johnsonii is, like other LAB, an aerotolerant anaerobe. Aerobic growth of this species
is accompanied by the production of millimolar amounts of H2O2 (38), which in part
results from the absence of several of the ROS-scavenging mechanisms present in other
LAB (Table 1.2). This H2O2 production is an intriguing characteristic which L. johnsonii
shares with several lactobacilli (94, 187) and streptococci (74, 85, 86). The enzymes
involved in H2O2 production in L. johnsonii had previously not been elucidated.
One of the primary goals of this thesis was to study the molecular mechanisms underlying
32
H2O2 production in L. johnsonii, e.g. to identify and characterize the enzymes that
catalyze the H2O2 producing reactions and decipher the role of H2O2 production in the
aerotolerance of L. johnsonii. In more generic terms the thesis also intends to provide
a more global understanding of molecular responses of L. johnsonii to the presence of
1
molecular oxygen.
In the first two experimental chapters of this thesis, we partially unravel the H2O2
producing reactions and aerobic metabolism in Lactobacillus johnsonii. In chapter 2,
we identify and characterize the main H2O2 producing enzyme in L. johnsonii, which
belongs to a novel NADH flavin reductase (NFR) enzyme family. In chapter 3 the
genome-wide transcriptional response of L. johnsonii to the presence of oxygen is
studied, leading to identification of a secondary H2O2 producing enzyme, which belongs
to the NADH oxidase enzymes and appears essential for aerotolerance in the absence
of NFR.
The relationship between specific growth dependencies of L. johnsonii in the presence
and absence of oxygen are subsequently studied in chapters 4 and 5. In chapter 4 we
demonstrate that oxygen is not only detrimental for L. johnsonii, but can also relieve
some of its growth dependencies. Endogenous acetate and CO2 production through
the oxygen-dependent POX-reaction overcomes the dependency of this bacterium on
exogenous C1 and C2- sources. In chapter 5, the growth dependency of L. johnsonii
for CO2 is further characterized by analyzing the genome-wide transcriptional response
to CO2 depletion. These analyses indicate that especially the pyrimidine biosynthesis
pathway is depending on a CO2 supply.
In chapter 6 the experimental results are discussed in the light of our current knowledge
of the physiology of these types of lactobacilli. In addition, we discuss the implications
of O2 and CO2 metabolism by L. johnsonii and other lactobacilli in the context of their
interaction with mammalian host organisms.
33
34
HemCat
lp_3578
-
-
-
HemCat
(query)
L. plantarum
WCFS1
L. lactis subsp.
cremoris MG1363
L. rhamnosus GG
L. casei BL23
E. faecalis V583
HemCat
ef_15971
1 TRXR, ef_1338
2 TRXR, ef_2738
3 PNDR, ef_2899
1 TRXR, lcabl_10620
2 TRXR, lcabl_08900
1 TRXR, lgg_00920
2 TRXR, lgg_00810
1 TRXR, llmg_1588
2 AHPR, llmg_0357
3 TRXR, llmg_0776
1 TRXR (query)
2 ferredoxin NAD(P)
reductase, lp_2585
TrxR2
lp_0761
AhpC/TSA
family protein,
ef_2932
TPX,
lcabl_08080
TPX,
lgg_00728
TPX,
llmg_0318
TPX, lp_2323
Tpx3
SPD_1464
1 GSHR, lcabl_23620
2 GSHR, lcabl_27950
3 DLD, lcabl_15390
4 DLD, lcabl_16690
1 GSHR, ef_3270
2 DLD, ef_1356
3 DLD, ef_1661
1 GSHR, lgg_02615
2 DLD, lgg_01323
3 TrxR, lgg_00920
1 GSHR, llmg_1702
2 DLD, llmg_0071
3 PNDR, llmg_2331
4 TRXB1, llmg_1588
1 GSHR, lp_1253
2 GSHR, lp_3267
3 GSHR, lp_1822
4 GSHR, lp_0369
5 DLD, lp_2151
GshR4
(accession nr. A1YAC0)
AhpC, ef_2739
AhpC lcabl_26730
AhpC,
lgg_02490
AhpC,
llmg_0356
-
AhpC5 (accession
nr. O66265)
1 NPR, lp_2544
2 NOX, lp_3449
3 NPR, lp_1445
4 NOX, lp_0760
5 NOX, lp_1941
6 NOX, lp_0766
7 NOX, pWCFS103_16
8 NOX, lp_1925
9 DLD, lp_2151
10 GSHR, lp_0368
1 NOX, llmg_0408
2 NOX, llmg_1770
3 hypothetical llmg_1249
4 PNDR, llmg_2331
5 DLD, llmg_0071
1 NPR lgg_00491
2 NOX lgg_00325
3 NOX lgg_00212
4 PNDR, lgg_00175
5 DLD, lgg_01323
1 NPR, lcabl_04690
2 NOX lcabl_ 02800
3 NOX lcabl_01750
4 DLD, lcabl_15390
1 NPR (query)
2 NOX, ef_1586
3 coenzyme A disulfide
reductase ef_2989
4 PNDR, ef_1932
5 DLD, ef_1356
NPR6
ef_1211
MnSOD,
ef_0463
MnSOD,
lcabl_20710
-
MnSOD
(query)
-
MnSOD7
llmg_0429
Table S1.1; Antioxidative/ROS-scavenging mechanisms in LAB genomes. Cut-off values for BlastP: maximum e-value: 10-10, minimum identical 20%, minimum query coverage
60%).
Supplementary materials

-
-
-
-
S. mutans UA159
L. sanfranciscensis
TMW 1.1304
L. delbrueckii subsp
bulgaricus ATCC
11842
L. johnsonii NCC
533
TPX, jw_1317
TPX, lj_1153
-
Hypoth.,
lsa_05160
Lipid hydroperoxide
peroxidase,
smu_924
TPX, stu_0990
TPX (query)
-
1 hypoth., lj_0042
2 PNDR, lj_1757
3 TRXR, lj_0852
1 GSHR, jw_3467
2 DLD, jw_0112
3 PNDR, jw_5040
4 PNDR, jw_5551
1 PNDR, ldb_0759
1 hypoth. lsa_2p00270
1 GSHR spd_0685
2 PNDR,spd_1415
3 DLD, spd_1025
1 GSHR, stu_0408
2 DLD, stu_1048
3 PNDR, stu_0557
1 GSHR, smu_140
2 GSHR, smu_838
3 DLD, smu_1424
4 DLD, smu_130
1 GSHR, l897_03320
2 DLD, l897_03940
AhpC, jw_1106
-
-
-
DLD, jw_0112
PNDR, lj_1757
PNDR, ldb_0759
NOX, lsa_05610
1 NOX, smu_1117
2 DLD, smu_1424
AhpC, smu_764
-
1 NOX, spd_1298
2 PNDR, spd_1415
3 DLD, spd_1025
1 NOX, stu_1281
2 PNDR, stu_0557
1 NOX, l897_0435
2 NPR, l897_06900
-
AhpC,
l897_08790
1 MnSOD,
jw_3879
2 SOD [Fe],
jw_1648
-
-
-
MnSOD,
smu_629
MnSOD
stu_0720
MnSOD,
spd_0667
MnSOD,
l897_05695
1 Hemin-dependent catalase (HemCat) from E. faecalis, (96), 2 Thioredoxin reductase (TRXR) L. plantarum WCFS1 (113), 3 Thiol peroxidase (TPX) from S. Pneumoniae D39
(116), 4 Glutathione reductase (GSHR) L. sanfranciscensis DSM 20451 (122), 5 Alkyl hydroperoxide reductase (AhpC), S. mutans NCIB 11723 (74), 6 NADH peroxidase (NPR) from
Enterococcus faecalis V583 (117), 7 Manganese-superoxide dismutase (MnSOD) L. lactis MG1363 (48), DLD = dihydrolipoamide dehydrogenase, PNDR = pyridine nucleotidedisulphide reductases/oxidoreductases, TPX = thiol peroxidase, GSHR= glutathione reductase, TRXR= thioredoxin reductase, NDH = NADH dehydrogenase, AHPR = alkyl
hydroperoxide reductase
1 TRXR, jw_0871
2 AHPR, jw_0599
-
S. thermophilus
LMG 18311
HemCat,
jw_1721
1 TRXR, lj_0852
2 hypoth., lj_0501
-
S. pneumoniae D39
E. coli K-12
1 TRXR, l897_06810
2 NDH, l897_08795
3 ferredoxin NADP reductase, l897_03465
4 DLD, l897_03940
1 TRXR, spd_1287
2 PNDR, spd_1393
3 PNDR spd_1415
1 TRXR, stu_1650
2 TRXR, stu_1417
3 DLD, stu_1048
1 TRXR, smu_463
2 AHPR, smu_765
3 TRXR, smu_869
4 DLD, smu_130
5 DLD, smu_1424
1 TRXR, lsa_05170
2 ferredoxin NADP
reductase, lsa_02530
1 TRXR, ldb_0613
2 TRXR, ldb_1586
-
S. pyogenes HSC5
1
35

36
Chapter 2
Hydrogen peroxide production in species of the
Lactobacillus acidophilus group, a central role for
a novel NADH dependent flavin reductase.
Rosanne Hertzberger1,3,4, Jos Arents1, Henk L. Dekker1, R. David Pridmore2,4, Christof
Gysler2,4, Michiel Kleerebezem3,5, M. Joost Teixeira de Mattos1.
1) Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park,
Amsterdam, The Netherlands
2) Nestlé Research Centre, Vers-chez-les-Blanc, Switzerland
3) NIZO food research, Ede, The Netherlands
4) Kluyver Centre for Genomics of Industrial Fermentation, The Netherlands
5) Host Microbe Interactomics Group, Wageningen University, Wageningen, The
Netherlands
Published in Applied and Environmental Microbiology, (2014), 80:2229-39

Summary
Hydrogen peroxide production is a well-known trait of many bacterial species associated
with the human body. In the presence of oxygen, the probiotic lactic acid bacterium
Lactobacillus johnsonii NCC 533 excretes up to 1 mM H2O2, inducing growth stagnation
and cell death. Disruption of genes commonly assumed to be involved in H2O2 production,
e.g. pyruvate oxidase, NADH oxidase and lactate oxidase, did not affect this. Here we
describe the purification of a novel NADH-dependent flavin reductase, encoded by two
highly-similar genes (LJ_0548 and LJ_0549) that are conserved in lactobacilli belonging
to the Lactobacillus acidophilus group. The genes are predicted to encode two 20 kDa
proteins containing FMN_red conserved domains. Reductase activity requires FMN, FAD
or riboflavin and is specific for NADH, and not NADPH. The Km for FMN is 30 ± 8
µM in accordance with its proposed in vivo role in H2O2 production. Deletion of the
encoding genes in L. johnsonii led to 40-fold reduced H2O2 formation, which could
only be restored by in trans complementation of both genes. Our work identifies a
novel, conserved NADH-dependent flavin reductase that is prominently involved in H2O2
production in L. johnsonii.
38
Introduction
Hydrogen peroxide (H2O2) production is a well-known capacity of several bacterial
species that are associated with the human body. Some of these H2O2-forming species
are opportunistic-pathogens or pathobionts, such as Streptococcus pyogenes (86),
Streptococcus mutans (74, 189), and Streptococcus pneumoniae (91, 190). Other H2O2
producing species or strains have been proposed to have probiotic properties, such as
Bifidobacterium bifidus (69) and Lactobacillus johnsonii (38), or are prevalent in the
commensal vaginal microbiota such as Lactobacillus crispatus, Lactobacillus jensenii and
2
Lactobacillus gasseri (94).
Accumulation of H2O2 mainly occurs in species that lack the main H2O2-scavenging
enzymes, such as catalase and NADH peroxidase. Analogously, when the genes encoding
these enzymes are deleted from the Escherichia coli genome, H2O2 is generated upon
oxygenation and accumulates in the extracellular growth medium (95, 191). Hydrogen
peroxide is mainly produced in central carbon and energy metabolism by oxidases,
including pyruvate oxidase (Pox), lactate oxidase (Lox) and NADH oxidases (Nox) (32).
For example, activity of lactate oxidase encoded by S. pyogenes is primarily responsible
for H2O2 production in cells that are depleted for glucose (192, 193), whereas H2O2
production by S. pneumoniae is due to pyruvate oxidase activity (91). In several
species, H2O2-producing NADH oxidases have been identified: for instance in Thermus
thermophilus (194), S. mutans (74) and Amphibacillus xylanus (72).
Species that accumulate H2O2 or other reactive oxygen species (ROS) upon exposure to
molecular oxygen generally have an energy metabolism that is adapted to anaerobic
environments. The proteins that catalyze the low-potential redox reactions in anaerobic
energy metabolism, such as fumarate and nitrate respiration, commonly carry lowpotential metal clusters and solvent-exposed flavin cofactors that readily react with
oxygen and contribute to the generation of ROS. H2O2 and superoxide (O2-) belong
to the strongest oxidant-species and can accelerate the rate of ROS generation, the
chemistry of which has been reviewed previously (6).
This study focuses on the H2O2 producing species L. johnsonii. This organism is applied
as a probiotic supplement in the food industry (176). The strain was isolated from
the human intestine, where it interacts with the host epithelium as well as with
other microbes (170, 171, 195). The gastro-intestinal (GI) tract is predominantly an
anaerobic niche, but the presence of oxygen gradients in the proximity of the mucosal
surfaces is well established (40, 41, 196). Hydrogen peroxide derived from species like
39

L. johnsonii may play a role in these environments. Several studies have speculated on
the effect that H2O2 may exert on the host as well as on the microbiome. Some authors
propose that it can directly damage the epithelium (62, 197) and cause cell death of
other bacteria (38, 190). Others suggest that H2O2 accumulation may contribute to
the maintenance of a normal and homeostatic microbiota. Especially for the vaginal
microbiota, strong evidence exists that women carrying H2O2-producing lactobacilli are
less prone to develop bacterial vaginosis (187, 198), which is a very common disease
and an independent risk factor for the acquisition of sexually transmitted disease and
preterm birth (199, 200).
Despite the data that support this hypothesis, the mechanism for the proposed
homeostatic effect of H2O2 producing lactobacilli in the microbiota remains largely
unknown. It has been suggested that H2O2 can contribute to the anti-inflammatory
effect of commensal and probiotic bacteria through its influence on the peroxisome
proliferator activated receptor-γ (PPAR-γ) which plays a central role in regulation of
intestinal inflammation and homeostasis (201, 202). Expression of PPAR-γ is induced in
vivo and in vitro by the presence of L. crispatus and is inhibited by the addition of either
catalase or glutathione, pinpointing H2O2 as the responsible factor for the observed
induction (50). A recent study on development of type 1 diabetes in rats substantiated
the role in immune modulation by bacterially derived H2O2. Here, H2O2 directly affected
the activity of indoleamine 2,3-dioxygenase which is an important immune modulator
(203).
Members of the L. acidophilus group are frequently encountereda the important microbial
groups involved in H2O2 production in the vaginal and GI-tract microbiota. This group of
lactobacilli encompasses several closely related species (168, 204), including those that
are proposed to confer probiotic effects to consumers (L. johnsonii, L. gasseri, and L.
acidophilus) as well as several important organisms in food fermentations (Lactobacillus
delbruecki subsp. bulgaricus, Lactobacillus kefiranofaciens and Lactobacillus helveticus).
Although many studies have reported on the H2O2 production by species of the L.
acidophilus group, our understanding of the enzymatic reactions and mechanisms
underlying these observations remains limited to the notion that NADH and flavin are
involved in the reaction (37, 93) and that it is catalyzed by a protein that is constitutively
expressed (94). The enzymes that catalyze the H2O2-generating reactions remain
uncharacterized, to date.
In this study, we identify a novel NADH-dependent flavin reductase as the primary source
40
for H2O2 in anaerobically grown L. johnsonii NCC 533, a member of the L. acidophilus
group, upon exposure to oxygen. The enzyme is encoded by two small consecutive
genes that show high similarity and are conserved throughout the L. acidophilus group.
Mutation of these genes in L. johnsonii NCC 533 led to a strain that failed to produce
H2O2 upon exposure to molecular oxygen.
Materials & Methods
Bacterial strains and culture conditions
2
L. johnsonii NCC 533 was obtained from the Nestec Culture Collection and cultured in
commercial MRS medium (Merck, Whitehouse Station, NJ, USA) (205) at 37°C under
static conditions, with minimal headspace for 16 hours. The deletion strains
strains NCC 9333,
NCC 9334, NCC 9337 and NCC 9359 were precultured in MRS containing 5 µg ml-1
erythromycin, while 5 µg ml-1 chloramphenicol was added for the NCC 9359 strains
carrying plasmid pDP1016, pDP1017 or pDP1019.
Growth and H2O­2 production
Cells were grown overnight in closed static tubes at 37°C in LAPTg medium. (20 g L-1
glucose, 10 g L-1 yeast extract, 10 g L-1 bacto peptone 10 g L-1 bacto tryptone plus 1 g L-1
Tween 80). This medium was used instead of the regular MRS medium for lactobacilli,
because the meat extract in MRS was found to interfere with the enzymatic assay for
H2O2. Cell density was determined by measurement of the optical density at 600 nm.
H2O2 concentrations were determined using the phenol red assay (described below).
Cell extracts
Bacterial cultures were grown in 1 L bottles that were filled to the top with MRS medium,
to minimize the headspace volume. Cultures were inoculated with 5 mL overnight
precultures in the same medium and incubated for 24 hours at 37°C with continuous
stirring. Cells were harvested by centrifugation (5’, 2600 x g, 4°C) and the cell pellets
were suspended in 50 mL of 50 mM potassium phosphate buffer (pH 7.0) with 2 mM
EDTA and 25 mM NaCl. Lysozyme (Sigma-Aldrich, St. Louis, MO, USA) was added to a
final concentration of 1 mg mL-1 and incubated for 30 minutes at 37°C. Subsequently,
cells were disrupted by 3 rounds of 1 minute sonication at 100W (Branson Ultrasonics,
Danbury, CT, USA) and cooled on ice water. Crude cell debris was removed from the
disrupted cell suspension by low-speed centrifugation (5’, 2600 x g, 4°C), followed
41

by ultracentrifugation of the supernatant (60’, 165000 x g, 4°C), generating the cell
extract (supernatant) that was used in subsequent purification steps and enzyme
assays. Protein concentrations of cell extracts were determined using the MicroBCA
(microbicinchoninic acid) assay kit (Thermo Fisher, Scientific Inc., Waltham, MA, USA).
Alternatively, 50 mL overnight anaerobic cultures in MRS were centrifuged, suspended
in 50 mM potassium phosphate buffer (pH 7.0) and transferred to screw-cap tubes
with 100 mg zirconium beads. Cells were disrupted in 3 rounds for 20 seconds and cell
debris was removed by centrifugation (10’, 21500 x g, 4°C). Protein concentrations of
cell extracts were determined using the MicroBCA assay kit (Thermo Fisher Scientific
Inc). These extracts showed a 2-fold lower enzyme activity overall. They were employed
for analysis of the activity, Km determination and SDS-gel.
Protein purification: ammonium sulfate precipitation, Q column, gel filtration and SDS gel
Cell extracts were placed in a beaker at 4°C and ammonium sulfate was added slowly
under continuous stirring until reaching intermediate steps (30, 50, 70 and 90%)
of saturation. Next, the cell extract was left without stirring on ice for 20 min and
subsequently spun down (10’ min, 12000 x g, 4°C). The precipitate was suspended in
50 mM potassium phosphate buffer (pH 7.0) and dialyzed overnight at 4°C against 3 L
20 mM Tris buffer, pH 8.0.
Anion-exchange chromatography was carried out with an Äkta FPLC system (GE
Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) fitted with a Hi-Trap Q
HP 5 mL column (GE Healthcare). As a loading buffer, 20 mM Tris, pH 8.0 was used at a
rate of 4 mL min-1 and a linear gradient was applied from 0 to 1 M NaCl in 20 column
volumes. The fraction showing NADH oxidase activity (see below) was concentrated
to 0.2 mL using a 10K Corning Spin-XUF6 column. This concentrated fraction was
subjected to further fractionation by size exclusion chromatography using a Superdex
200 HR 10/30 column (GE Healthcare) in 20 mM Tris buffer (pH 8.0) with 250 mM NaCl
at 0.5 mL min-1.
For the SDS-gel electrophoresis, cell extracts were boiled for 5 minutes with SDS
sample buffer (50 mM Tris-HCl pH 6.8, 100 mM dithiothreitol [DTT], 2% SDS, 0.1%
bromophenol blue, 10% glycerol) and loaded on a 15% SDS gel with a 7% stacking
gel on a Hoefer system (Thermo Scientific). The amount of sample that was applied was
corrected for the variations in OD600 of the culture. The prestained marker Pageruler
Plus from Fermentas (Thermo Scientific) was used. Gels were stained with PageBlue
42
Protein Staining Solution (Fermentas Coomassie G-250 dye).
Enzyme activity assay
NADH dependent flavin reductase activity in crude extract (for preparation, see
above) was analyzed by determination of the NADH dissipation, as well as through
the determination of the final H2O2 concentration. NADH oxidation was measured by
monitoring the absorption at 340 nm at 37°C in a 200 µL reaction mixture with 500 µM
NADH or 500 µM NADPH. As a flavin source either 250 µM flavin adenine dinucleotide
2
(FAD) or 25 µM FAD, flavin mononucleotide (FMN) or riboflavin was added. The reaction
mixture was buffered by 50 mM potassium phosphate buffer at pH 7.0.
For H2O2 measurement, the same reaction mixture in 200 µL volume was used with a
lower NADH concentration (250 µM) to prevent oxygen from becoming the limiting
substrate. After 10 minutes of incubation, the H2O2 concentration was determined
with a phenol red enzymatic assay by transferring 20 µL sample to 180 µL of a reaction
mixture containing 5 µg mL-1 horseradish peroxidase (Roche, Penzberg, Germany) and
30 µM phenol red in water. After 5 minutes of reaction, pH was increased by the
addition of 10 µL 1M NaOH. Absorption was determined at 620 nm.
These enzymatic activity assays were employed to detect protein activity in the fractions
that were obtained from the three protein purification steps. Furthermore, the assays
were used to determine the enzyme activity level in cell extracts of mutant strains. In
the latter instance, 25 µM FMN was used as the flavin source. Enzyme activities are
expressed as specific activity per milligram of protein in the cell extract per minute and
were measured in triplicate.
The specificity constant Km was determined by measuring NADH consumption
rate of the cell extract in the presence of various concentrations of FAD, FMN
and riboflavin (2.5 – 250 µM). The Km is calculated by fitting a hyperbolic curve
(Vmax * Cs / (Km­ + Cs), with Cs for flavin concentration and optimization for Vmax and
Km using the Solver function in Microsoft Excel. (R2 >0.96). Averages and standard
deviations are calculated from technical triplicates.
Digestion, MS analysis, and protein identification
Semipurified protein samples were digested with an in-house protocol using 1 mg of
trypsin (modified to prevent autodigestion, Promega, Madison, WI, USA) per 50 mg of
43

protein in a 0.1 M Tris pH 7.5 buffer following alkylation of the cysteine residues with
DTT and iodoacetamide. Trypsin digestion was stopped after 16 hours by the addition
of 10% trifluoroacetic acid (TFA) to a final concentration of 1%. Tryptic peptides were
purified using an 80 µg capacity OMIX tip (Varian, Agilent) and collected in a volume of
30 µL 50% acetonitrile (ACN) - 0.1% TFA.
Mass spectrometry analysis of the peptide samples was performed with a Micromass
Q-TOF1 (quadrupole time of flight) mass spectrometer (Micromass, Waters, Milford, MA,
USA) coupled to a nano-liquid chromatography (nano-LC) system (LC Packings, Dionex,
Sunnyvale, CA, USA). The peptides were separated on a nano-analytical column (75 µm
i.d., 25 cm length C18 PepMap, Dionex) using a gradient of 0–50% acetonitril and 0.1%
formic acid. The LC eluent flow of 300 nL min-1 was directly infused into the Q-TOF1
spectrometer, operating in data-dependent MS and tandem MS (MS/MS) modes. Lowenergy collision-induced dissociation (CID) of selected precursor ions was used to obtain
fragmentation spectra of the peptides. After processing the raw data with the Masslynx
software (Micromass, Waters) the resulting peaklist (.pkl file) was used to search in the
NCBInr database with MASCOT online (Matrix Science, Boston, Ma, USA).
The search parameters were: a fixed modification of carbamidomethyl for cysteine,
variable modifications of oxidized methionine, trypsin with the allowance of one
missed cleavage, peptide and MS/MS tolerance ±0.3 Da and peptide charge state +1.
Probability based MASCOT scores were used to evaluate the protein identifications.
Construction of L. johnsonii deletion strains
An overview of the mutants and plasmids used in this study can be found in table
2.1. An overview of all primers can be found in Supplementary material Table S2.1.
The genome sequence of L. johnsonii is deposited in GenBank under accession no.
AE017198 (176).
44
Table 2.1 List of strains and plasmids used in this study.
Strain
Description
NCC 533
Lactobacillus johnsonii strain from the Nestec Culture Collection
NCC 9333
Δpox (chapter 4)
NCC 9334
ΔLJ_1826 (predicted to encode lactate oxidase; Lox)
NCC 9337
ΔLJ_1254 and LJ_1255 (predicted to encode NADH oxidase; Nox)
NCC 9359
ΔLJ_0548 and LJ_0549 (predicted to encode NADH flavin reductase)
pDP749
pDP889
Temperature sensitive, allele exchange plasmid for L. johnsonii NCC 533
(185)
pDP749 construct for lox (LJ_1826) deletion
pDP902
pDP749 construct for nox (LJ_1254 and LJ_1255) deletion
pDP1010
pDP749 construct for LJ_0548 and LJ_0549 deletion
pDP794
pDP1016
pNZ124 based expression plasmid with LJ_0045 promoter and LJ_1125
terminator
pDP794 with LJ_0548 expression plasmid
pDP1017
pDP794 with LJ_0549 expression plasmid
pDP1019
pDP794 with LJ_0548 and LJ_0549 expression plasmid
2
The construction of the pox-deletion strain NCC 9333 is described in the Materials &
Methods section of chapter 4. The deletion of the gene LJ_1826, predicted to encode a
lactate oxidase enzyme, was achieved similarly: the 5’ homology region of the LJ_1826
gene was amplified from L. johnsonii NCC 533 genomic DNA using primers A and B.
The 1077 bp amplicon was SacI-BamHI digested and cloned in SacI-BamHI digested
pDP749, yielding an intermediate plasmid. The 3’ region of the LJ_1826 gene was
amplified using the primers C plus D, the 1170 bp amplicon digested with PstI-KpnI and
cloned in the similarly digested intermediate plasmid to yield the lox-deletion plasmid
pDP889. Plasmid pDP889 isolated from L. lactis was used to transform NCC 533 (185)
and loop-in/loop-out gene replacement was achieved as described previously (206). The
deletion was confirmed by PCR analysis and the deletion strain was named NCC 9334.
In L. johnsonii NCC 533, NADH oxidase is predicted to be encoded by the genes
LJ_1254 and LJ_1255. Deletion of LJ_1254 and LJ_1255 was achieved in the same way
as for the LOX-encoding gen: the 1008 bp at the 5’ end region of the LJ_1255 gene
was amplified using primers E and F, and the 993 bp at the 3’ end region of the LJ_1254
gene was amplified using primers G plus H. These amplicons were cloned into pDP749
to give plasmid pDP902 and used to produce the nox deletion strain NCC 9337.
The deletion of LJ_0548 and LJ_0549 was achieved by amplification of 1062 bp at the
45

5’ end region of the LJ_0548 gene with primers I plus J and the 1098 bp at the 3’ end
region of the LJ_0549 gene was amplified using primers K plus L. These were cloned
into pDP749 to give plasmid pDP1010 which was used to produce the LJ_0548 and
LJ_0549 deletion strain NCC 9359.
Construction of the L. johnsonii overexpression strains
The L. johnsonii expression plasmid pDP794 was constructed as follows: the predicted
bidirectional terminator situated between the LJ_1125 and LJ_1126 genes of NCC 533
was amplified with the primers O and P. This 359 bp amplicon was digested with the
restriction enzymes HindIII and XhoI and cloned into similarly digested pNZ124 (207)
to yield pNZ124- LJ_1125 trm. The LJ_0045 D-lactate dehydrogenase promoter was
amplified using NCC 533 chromosomal DNA as a template using the primers R plus
S. This 215 bp amplicon was digested with the restriction enzymes BglII plus SacI and
cloned into similarly digested pNZ124- LJ­­_1125 trm to produce plasmid pDP794. This
plasmid, with the promoter region of the LJ_0045 lactate dehydrogenase gene and
a bidirectional terminator, was used for overexpression of the LJ_0548 and LJ_0549
genes.
For the construction of these overexpression plasmids the following cloning steps
were performed: For pDP1016 the gene LJ_0548 was amplified using primers T plus
V. The 597 bp amplicon was digested with SphI and HindIII and cloned into SphI plus
HindIII digested pDP794 to give plasmid pDP1016. For pDP1017, the LJ_0549 gene
was amplified using primers U plus W. The 583 bp amplicon was digested with SphI
and HindIII and cloned into SphI plus HindIII digested pDP794 to give plasmid pDP1017.
For pDP1019 the genes LJ_0548 and LJ_0549 were amplified using primers T plus W,
the 1132 bp amplicon was digested with SphI and HindIII and cloned into SphI plus
HindIII digested pDP794 to give plasmid pDP1019. These cloning procedures yielded
plasmids on which expression of LJ_0548 and/or LJ_0549 is controlled by the strong ldh
promoter. Genetic maps of plasmids pDP1016, pDP1017 and pDP1019 were created
using Clone Manager (Supplementary materials Figure S2.1).
Growth in batch culture
Aerotolerance of NCC 9359 was compared to wildtype L. johnsonii in continuously
stirred vessels with 400 mL MRS medium. Batches were sparged with specific gas
mixtures containing 5% CO2 and either no oxygen (0% oxygen, anaerobic) or normal
46
oxygen levels (20% oxygen, aerobic). Cultures were grown at 37°C with continuous
mixing (ca. 200 rpm) and pH was maintained at 6.5 by automated 4M NaOH titration.
Cell densities were determined by measuring the optical density at 600 nm (OD600).
Maximum specific growth rate was determined by fitting an exponential trend line
through the data points with a minimal R2 of 0.99.
Organic acid measurement by HPLC
Extracellular metabolite concentrations were determined as described previously (208)
2
using high pressure liquid chromatography (HPLC, LKB and Pharmacia, Oregon City,
OR, USA) fitted with a Rezex organic acid analysis column (Phenomenex, Torrance, CA,
USA) at 45°C and an RI 1530 refractive index detector (Jasco, Easton, MD, USA). The
mobile phase consisted of a 7.2 mM H2SO4 solution. Chromatograms were analyzed
using AZUR chromatography software (St. Martin D’Heres, France).
Statistical analysis
Statistical significance was determined using a Student’s two tailed t-test for unequal or
equal variance. An F-test was employed to verify whether variances could be considered
equal (p>0.05) or unequal (p<0.05).
Results
H2O2 accumulation results in premature growth stagnation during aerobic growth of
L. johnsonii NCC 533
To assess the growth behavior of L. johnsonii NCC 533 in anaerobic and aerobic
conditions, LAPTg medium was inoculated with an overnight culture and incubated at
37°C either in a static tube with minimal headspace (anaerobic) or under continuous
shaking with 10 volumes headspace (aerobic). Growth rates of aerobic and anaerobic
cultures were similar up to an OD600 of 1.0 (Figure 2.1A). However, aerobic cultures
accumulated up to 1 mM H2O2 during growth (Figure 2.1B), leading to growth
stagnation at an approximate density of OD600 1.5. This growth stagnation could be
completely abolished by the addition of 0.5 mg mL-1 catalase to the medium, which
prevented the accumulation of H2O2. These findings show that oxidative stress resulting
from endogenous H2O2 production is the main cause for the observed growth arrest of
L. johnsonii NCC 533 under aerobic conditions.
47

A
B
1,4
4
1,2
1
H2O2 (mM)
OD 600
3
2
1
0,8
0,6
0,4
0,2
0
0
2
4
time (h)
6
8
10
0
0
2
4
time (h)
6
8
10
Figure 2.1: Growth and H2O2 concentration of L. johnsonii NCC 533 in LAPTg medium under anaerobic (square
symbols), aerobic conditions (circular symbols) or aerobic conditions with 0.5 mg ml-1 catalase added to the
medium (triangular symbols). Culture densities were determined by optical density measurement at 600 nm
(Panel A) and H2O2 concentrations were determined by the phenol red enzymatic assay (Panel B). The data
represent duplicate experiments ± standard error of the mean.
H2O2 production is not dependent on predicted pyruvate oxidase, lactate oxidase or
NADH oxidase encoding genes.
The main contributor to H2O2 production in lactic acid bacteria (LAB) has been proposed
to be the oxygen dependent lactate utilization pathway, which oxidizes lactate via
pyruvate and acetyl-phosphate to acetate, generating CO2, ATP, NADH and H2O2 (83,
86). The redox balance in this pathway is proposed to be restored by dissipation of the
NADH via an NADH-oxidase dependent reaction that generates either H2O2 or water.
The oxygen-dependent lactate utilization pathway thereby encompasses three potential
H2O2 producing reactions: (i) pyruvate oxidation (catalyzed by the Pox enzyme), (ii)
lactate oxidation (catalyzed by the Lox enzyme) and (iii) NADH oxidation (catalyzed by
the Nox enzyme).
To assess the contribution to the observed H2O2 production of the genes predicted
to encode these enzymes in L. johnsonii, mutant derivatives of the wild-type strain
were constructed that lack the lactate oxidase encoding gene LJ_1826 (NCC 9334), the
pyruvate oxidase encoding gene LJ_1853 (NCC 9333) or the NADH oxidase encoding
genes LJ_1254 and LJ_1255 (NCC 9337). The H2O2 production capacity of the mutants
was compared to that of the wild-type strain. To this end, all strains were grown
anaerobically (static cultures) in LAPTg medium to an OD600 of ~0.7 and subsequently
transferred to aerobic conditions (shake flask incubation). H2O2 production was
measured after 1 and 2 hours of incubation. Deletion of the predicted pox, lox, or nox
genes did not significantly affect the level of H2O2 production after 1 hour in these
48
strains in comparison with the level produced by the parental strain NCC 533 (Figure
2.2, all p-values >0.05). Exposure to oxygen for two hours resulted in small differences
between H2O2 levels between the strains: the pox mutant produced less (0.48 mM
vs 0.53 mM in the wild type, p<0.05) and the Δnox produced more H2O2 (0.56 mM,
p<0.05). It appears justified to conclude that the oxidative lactate-utilization pathway
is not responsible for the greater part of the H2O2 production, suggesting that an
alternative metabolic conversion may account for the H2O2 production. This suggestion
is in agreement with the observation that in the presence of oxygen, no substantial
production and excretion of acetate occurs (chapter 4).
2
Previously, it has been suggested that L. delbrueckii, a close relative of L. johnsonii,
produces H2O2 via an NADH dependent reaction that is enhanced by the addition of a
flavin source (37, 93, 94). To identify the protein and gene involved in such proposed
enzymatic reaction in L. johnsonii we initiated its purification, using enzyme activity
assays to track the enzyme during purification.
0,7
0,6
mM H2O2
0,5
0,4
0,3
0,2
0,1
0
NCC 533
(wt)
NCC 9333
(Δpox)
NCC 9334
(Δlox)
NCC 9337
(Δnox)
Figure 2.2: H2O2 production of L. johnsonii NCC 533 and the derivatives NCC 9333 (Δpox), NCC 9334 (Δlox) and
NCC 9337 (Δnox). Anaerobic logarithmic phase cultures were transferred to shake flask and incubated at 37°C.
After 1h (open bars) and 2h (closed bars) cells were removed by centrifugation and H2O2 concentrations were
determined in the culture medium, using the phenol red / peroxidase enzymatic assay. Data represent average
of three independent experiments.
Cell extracts of L. johnsonii NCC 533 contain NADH-dependent flavin reductase
activity.
Cell extracts of L. johnsonii NCC 533 contain NADH consumption activity when a flavin
compound is added as a supplement to the assay’s reaction mixture (Figure 2.3A). The
activity requires the addition of either FAD, FMN or riboflavin and does not show any
activity with NADPH instead of NADH. Addition of 10-fold lower FMN concentration
resulted in a significantly lower enzymatic rate (0.21 and 0.12 µmol / mg protein / min,
p<.05). Following this observation, we further explored enzyme kinetics with different
flavin sources at various concentrations. Michaelis-Menten-like kinetics were observed
49

when the flavin concentration is varied (see Materials & Methods for method of Km
calculation), indicating that flavin is a direct substrate for the enzyme. The Km of free
flavin does not significantly differ for the various flavins used: 30 µM ± 8 for FMN, 64
µM ± 21 for FAD and 41 µM ± 10 for riboflavin (p<.05).
B
0,3
0,25
0,25
0,2
Endpoint mM H2O2
µmol NADH/ mg extract / min
A
0,2
0,15
0,1
B
0,05
0
250 µM
FAD
250 µM
riboflavin
250 µM
FMN
NADH
NADH
25 µM FMN no flavin
0,15
0,1
0,05
NADPH
25 µM FMN
0
25 µM
FAD
25 µM
Riboflavin
25 µM
FMN
NADPH,
25 µM FMN
Figure 2.3A and B: Typical NADH dependent flavin reductase activity in L. johnsonii cell extract. NADH consumption
rates were measured by absorption at 340 nm (Panel A). Endpoint H2O2 concentrations were determined using the
phenol red assay (Panel B). Either 500 µM NADH was used, substituted by 500 µM NADPH where indicated (panel
A) or 250 µM NADH substituted by 250 µM NADPH where indicated (Panel B). 250 µM or 25 µM of either FAD,
FMN or riboflavin was added as a flavin source. Protein concentration in the cell abstracts was determined by the
MicroBCA assay. Data represent the average of technical triplicates ± standard deviation and are representative
of cell extracts derived in comparable experiments.
The reaction is likely to involve a two electron transfer reaction since a considerable
amount of H2O2 is formed as an end product, regardless of the flavin form that is added.
When the FMN level in the assay is lowered to 25 µM, the H2O2 concentration exceeds
the flavin concentration more than five fold (135 µM H2O2 ± 33 µM), indicating that the
free flavin is recycled during the reaction (Figure 2.3B). Altogether, these observations
allow the classification of the protein(s) responsible for the measured activity as an
NADH-dependent flavin reductase.
Purification and identification of the NADH-dependent flavin reductase activity.
In order to identify the protein(s) responsible for the NADH-dependent flavin reductase
activity, cell extract of wild type L. johnsonii NCC 533 was subjected to the following
purification steps; (i) ammonium sulfate precipitation, (ii) Q column chromatography
and (iii) gel filtration (see Materials & Methods for details). After the ammonium sulfate
precipitation, the 50%-70% fractions clearly display most NADH consumption. After
the subsequent fractionation of this fraction using anion exchange chromatography,
highest activity clearly eluted after 83 an 86 ml (see Supplementary materials, Figure
S2.2). These fractions were combined and were subsequently further separated by
50
size exclusion chromatography (Superdex2000). Only a single fraction eluted during
this chromatography step that displayed clear H2O2 production in the enzymatic assay
(Figure 2.4). The size of the enzymes in this fraction were, based on its elution time,
estimated to be ~18kDa. The enzyme(s) in this fraction that showed NADH flavin
reductase activity were only partially purified, since multiple bands were apparent when
it was loaded on SDS-gel (results not shown). The active fraction obtained was digested
with trypsin and analyzed using LC-MS/MS, using the fraction preceding this active
fraction (and containing no activity) as a comparative negative-control. In the active
fraction 25 L. johnsonii proteins could be assigned with a probability based score of
2
P<0.05. The protein that was predicted with highest probability was the hypothetical
protein LJ_0548 to which 8 peptides could be assigned with a total coverage of 63%,
indicating a high abundance in the fraction. Three peptides could be assigned to the
hypothetical protein LJ_0549 (33% sequence coverage) which is encoded by LJ_0549,
the LJ_0548 neighboring gene. Noteworthy, the predicted protein sequences of LJ_0548
(accession number Q74HL7) and LJ_0549 (accession number Q74HL8) both contain a
conserved FMN reductase domain, supporting the role of these gene-products in the
NADH dependent flavin reductase activity. Furthermore, no peptides belonging to these
two proteins were detected in the fraction that did not show any NADH flavin reductase
activity but eluted close to the active fraction (negative-control). Taken together, these
observations pointed toward the involvement of the LJ_0548 and LJ_0549 genes in the
NADH flavin reductase activity of cell extracts of L. johnsonii.
80
0,4
0,35
75
0,25
A 280
0,2
65
0,15
0,1
60
0,05
55
50
H 2O2 (mM)
0,3
70
0
0
5
10
15
20
Volume (ml)
25
30
-0,05
35
Figure 2.4: Size-exclusion chromatogram (Superdex200) of final purification step. Protein concentration is
determined by absorption at 280 nm (black line). The eluting proteins were collected in fractions of 1 ml and
tested for NADH flavin reductase activity by addition of 500 µM NADH and 250 µM FAD. H2O2 concentration was
determined after 10 min (symbols).
A mutant derivative of L. johnsonii NCC 533 was constructed that lacks the genes
LJ_0548 and LJ_0549 (NCC 9359). The cell extracts of this mutant strain completely
51

lacked flavin dependent NADH reductase activity that was detected in the extracts
obtained from the wild-type strain, nor could any H2O2 be detected in the reaction
mixture supplemented with the mutant strain extract. These results indicate that the
LJ_0548 and/or LJ_0549 encode the NADH dependent flavin reductase activity. The
LJ_0548-0549 deletion strain (NCC 9359) was complemented by providing one or both
of the deleted genes in trans on a plasmid under expression control of the strong,
constitutive D-lactate dehydrogenase gene promoter (LJ_0045; ldhDp). Cell extracts
derived from the NCC 9359 strain harboring either the LJ_0548- (pDP1016) or the
LJ_0549- (pDP1017) expression plasmid did not show any additional bands on an SDSprotein gel. These cell extracts also did not show any significant NADH-dependent flavin
reductase activity. Conversely, the extract derived from the strain harboring the plasmid
expressing both the LJ_0548 and LJ_0549 genes (pDP1019) displayed an additional
band of 20 kDa on an SDS-gel (Supplementary materials, Figure S2.3) and an NADH
consumption rate that was more than 7-fold higher compared to the rate measured in
extracts derived from the wild-type strain (Figure 2.5A). The level of H2O2 production
driven by the extract derived from the strain overexpressing LJ_0548 and LJ_0549 was
comparable to the level produced by the extract from the wild-type, which reflects the
maximal level of H2O2 production that can be obtained in this assay as a consequence
of the limited amount of NADH provided in the reaction mixture (Figure 2.5B). These
results show that the LJ_0548-0549 operon encodes the observed NADH-dependent
flavin reductase activity.
H2O2 production of the LJ_0548 and LJ_0549 deletion strain
Having identified LJ_0548 and LJ_0549 as coding for the enzymes responsible for
NADH-dependent flavin reductase activity in the cell extracts of L. johnsonii NCC 533,
we studied the in vivo contribution of this activity to the aerobic physiology of this
bacterium by comparing the wild type to its nfr-deletion derivative (ΔLJ_0548-LJ_0549,
NCC 9359). Maximum specific growth rate of the mutant in shake flask was similar to
the wild type and its metabolism remained homolactic (result not shown). However,
when exposed to stronger aeration (750 ml/min, 75% N2, 20% O2, 5% CO2), the
nfr-deletion derivative displayed a reduced growth rate compared to wild type strain,
whereas exposure to the anaerobic gas-mixture equivalent of this regimen (750 ml/min
95% N2, 5% CO2) did not result in a difference between the nfr-mutant and its wild
type counterpart (Supplementary material, Figure S2.4).
52
A
A
B
B
0,18
0,16
1
0,14
0,8
0,12
mmol H2O2
µmol NADH/ mg extract / min
1,2
0,6
0,4
2
0,06
0,04
0,2
0
0,1
0,08
0,02
NCC 533
NCC
9359
NCC
9359
+pDP
1016
NCC
9359
+pDP
1017
NCC
9359
+pDP
1019
0
NCC 533 NCC
9359
NCC
9359
+pDP
1016
NCC
9359
+pDP
1017
NCC
9359
+pDP
1019
Figure 2.5: Typical NADH dependent flavin reductase activity in cell extracts of wild-type L. johnsonii NCC 533
and its LJ_0548 - LJ_0549 (NCC 9359) mutant derivative, with and without complementation by plasmid-borne
expression of one (pDP1016 and pDP1017) or both (pDP1019) of the deleted genes. Standard assay conditions
were employed, containing 500 µM NADH and 25 µM FMN (NADH consumption rates) and 250 µM NADH and 25
µM FMN (to determine H2O2 concentration). NADH consumption rates were measured by absorption at 340 nm
(panel A). H2O2 concentrations were determined after 10 minutes of reaction using the phenol red assay (panel
B). Protein concentrations in the cell extracts were determined by MicroBCA assay. Data represent average of
three technical replicates ± standard deviation and are representative for activity measured in multiple (>3) cell
extracts.
To test H2O2 production, the wild-type strain (NCC 533) and its LJ_0548-0549 deletion
derivatives were grown anaerobically to mid-logarithmic phase (OD600 ~0.8) at 37°C
and were then transferred to aerobic (shake-flask) conditions. H2O2 in the spent
medium of each of the cultures was assessed after 1 hour of oxygen exposure (Figure
2.6). Notably, the LJ_0548-0549 deletion (strain NCC 9359) resulted in complete loss of
the capacity to produce H2O2 under these conditions, while substantial amounts of this
reactive oxygen molecule were detected in the NCC 533 culture exposed to the same
conditions. Moreover, the NCC 9359 mutant strain that was in trans complemented
with plasmid borne expression of either LJ_0548 (pDP1016) or LJ_0549 (pDP1017) did
not produce detectable H2O2 levels, whereas the strain complemented with plasmidborne expression of both LJ_0548 and LJ_0549 (pDP1019) displayed a restored H2O2
production capacity, comparable to that observed in the parental strain NCC 533.
These data confirm that the L. johnsonii NCC 533 NADH-dependent flavin reductase is
encoded by the LJ_0548-0549 cluster and that this activity is the major H2O2 producing
system expressed under the conditions employed here.
In order to test if LJ_0548 and LJ_0549 play a role in anaerobic fumarate respiration of
L. johnsonii, the external metabolite profiles of wild type NCC 533 and NCC 9359 (the
53

LJ_0548 and LJ_0549 deletion derivative) were compared. After 7 hours of anaerobic
growth in MRS medium supplemented with 10 mM fumarate, cells were removed by
centrifugation and external metabolites were analyzed using HPLC. No change in the
concentration of fumarate was observed and no succinate formation was detected. Both
aerobic and anaerobic metabolism of the mutant strains remained entirely homolactic
(results not shown).
0,25
H2O2 (mM)
0,20
0,15
0,10
0,05
0,00
NCC 533 NCC 9359 NCC 9359 NCC 9359 NCC 9359
+ pDP1016 + pDP1017 + pDP1019
Figure 2.6: H2O2 production in L. johnsonii NCC 533 and NCC 9359, with or without complementation of LJ_0548 an
LJ_0549 under aerobic conditions. Anaerobic logarithmic phase cultures of wild type L. johnsonii and its deletion
derivative were transferred to shake flask and incubated at 37°C. After 1h cells were removed by centrifugation
and H2O2 concentrations were determined in the culture medium, using the phenol red assay. Data represent
average of three independent experiments ± standard deviation.
Sequence analysis of LJ_0548 and LJ_0549, and their conservation among bacteria
Using the wealth of genomic sequence availability, the prevalence and context of the
LJ_0548 and LJ_0549 single genes as well as the combination of the two consecutive
genes were analyzed using diverse genome comparison tools.
The LJ_0548 and LJ_0549 genes are predicted to encode proteins of 178 and 184
residues, respectively, that share substantial similarity (40% identity and 59% similarity
at amino acid sequence level). The protein domain signature recognition module
Interproscan (209) revealed that both genes have a highly conserved FMN reductase
domain (PFAM domain 03358), covering the N-terminal 145 residues. In the PANTHER
classification system program (210), LJ_0548 was classified as a chromate reductase,
a group of enzymes that has been annotated as such due to their potential use in
chromate bioremediation (211). The ortholog in Pseudomonas putida has been shown
to catalyze the transfer of electrons from NADH to the quinone pool (212). The crystal
structure of the E. coli gene annotated as chromate reductase clearly demonstrates the
amino acids that constitute the flavin binding site (213). A ClustalW multiple sequence
54
alignment shows that four out of the eleven residues of this binding site are conserved
in both LJ_0548 and LJ_0549, i.e. Ser18, Asn20, Glu82 and Ser 117 (Supplementary
Materials Figure S2.5).
The LJ_0548-0549 locus in L. johnsonii NCC 533 appears to be conserved in all other
members of the L. acidophilus group. Examination of the genetic context in more
distant species using the STRING module (214) revealed that the closest homologues
of LJ_0548 and LJ_0549 are encountered as consecutive genes in several different
species (see Figure 2.7), including L. plantarum, and several species belonging to the
2
Streptococcus, Enterococcus and Pediococcus genera. In these examples, the first of
these two genes is similar in sequence and size to either LJ_0548 or LJ_0549 and
is followed by a second, larger gene, of which the N-terminal residues (~200) are
homologues to LJ_0548. This type of arrangement is also present in a more distant
species from the Actinobacteriaeae class, Atopobium parvulum, which is a species often
found in the human oral cavity(215). In all aforementioned species, the homologues of
LJ_0548 and LJ_0549 are annotated as fumarate reductases, NADH dehydrogenases
or flavin reductases, but to the best of our knowledge there is no experimental data to
support these annotations.
55
Figure 2.7: Genetic context conservation of LJ_0548 and LJ_0549 found using the STRING module.

56
Discussion
Our work has identified a novel NADH-dependent FMN reductase in L. johnsonii
NCC 533, that is encoded by two adjacent genes (LJ_0548 and LJ_0549) and acts
as the major H2O2 producing system in this bacterium. L. johnsonii is a lactic acid
bacterium that belongs to the phylogenetically closely related L. acidophilus group
(168, 216), which includes several strains of Lactobacillus species that are marketed
as probiotic supplements like L. acidophilus, L. johnsonii, L. jensenii, L. crispatus and
L. gasseri, but also encompasses the well-known yoghurt bacterium L. delbrueckii. It
2
has been established that these species endure oxidative stress as a consequence of
endogenously produced H2O2 (35-37). The enzymes identified here have characteristics
that are in agreement with those observed previously in strains of the L. acidophilus
group, including that the enzyme reaction consumes NADH, uses flavin as a cofactor, is
constitutively expressed and is not produced in response to molecular oxygen (37, 93,
94). In addition, the maximal reaction rate measured in cell extracts of L. johnsonii is
in the same order of magnitude as the rate previously reported for L. delbrueckii (37).
Apparent Michaelis-Menten kinetics were observed when the concentration of flavin
was varied in the enzymatic assay. However, the H2O2 produced in the assay reaches a
higher level than the total initial concentration of flavin added to the assay, indicating
that free oxidized flavin serves as a substrate in this reaction but the reduced flavin is
subsequently oxidized and reused. This is in agreement with the behavior of reduced
flavins that spontaneously react with oxygen, yielding H2O2.
Despite the recognition of this enzyme activity in various L. acidophilus group species,
the molecular characteristics and genetic determinant(s) of this activity have not been
described to date. Therefore, our study is the first to identify and characterize this novel
enzyme family and its encoding genes. The enzyme is shown to be responsible for the
major H2O2 production in an industrially relevant member of the L. acidophilus group,
L. johnsonii. This capacity has previously been proposed to influence gut homeostasis
and anti-inflammatory activity of this group of organisms (50) and the identification of
the responsible genes and the construction of the corresponding deletion strain may
accelerate the establishment of this presumed function in vivo, which is assumed to
play a role in the bacterium’s protective effect against vaginal disease (187).
The Vmax and Km values of the different flavins that were tested in the enzymatic assay
did not differ strongly. The Km value for flavins were found in the range of 30-50 µM.
Intracellular flavin concentrations in E. coli and Shewanella oneidensis were reported
57

in the order of 0.5 µmol per gram protein (217), which in combination with the
“rule-of-thumb” estimate of ~200 g/l as the concentration of intracellular protein in
prokaryotes (218) implies that the intracellular flavin concentration would be ~100 µM.
The partitioning of flavin bound to protein and available as electron acceptor for the
proteins we describe here is unknown. One study on intracellular free FAD concentration
in Amphibacillus xylanus finds 13 µM using HPLC (219). Such a flavin concentration in L.
johnsonii would be sufficient for its proposed in vivo role. Given the clear confirmation
of the in vivo role of Nfr by the physiology of the nfr-deletion mutants, we consider it a
valid conclusion that analogous to the previously found concentration in A. xylanus the
free flavin levels in L. johnsonii suffice for significant H2O2 production.
Surprisingly, the genes that are considered to be responsible for H2O2 production in other
LAB were shown to not contribute significantly to this phenotype in L. johnsonii, since
the genes predicted to encode lactate, pyruvate or NADH oxidases could be deleted
without consequences for the H2O2 production in this species. The homologs of LJ_0548
and LJ_0549 have been annotated as fumarate reductase, NADH dehydrogenase or
NAD(P)H dependent FMN reductase. The LJ_0548 and LJ_0549 proteins are predicted
to be small flavoproteins that are highly similar (40%). The size of the denatured protein
components in the cell extract of the LJ_0548-0549 overexpressing strain on the SDS-gel
is 20 kDa, corresponding to the size of the gene product of either LJ_0548 or LJ_0549
as inferred from their gene sequence. Its elution from the gel filtration column suggests
that the active protein has a size of ~18 kDa, which would mean that only one of either
LJ_0548 or LJ_0549 would be required for activity. However, for complementation of
the LJ_0548-0549 deletion strain, both genes appear to be required, whereas in trans
complementation with a plasmid harboring either one of the genes failed to result in
detectable protein expression (SDS-PAGE) or functional complementation. Although
we can conclude that both genes are required to produce the functional enzyme, its
exact composition remains unclear.
The observation that the deletion derivative NCC 9359 produces small amounts of H2O2
upon prolonged exposure to oxygen indicates that besides the NADH flavin reductase
identified here, other H2O2 producing enzymes may exist in this species. Nevertheless,
the enzyme identified here appears to be the major contributor to the H2O2 production
capacity in this species. Possibly, the additional H2O2 producing reactions involve
oxidases, like the aforementioned pyruvate, lactate and NADH oxidase, which may
contribute to H2O2 production upon extended oxygen exposure. However, it is unlikely
that the conditions of oxygenation used in this study, both in terms of its duration and/or
58
oxygen tension, will be encountered in the GI-tract, which is thought to be the natural
habitat of L. johnsonii. We propose therefore that the constitutive flavin reductase is the
primary source of H2O2 in an environment where microbes predominantly encounter
anaerobic (intestinal lumen) conditions and only sporadically encounter lower and more
variable concentrations of oxygen when they are present in closer proximity to the
intestine mucosa. In contrast, prolonged exposure to aerobic conditions and/or higher
oxygen tensions can occur during industrial processing, which may elicit the activation
of alternative H2O2 production reactions as our preliminary observations imply.
2
L. johnsonii has been proposed to have lost numerous genes and pathways during its
adaptation to the nutrient-rich environment of the intestinal tract (176). Nevertheless,
the newly discovered NADH dependent flavin reductase appears to be constitutively
expressed, suggesting that it plays an important role in the lifestyle of L. johnsonii in its
natural environment. Since lactate fermentation from glucose is entirely redox-neutral,
it is unclear in what metabolic step the NADH is generated that is consumed in the
reaction catalyzed by the LJ_0548-549 enzyme. We hypothesize that the additional
electrons are generated in the metabolism of one of the many vitamins, peptides and
amino acids that are consumed by L. johnsonii in addition to glucose.
Although the results presented here do not rule out that this newly identified flavin
reductase serves a metabolic purpose in which H2O2 is a side product, we suggest that
the production of H2O2 in itself has a biological function. For example, it may contribute
to the antimicrobial capacities of L. johnsonii that may be of great importance for the
organism to maintain its niche / position within the densely populated microbiota (38).
Moreover, H2O2 may serve as a chemical signal in host-microbe interactions, as it has
been proposed to influence PPAR-g, one of the major regulators of inflammation
in the intestinal epithelium (50). Alternatively, the reduced aerotolerance of the nfrdeletion derivative as compared to its wildtype counterpart suggests that the reaction
catalyzed by this enzyme may enable L. johnsonii to prevent or reduce oxidative stress.
If the flavins that are reduced by these proteins, form the most readily oxidized parts
in the cytoplasm and can effectively capture oxygen, the activity of these flavins may
prevent other, more damaging effects of oxygen, such as the direct oxidation of ironsulfur clusters (220, 221) or the formation of semiquinones (212). Also in L. johnsonii,
the controlled production of H2O2 may be preferred over the uncontrollable other
effects oxygen might exert. The role that this NADH dependent flavin reductase plays
in oxidative stress, H2O2 scavenging and aerotolerance of L. johnsonii is the subject of
further studies.
59

Acknowledgments
This work was supported by Nestlé Research Centre, Vers-chez-les-Blanc, Switzerland.
We would like to acknowledge Anne-Cécile Pittet for her technical assistance in
construction of the mutants and Filipe Branco dos Santos for his valuable input on the
purification of the NADH flavin reductase activity.
60
Supplementary materials
Table S2.1: Primers used for construction of L. johnsonii mutant strains (chapter 2).
Region
ID
Sequence
LJ_1826 5’ homology 5’ primer
A
CTACTCCAGAAGAAGTCG
LJ_1826 5’ homology 3’ primer
B
ATATATGGATCCAAGGGTGAAGGACAAAGC
LJ_1853 3’ homology 5’ primer
C
GGGTTTTGCATTCCAGTC
LJ_1853 3’ homology 3’ primer
D
ATATATGGTACCTTGGCCAAAAGTTGGAGC
LJ_1254- LJ_1255 5’ homology 5’
E
ATATATGAGCTCAATACTCAATGTAAGCGC
LJ_1254- LJ_1255 5’ homology 3’
F
ATATATGGATCCTGATTGCAGGTCCACCTG
LJ_1254- LJ_1255 3’ homology 5’
G
ATATATGAATTCTGATTTAGTAGCTGCTGG
LJ_1254- LJ_1255 3’ homology 3’
H
ATATATGGTACCCAGAAACTATGAAGGCTC
LJ_0548- LJ_0549 5’ homology 5’
I
ATATATGAGCTCGCTGCAAATGAAGGGCTAGA
LJ_0548- LJ_0549 5’ homology 3’
J
ATATATGGATCCGCGTTGCTACCTACAATGGC
LJ_0548- LJ_0549 3’ homology 5’
K
ATATATGAATTCTTTAATTGGTCATGCTGCAG
LJ_0548- LJ_0549 3’ homology 3’
L
ATATATGGTACCAGCTCGCCTTCACTACGGAG
LJ_1125 term 5’
O
ATATATAAGCTTTGCCAATGGATAACCAGG
LJ_1125 term 3’
P
ATATATCTCGAGAATCTCTCTTGGACTTGC
LJ_0045 promoter 5’
R
ATATATAGATCTCATTATCATAAGGCACCC
LJ_0045 promoter 3’
S
ATATATGAGCTCGCTAGCGCATGCATTAAACCTCCGTC
SphI site before LJ_0548
T
ATATATGCATGCAACTCTTTGCCATTGTAGG
SphI site before LJ_0549
U
ATATATGCATGCAATTACTAGCAATTGTTGG
HindIII site after LJ_0548
V
ATATATAAGCTTAATTTCATGGGTCGTTCCTC
HindIII site after LJ_0549
W
ATATATAAGCTTGGTTTTAACTTATTTTTGAGCTTG
2
61
62
ori
RepC
2500
3000
Term
2000
3860 bps
LJ0548
1000
HindIII
500
1500
pDP1016
3500
XhoI
MunI
MunI
RepA
SspI
p-LJ0045
CAT
MunI
BglII
PstI
NheI
SphI
NruI
BglII
StuI
NcoI
SspI
NdeI
ori
RepC
2500
3000
SalI
Term
2000
3867 bps
1000
SspI
HindIII
SphI
MunI
EcoRI
BglII
StuI
NcoI
EcoRI
NcoI
p-LJ0045
CAT
MunI
PstI
LJ0549
500
1500
pDP1017
3500
XhoI
MunI
MunI
RepA
Figure S2.1: Genetic maps of LJ_0548 and/or LJ_0549 overexpression plasmids.
SspI
NdeI
SalI
XhoI
MunI
MunI
ori
RepC
SspI
NdeI
HindIII
PstI
1000
SspI
MunI
LJ0548
p-LJ0045
CAT
MunI
EcoRI
NcoI
EcoRI
LJ0549
2000
4416 bps
pDP1019
4000
Term
3000
RepA
SalI
BglII
PstI
NheI
SphI
NruI
BglII
StuI
NcoI

NADHconsumptionrate (µM / s)
70
60
50
40
30
2
20
10
30%-50%
70% - 90%
90%+
50
40
fraction 29
fraction30
30
A
280
1000
900
800
700
600
500
400
300
200
100
0
50%-70%
0
20
10
0
50
100
Eluted volume (ml)
150
-10
NADH consumption rate (uM/s)
0
Figure S2.2: Purification of NADH flavin reductase activity in cell free extract of wild type L. johnsonii. NADH
consumption rate (in the presence of 250 µM FAD) is determined in fractions obtained during ammonium
sulphate precipitation (panel A). The fraction with highest activity is subsequently used for anion exchange
chromatography. All fractions eluting from the Q column (panel B) are tested for NADH flavin reductase activity.
On the left axis, absorption at 280 nm is shown which is an indicator of protein concentration. On the right axis,
NADH consumption rate is shown in the eluting fractions.
Figure S2.3: SDS gel of cell free extracts of L. johnsonii NCC 533 (wt, lane 1), NCC 9359 + pDP 1016 (Δnfr + LJ_0548,
lane 2), NCC 9359 + pDP 1017 (Δnfr + LJ_0549, lane 3), NCC 9359 (Δnfr, lane 4), NCC 9359 + pDP 1019 (Δnfr +
LJ_0548-LJ_0549, lane 5), PageRulerTM marker (lane 6).
63

1,2
1
µ (h-1)
0,8
0,6
0,4
0,2
0
anaeroob
aerobic
Figure S2.4: Growth rate of L. johnsonii NCC 533 (grey bars) and its nfr-deletion derivative NCC 9359 (white bars)
in MRS-medium in stirred pH controlled sparged with 750 ml/min of N2 + 5% CO2 (anaerobic) or N2+ 20% O2+ 5%
CO2 (aerobic). Growth rates were determined as explained in Materials & Methods. Data are average of triplicate
experiments ± standard deviation.
L. johnsonii LJ_0549
L. johnsonii LJ_0548
E. coli K12 ChrR
----MKLLAIVGTNADFSYNRFLDQFMAKRYKDQAEIEVY-EIADLPRFK 45
----MKLFAIVGSNADHSYNRDLLNFIKKHFTDRYDIELG-EVKDLPMFK 45
MSEKLQVVTLLGSLRKGSFNGMVARTLPKIAPASMEVNALPSIADIPLYD 50
:::.:::*: . *:* : . : *
:::
.: *:* :.
L. johnsonii LJ_0549
L. johnsonii LJ_0548
E. coli K12 ChrR
KEAQP----DSKVEEFKNKIREADGVIFATPEYDHGIPSALKSAMEWTGS 91
EGVKE----PAAVASFAKKVADADAVLISTPEQQHSVPSSLKSALEWLSS 91
ADVQQEEGFPATVEALAEQIRQADGVVIVTPEYNYSVPGGLKNAIDWLSR 100
.:
: * : ::: :**.*:: *** ::.:*..**.*::* .
L. johnsonii LJ_0549
L. johnsonii LJ_0548
E. coli K12 ChrR
HAQGNADVMKMKPAMVLGTSYGIQGASRAQEEMREILLSPDQSANVLPGN 141
AEHP----FKDKPVVIVGTSVLPQGSARGQSHLKLVLSSPGFGAKVFNGD 137
LPDQP---LAGKPVLIQTSSMGVIGGARCQYHLRQILVFLD--AMVMNKP 145
.
: **.:: :*
*.:* * .:: :*
. * *:
L. johnsonii LJ_0549
L. johnsonii LJ_0548
E. coli K12 ChrR
EVLIGHAADKFDKNTGDLLDQETIHAIDLAFNNFVKFVEQAQK 184
EFMMGTAPEQFDENGN--LPAGTVKFLDHFFDEFDSFYAEVSK 178
EFMGGVIQNKVDPQTGEVIDQGTLDHLTGQLTAFGEFIQRVKI 188
*.: *
::.* : . :
*:. :
: * .* ...
Figure S2.5: ClustalW2 comparison of LJ_0548 / LJ_0549 to chromate reductase (ChrR) from E. coli. Highlighted
in grey are the residues that constitute the flavin binding site, highlighted in yellow are the similarities in these
residues in the L. johnsonii genes.
64
2
65
Chapter 3
Transcriptome response in Lactobacillus johnsonii
identifies an oxygen induced NADH oxidase that
contributes to H2O2 production

Summary
Oxidative stress due to endogenous hydrogen peroxide production by Lactobacillus
species is a well-known issue in the food industry. In this study, the transcriptional
response to oxygen was analyzed in Lactobacillus johnsonii, one of the H2O2-producing
strains used in the food industry. Aerobic growth conditions led to a more than two-fold
repression of 45 gene-specific transcripts as compared to anaerobic growth, whereas
transcripts of 6 genes were more than two-fold induced. Among the higher expressed
genes were two genes that displayed significant homology to NADH-dependent
oxidoreductase (NOX). The transcriptional regulation of the nox promoter by oxygen
was verified using a GUS-reporter construct, whereas the nox promoter activity did not
appear to respond to other oxidative conditions, e.g. exposure to sublethal levels of H2O2.
Experiments in chapter 2 showed that H2O2 production by L. johnsonii largely depends
on genes encoding an NADH flavin reductase (NFR). However, here we show that an
NFR deficient strain could regain its H2O2 producing capacity upon prolonged oxygen
exposure, which was hypothesized to involve the oxygen induced nox locus, of which
the transcription appeared to be more strongly induced by oxygen in the NFR-deficient
strain as compared to its parental strain. Indeed, deletion of the NOX-encoding locus in
the NFR-deficient background, resulted in a strain that could no longer produce H2O2.
Moreover, the NFR-NOX deficient strain (nfr­-, nox-locus) displayed strongly impaired
aerobic growth and oxygenation induced rapid H2O2 independent growth stagnation.
We conclude that H2O2 production in L. johnsonii is primarily dependent on NFR but
also involves the oxygen-inducible NOX. Moreover, our results imply that the capacity to
produce H2O2 plays a prominent role in oxygen tolerance of L. johnsonii.
68
Introduction
The lactobacilli belonging to the L. acidophilus group play a key role in the food industry.
They are used in cheese fermentation (e.g., L. helveticus), kefir fermentation (e.g., L.
kefirofaciens) and in yoghurt fermentation (e.g., L. delbrueckii subspecies bulgaricus).
Especially the latter application is of great economic importance and represents a
rapidly growing multibillion market. In these dairy fermentations the lactobacilli
primarily contribute to the acidification of the milk by the production of lactic acid, but
also contribute to the product’s texture and flavor by producing exopolysaccharides,
and specific volatile components like acetaldehyde and diacetyl, respectively (222).
In addition to these features, several species belonging to the L. acidophilus group
are marketed as probiotics that, when consumed in adequate amounts, can convey a
health benefit to the consumer (223).
3
For reliable industrial application of lactobacilli, their stress tolerance and functional
robustness under industrial conditions is of great importance. To effectively initiate and
complete fermentation processes, it is required that the Lactobacillus starter cultures
survive the stressful industrial production and processing conditions. Similarly, sustained
viability during product-processing and shelf-life of probiotic-containing products is
crucial for this product-category to deliver their health benefit to the consumer (224,
225). Thereby, robustness and sustained viability of probiotic cultures under industrial
and product conditions is essential to ensure their efficacy. In the life cycle of probiotic lactobacilli, from industrial production to in situ delivery
in the intestinal tract, oxidative stress exposure is considered an important cause of
viability loss (35). The product environment commonly contains relatively high levels of
oxygen (46), in which the lactic acid bacteria that belong to the L. acidophilus group
produce significant amounts of H2O2 (37, 38, 94) even at chilled temperatures (36).
Lactic acid bacteria are fermentative microorganisms that lack the endogenous capacity
for respiration, but many species and strains of this group are relatively aerotolerant and
grow well in aerobic environments (see chapter 2). LAB employ several mechanisms for
reactive oxygen species (ROS) detoxification to relieve oxidative stress and prevent its
corresponding damaging effects on cellular constituents (32). These mechanisms include
enzymes that target the ROS hydrogen peroxide (H2O2), such as the heme-requiring
catalase expressed by L. sakei (226), and the heme-independent catalase of L. plantarum
(100). The latter species is also protected against ROS, including superoxide (O2-) induced
cell-damage by its capacity to accumulate high intracellular levels of manganese that
69

can directly scavenge superoxide molecules (141). Superoxide detoxification can also
be achieved by the enzyme activity of superoxide dismutase (SOD), which was shown
to play a key-role in oxygen tolerance in Lactococcus lactis (188). In addition, several
lactic acid bacteria, contain glutathione and thioredoxin reductases (113, 114, 122),
peroxidases and/or alkyl hydroperoxide reductases (74, 75, 227), which can effectively
remove reactive oxygen species from the cell and maintain reducing conditions in the
cell’s cytoplasm. Apart from the thioredoxin reductase system, the lactobacilli belonging
to the L. acidophilus group commonly lack these ROS-scavenging activities, which is an
important determinant in the H2O2 accumulating capacity that is seen in many species
and strains of this group of the lactobacilli (37, 93, 94).
A constitutively expressed NADH flavin reductase (NFR), composed of two small
(20kDa) flavoproteins, was shown to be the major source of the accumulating H2O2
when anaerobically growing cells were exposed to molecular oxygen. The formation of
H2O2 induces premature growth stagnation and cell death in L. johnsonii under aerobic
growth conditions (chapter 2) (228). Conversely, L. johnsonii may also benefit from
oxygen exposure, which can drive the endogenous pyruvate oxidase-dependent (POX)
metabolic pathway that produces C1- and C2- metabolic intermediates that are required
for growth. This growth stimulatory effect of oxygen was clearly demonstrated by the
observation that L. johnsonii strictly depends on environmental supplies of acetate (C2) and inorganic carbon (C1-) sources when grown under strict anaerobic conditions
(chapter 4). Oxygen therefore plays a paradoxical role in the lifestyle of L. johnsonii: its
presence results in H2O2 accumulation which threatens viability, but at the same time
alleviates some of its environmental growth requirements.
Several studies have focused on the transcriptional response of LAB upon their exposure
to oxygen, revealing the activation of oxidative stress-related genes, including those
encoding ROS-scavenging enzymes, such as the thioredoxin reductase in L. plantarum
(113) and the heme-catalase in L. sakei (226). Moreover, aerobic growth of L. lactis
activated the expression of genes encoding superoxide dismutase, alkyl hydroperoxide
reductase, glutathione reductase and NADH oxidase (noxE), which have all been
shown to play a role in oxidative stress tolerance (229). Therefore, the elucidation of
transcriptional responses to oxygen exposure can help to decipher the mechanisms
by which these LAB can handle oxidative stress generated by oxygen exposure.
Notably, the oxygen-induced NADH oxidases of Streptococcus mutans were initially
proposed to contribute to its H2O2 production (124), but were in subsequent studies
also shown to play a prominent role in aerotolerance. This aerotolerance contribution
70
of the NOX function was associated with the alkyl hydroperoxide reductase activity,
which is encoded by the ahpF gene that is genetically linked and co-expressed with the
nox1 gene (74, 76). A similar relationship between aerotolerance and H2O2 producing
enzymes has also been established in L. johnsonii in which the deficiency of the H2O2
producing NFR resulted in a reduced growth rate under conditions encompassing highlevel oxygen exposure.
In this study, we analyze the genome-wide transcriptional response of L. johnsonii to
oxygen exposure. Amongst the three most upregulated genes under aerobic conditions
we found two genes that display strong homology to NADH oxidoreductases (nox-locus).
By means of a GUS-reporter assay it could be confirmed that the gene’s promoter activity
is controlled (activated) by the presence of oxygen. Although the nox-locus deletion
derivative of L. johnsonii had no apparent phenotype with respect to aerobic growth
3
or H2O2 production, the introduction of this deletion in the L. johnsonii background
that lacks NFR led to a strain that has completely lost its capacity to produce H2O2.
Moreover, the latter strain appeared to be significantly less aerotolerant as compared to
the wild-type or either of the single deletion derivatives (NOX- or NFR-deficient strains).
These results indicate that the H2O2 producing activities catalyzed by NFR and NOX are
important for aerotolerance of L. johnsonii.
Materials & Methods
Strains and growth conditions of L. johnsonii
Lactobacillus johnsonii NCC 533 was obtained from the Nestec Culture Collection and
routinely cultured in MRS medium (205) at 37°C under anaerobic conditions, with
minimal headspace (unless indicated otherwise). An overview of all strains used in this
study is presented in Table 3.1. Erythromycin and chloramphenicol were supplemented
at 5 µg /ml when appropriate. For preculturing of NCC 9360 (nfr-, nox-locus) 500 µg/
ml cysteine was added to sequester oxygen from the growth medium.
For anaerobic conditions, cells are grown at 37°C in static 15 ml Falcon tubes with
minimal headspace. For aerobic conditions, Erlenmeyers were used that have a
headspace volume that is minimally 10-fold larger than the growth medium volume,
and were agitated on a rotating incubator at 200 rpm. Cells are inoculated at an OD600
of approximately 0.05 in fresh medium. The regular Lactobacillus medium MRS is used
(205) or, where indicated, MRS is substituted for LAPTg (20 g/L glucose, 10 g/L yeast
extract,(217), 10 g/L bacto peptone, 10 g/L bacto tryptone plus 1 g/L Tween 80). This
71

medium was used instead of the regular MRS-medium for lactobacilli, because the
meat extract in MRS was found to interfere with the enzymatic assay for H2O2.
Table 3.1: Strains and plasmids used in this study.
Strains and
plasmids
Genotype
NCC 533
Wild type L. johnsonii
(176)
NCC 9337
EmR, Δnox (LJ_1254-LJ_1255), predicted to encode
NADH oxidase
Chapter 2
NCC 9359
EmR, Δnfr (LJ0548-LJ0549) encoding NADH flavin
reductase
Chapter 2
NCC 9360
EmR, Δnox, Δnfr (ΔLJ_1254-LJ_1255, ΔLJ_0548-LJ_0549)
This study
pDP600
a chloramphenicol-resistant version of pG+host9
containing a complete pBluescript array of unique
restriction sites
(206)
pDP893
pDP600- PpgiA - flpSC - LJ_1125 trm
This study
pNZ5372
CmR, pNZ273 derivative containing the promoterless
gusA gene under control of the LJ_1255 promoter
(Pnox)
This study
pNZ273
CmR, promoter-probe vector, containing a promoterless
gusA gene
(207)
pNZ4040
CmR, pNZ273 derivative containing the promoterless
gusA gene under control of the epsB40 promoter (Peps)
(240, 241)
For transcriptome analysis, cultures were inoculated at an OD of 0.026 ± 0.005 and
grown under more tightly controlled conditions in continuously stirred vessels with a
400 ml working volume of MRS medium. The cultures were sparged with specific gas
mixtures containing 5% CO2 and either no oxygen (0% oxygen, anaerobic) or normal
oxygen levels (20% oxygen, aerobic). Cultures were grown at 37°C with continuous
mixing (ca. 200 rpm) and pH was maintained at pH 6.5 by automated 4M NaOH titration.
Cell densities were determined by measuring the optical density at 600 nm (OD600).
RNA extraction, labelling, hybridization and data analysis
Cells were harvested at an OD600 of 0.15 ± .03 from 50 ml of culture by cold centrifugation
(5’, 2600xg, 4°C). Cell pellets were resuspended in 0.5 ml ice-cold Tris-EDTA buffer and
transferred to screw-cap tubes with 0.5 gram zirconium beads (0.1 mm), 0.25 ml acidic
phenol, 30 µl 10% SDS and 30 µl 3 M sodium acetate. After mixing, the samples were
72
immediately frozen in liquid nitrogen and stored at -80°C until further use.
Cells were disrupted by bead-beating in 3 rounds of 40 seconds in a Savant FastPrep
FP120, with in between cooling on ice. Cell debris was removed by centrifugation (20817
x g, 10’, 4°C) and residual phenol was extracted by addition of ice-cold chloroform
followed by centrifugation. RNA was isolated using a High Pure RNA Isolation Kit
(Roche Diagnostics, Mannheim, Germany). RNA purity and yield was determined by
comparison of absorption at 260 and 280 nanometer (Ultrospec 3000, Pharmacia
Biotech, Roosendaal, The Netherlands). RNA quality control was carried out using the
RNA 6000 Nano Assay in an Agilent 2100 Bioanalyzer (Agilent technologies, Palo Alto,
Ca, USA). The Cyscribe Post-labeling kit was used to synthesize cDNA using 5 µg of
total RNA, which was subsequently labeled according to the manufacturer’s protocol
(Amersham Biosciences, Amersham, UK). Samples in which the CyDye labeled cDNA
3
concentration was below 24 ng/µl were concentrated prior to cDNA synthesis using a
Hetovac VR-1 (Heto Lab Equipment A/S, Birkerod, Denmark).
A hybridization scheme was designed that allowed duplicate comparisons between
the transcriptome profiles of aerobic and anaerobic grown cultures. 60 Oligomer
microarrays (Agilent technologies) were used with 12 ± 2.5 probes per gene and 21841
probes in total (GEO accession number GPL18009). These arrays were employed as
previously described (173). In short, two differentially labeled cDNAs (300 ng) were
mixed (final-volume 25µl), incubated at 95° C for 3 minutes and subsequently cooled
to 68° C. To these mixed cDNAs 25 µl Slidehyb#1 hybridization buffer (Ambion, Austin,
USA) and 2X Hi-RPM hybridization buffer (Agilent Technologies) were added and 40
µl of the resulting solution was applied on a 8 * 15K slide preheated at 68°C. Slides
were hybridized at 65°C, rotating at 10 rpm for 16 hours in an Agilent hybridization
oven (Agilent technologies). Subsequently, slides were washed with wash buffer 1
(Agilent technologies) at room temperature for 1 minute and wash buffer 2 (Agilent
technologies) at 37 °C. The slides were dried using nitrogen gas and scanned with
a ScanArray Express 4000 scanner (Perkin Elmer, Wellesley, MA). Image analysis and
processing were performed using the ImaGene Version 7.5 software (BioDiscovery Inc.,
Marina Del Rey, CA, USA). The microarrays were scanned at different intensities. For
each of the individual microarrays the best scan was selected on the basis of signal
distribution (combination of a low number of saturated spots and a low number of
low signal spots). The data were normalized using Lowess normalization as available
in MicroPrep (230). The data were corrected for inter-slide differences on the basis
of total signal intensity per slide using Postprep (230). The median intensity of the
73

different probes per gene was selected as the gene expression intensity. CyberT was
used to compare the different transcriptomes, taking into account the duplicates (dye
swaps) of each of the conditions (231). This analysis resulted in a gene expression ratio
and false discovery rate (FDR) for each gene. Differential gene expression values of
expression-ratios with FDR values <0.05 were considered to be statistically significant.
All microarray data is MIAME compliant and is available in GEO (accession number
GSE52876).
Hydrogen peroxide measurements
H2O2 concentration was determined by addition of 25 µl cell-free culture media to 175
µl reaction mix containing 5 µg/mL horse radish peroxidase and 30 µM phenol red in
water. After 5 minutes of reaction, the pH was increased by the addition of 5 µL 1M
NaOH, and absorption was determined at 620 nm and compared to a standard curve
(232).
Deletion mutants: Construction of NCC 9360 (Δnfr, Δnox)
The construction of the L. johnsonii single deletion derivatives ΔLJ_1254-LJ_1255 (Δnox,
NCC 9337) and ΔLJ_0548-LJ_0549 (Δnfr, NCC 9359) were described in chapter 2.
For this study a double deletion derivative that lacks both LJ_1254-LJ_1255 (nox) and
LJ_0548-LJ_0549 (nfr) was constructed.
To obtain the double deletion derivative, the ery-cassette was removed from NCC
9359 (Δnfr) by the plasmid-borne expression of the Saccharomyces cerevisiae flpgene (flpSC), using pDP893. The construction of pDP893 employed Lactococcus
lactis MG1363 as an intermediate cloning host. It was constructed by amplifying
the pgiA promoter (PpgiA) using L. lactis MG1363 genomic DNA as a template,
with
the
primers
A
(ATATATACTAGTACCCTTAAAAGTGTTAGGAG)
(ATATATAAGCTTGAGCTCGCTAGCGCATGCTAATTCCTTTCAATTTCTCGC),
and
B
the
resulting amplicon was digested with SpeI and HindIII and ligated in similarly
digested pDP600 (Cmr, Emr) (206) generating pDP600-PpgiA. The predicted
bi-directional terminator positioned between the LJ_1125 and LJ_1126 genes of
NCC 533 (TLJ1125) was amplified using genomic DNA of this L. johnsonii strain as
a template and the primers C (ATATATAAGCTTTGCCAATGGATAACCAGG) and D
(ATATATCTCGAGAATCTCTCTTGGACTTGC). The resulting amplicon was digested with
HindIII and XhoI and cloned into similarly digested pDP600-PpgiA, to yield pDP600-
74
PpgiA-TLJ1125. The S. cerevisiae SC288c 2 micron plasmid (233) was isolated and
digested with SphI and XbaI, and the 1.46 kb fragment containing the flp gene was
isolated and cloned in similarly digested pDP600-PpgiA-TLJ_1125, yielding plasmid
pDP893. This plasmid encodes the flp gene under transcriptional control of the pgiA
promoter and is followed by the TLJ1125 terminator.
Plasmid pDP893 was isolated from L. lactis MG1363 and used to transform NCC 9359.
Transformants were cultured in MRS medium supplemented with chloramphenicol,
at 32°C for 5 serial passages (permissive temperature for plasmid replication), then
plated to isolate single, chloramphenicol resistant colonies, which were subsequently
replica plated to confirm erythromycin sensitivity. The selected colony with the required
antibiotic resistance phenotype (Cmr, Emr) was cultured in MRS at 37°C for three serial
passages, and subsequently plated to identify single colonies that are chloramphenicol
3
and erythromycin sensitive by replica plating on plates containing these antibiotics. The
selected strain was designated NCC 9359-FO (FLP-out). NCC 9359-FO was transformed
with pDP902 to achieve the deletion of the nox-locus as described in chapter 2. The
deletion was confirmed using primers flanking the target regions. The Δnfr, Δnox
derivative of L. johnsonii NCC 533 was designated NCC 9360.
Construction of GUS reporter plasmids
To study the expression driven by the nox­-locus promoter, the region upstream the
LJ_1255 gene was amplified using genomic DNA isolated from L. johnsonii NCC
533, and the primers E (ATATTGGATCCCCAGTTGATGAAGTTTTGAAATTCG) and F
(CATAAGAATTCCACCATGTTTAAAAGTTACTTTGTCGG). The resulting 184 bp amplicon
was digested with EcoRI and BamHI and ligated into similarly digested pNZ273 (207)
yielding plasmids pNZ5372, which carries a promoterless copy of the β-glucuronidase
encoding gusA gene under control of the LJ_1255 promoter (Pnox). Plasmid constructions
were performed in the intermediate cloning host Lactococcus lactis and subsequently
transformed to wildtype L. johnsonii (NCC 533) and its nfr-locus deletion derivative
(NCC 9359).
GUS assay
GUS-activity was determined in exponentially growing cultures with OD600 of ~0.3 and
~0.6, taken from the aforementioned batch reactors. Cells were pelleted by centrifugation
(2600 * g, 5’, 4°C), washed with 50 mM potassium phosphate buffer pH 7,0 and
75

again pelleted by centrifugation (2600 * g, 5’, 4°C). Cells were resuspended in 50 mM
potassium phosphate buffer pH 7,0 with 10 mM ß-mercaptoetha­nol, 1 mM EDTA and
0,1% Triton X-100 (GUS-buffer). Initial sample volumes were adjusted to normalize the
amount of cells at a final OD600 of 4. The cell suspensions were transferred to screw cap
tubes with 100 mg 0.1 mm zirconium beads and disrupted by bead-beating in three
cycles of 20 seconds. Cell debris was removed by centrifugation (21500 * g, 10’, 4°C)
and kept on ice. The protein concentration was determined with a microBCA assay
(Thermoscientific).
Glucuronidase activity was determined in a 96-wells plate, by adding 100 µl of the cell
lysate to 150 µl of the aforementioned GUS-buffer (preheated at 37°C). The reaction
was initiated by the addition of 5 µl of the substrate, para-nitro-ß-D phenyl-glucuronide
(PNP-gluc) dissolved in 50 mM potassium phosphate buffer pH 7,0. The mixture was
kept at 37°C and the rate of increase of absorption at 405 nm was used to calculate
the specific activity per mg protein per minute. A molar absorption coefficient of 18000
ml/mmol.cm was used.
Results
Transcriptome comparison of aerobic and anaerobic L. johnsonii cultures.
The whole genome transcriptional response of L. johnsonii to oxygen was determined
using microarray based transcriptome analysis, comparing early-logarithmic aerobic
and anaerobic growing cultures. RNA was isolated from these cultures and whole
genome transcriptome profiles were obtained using oligonucleotide based microarrays
(see Materials & Methods section for details). Data analysis focused on genes that
were significantly regulated by oxygen (FDR<0.05) and displayed a more than 2-fold
expression change between the two conditions. Using these criteria, the expression of
45 genes appeared to be repressed whereas the expression of only 6 genes was induced
in aerobic cultures compared to anaerobic cultures (see Table 3.2 and Supplementary
materials Table S3.1).
Regulation of prophage-related genes
Of the 45 downregulated genes, 23 belong to the two L. johnsonii prophages Lj928
and Lj965 (234). It was previously reported that these prophages are transcriptionally
silent, and in Northern Blot analysis only transcription of the LJ_1454 gene could be
detected (235). The transcriptome data confirmed that the prophage gene expression
76
Table 3.2: Transcriptome comparison of aerobic and anaerobic logarithmic cultures of L. johnsonii. All transcripts
that were expressed at least two-fold higher under aerobic conditions, with an FDR<0.05 are included. Gene
identities are based on the genome annotation (176) or were determined by BlastP alignment.
Gene ID
Fold regulation
FDR
Aerobic / anaerobic
Annotation
LJ_0814
2,4
2,8E-03
Hypothetical protein
LJ_1255
2,3
4,52E-05
possible NADH-dependent
oxidoreductase1
LJ_1254
2,3
8,98E-05
Possible NADH-dependent
oxidoreductase1
LJ_1454
2,2
2,6E-03
Lj928 prophage protein
LJ_1615b
2,1
8,6E-03
hypothetical protein
LJ_0480
2,1
1,6E-03
Thioredoxin
3
1 homology ascertained with BlastP; E-value < e-40 with NADH-dependent oxidoreductase of L. salivarius NIAS840
is low (9.7 and 7.6-fold below average expression of all genes for Lj928 and Lj965,
respectively). Nevertheless, aerobic growth led to a more than 2-fold repression of 20
of the 44 genes that belong to prophage Lj965 and 26 of the 51 genes belonging to
prophage Lj928. Notably, the LJ_1454 gene of prophage LJ928, ranks amongst the 6
genes with the most prominent expression difference between anaerobic and aerobic
conditions. Despite the overall low-expression of the prophage associated genes under
both conditions tested, these genes appear to be subjected to significant environmental
control.
Aerobic conditions elicit upregulation of thioredoxin but not of thioredoxin reductase
One of the 6 genes expressed at a higher level in the presence of oxygen was the
thioredoxin encoding gene LJ_0480 (Trx; 2.1 ± 0.0016). Reduced thioredoxins can
participate in numerous redox-reactions in cellular processes, including the scavenging
of reactive oxygen species. Besides LJ_0480, the L. johnsonii genome encodes a second
copy of this gene (LJ_1665) which was also upregulated under aerobic conditions,
albeit with a fold-change of 1.93 ± 0.0028 (fdr<.05), explaining why it was not
identified using the selection criteria employed (minimum twofold regulation). Oxidized
thioredoxins are regenerated by the activity of thioredoxin reductases (TrxR) (110, 236).
For several lactic acid bacteria, transcriptional regulation of these TRX/TRXR systems
upon oxygen or H2O2 exposure has been reported (113-115). Four different ORF’s in the
L. johnsonii genome have significant homology with the Lactococcus lactis thioredoxin
reductase TrxB1 (115), i.e., LJ_0852, LJ_0501, LJ_0042 and LJ_1757. However, only
77

the LJ_0852 encoded protein contains the canonical CXXC motif of the thioredoxin
reductase catalytic site (110) and none of these thioredoxin reductase homologues
displayed a significant degree of differential expression upon the exposure to molecular
oxygen. These observations seem to imply that 3 of the 4 TRXR homologues are unlikely
to encode functional thioredoxin reductases (lack of the canonical CXXC motif). The
fourth TRXR homologue may be a key-component of the thioredoxin-recycling system
although its expression appeared to be unaffected by oxygen.
Transcription of genes homologous to NADH oxidase is upregulated by oxygen.
Two of the genes that were expressed at a higher level under aerobic conditions
(LJ_1254 and LJ_1255 2.28 and 2.32 fold, respectively) encode proteins that display
significant homology with NADH oxidases (flavin oxidoreductases) from lactobacilli. To
further establish the transcriptional regulation of the NOX encoding locus of L. johnsonii
(LJ_1254-1255) the putative promoter region of this locus (PLJ_1255; 184 nucleotides
upstream of the LJ_1255 start-codon) was cloned upstream of the promoterless gusA
reporter gene in the promoter-probe vector pNZ273 (207), resulting in pNZ5372. GUSactivity in cell free extracts from anaerobically and aerobically growing L. johnsonii
NCC 533 harboring pNZ5372 was determined. As a constitutive control a L. johnsonii
NCC 533 derivative harboring pNZ4040 was employed. This plasmid is a pNZ273
derivative in which the gusA expression is under control of the constitutive PepsB40
promoter (237). The GUS measurements clearly established that the LJ_1255 promoter
was approximately 2- to 2.5- fold induced in aerobic growth conditions compared to
anaerobic growth conditions, which is in good agreement with the level of induction
of the nox-locus assessed by transcriptome analysis (Table 3.3). In contrast, addition of
sublethal amounts of H2O2 to the growth medium (0.2 mM) did not induce the activity
of the LJ_1255 promoter significantly (results not shown), indicating that the induction
of the nox-locus is induced by exposure to molecular oxygen rather than by oxidative
stress.
A peculiar genetic organization of the LJ_1254-1255 locus
LJ_1254 and LJ_1255 are predicted to encode two overlapping polypeptides of 307
and 86 amino acid residues, respectively (Figure 3.1A). The overlap between the two
ORF’s encompasses a 13 bp repeat sequence (Figure 3.1B). The LJ_1255 gene in the L.
johnsonii NCC 533 clearly has a stop-codon, suggesting that the LJ_1254-1255 locus of
L. johnsonii encodes a disrupted pseudogene. By sequencing transcript-derived cDNA
78
Table 3.3: GUS-activity in mmol / mg protein / min determined in aerobic or anaerobic logarithmic cultures with or
without 0.2 mM H2O2. In each experiment, GUS-activity was determined in two samples taken during logarithmic
phase (OD ~0.3 and OD ~0.6). Depicted are averages of two independent experiments (totaling 4 samples) ±
standard deviation. In case of the positive control L. johnsonii + pNZ4040 and wildtype L. johnsonii two samples
of one individual experiment are depicted ± standard error.
Aerobic batch
Anaerobic batch
L. johnsonii NCC 533 + pNZ5372
8.14 ± 1.1
3.9 ± 1.2
L. johnsonii NCC 9359 (Δnfr-locus) + pNZ5372
9.3 ± 0.8
2.5 ± 0.4
L. johnsonii NCC 533 + pNZ4040
21.2 ± 0.4
21.3 ± 4.5
L. johnsonii NCC 533 (wildtype)
.08 ± 0.01
.03 ± 0.01
sequences, we confirmed the tandem organization of the two genes and their
transcription on a single mRNA molecule (results not shown). We assume that LJ_1254
3
may encode an NADH oxidase, whereas the LJ_1255 encoded short protein is unlikely
to be functional as such, but may function in conjunction with the LJ_1254 protein.
Within the genetically closely related species of the L. acidophilus group, these two
genes are poorly conserved and appear to be randomly distributed in strains belonging
to different species. Of the 24 genome sequences available for L. gasseri, one encodes a
LJ_1254 homologue, whereas the published genomes of L. crispatus and L. delbrueckii
have no regions that are homologous to either of the two genes. Only one of the 11
genomes sequences available for L. acidophilus has a homologue of LJ_1254 with a
similar length (LBA_1418 in L. acidophilus NCFM). Upstream of this region a smaller
gene is found with homology to LJ_1255 (LBA_1421). However, these two genes are
interspersed by two other genes, of which one (LBA_1420) encodes a transposase (See
Supplementary materials, Table S3.2 and Figure S3.1).
In other LAB, LJ_1254 homologs are found in two published L. lactis genomes, whereas
the most intensively studied strain of Lactococcus lactis (MG1363) appears to lack
this gene. Several of the published L. plantarum strains have genes with homology
to LJ_1254; the most intensively studied strain WCFS1 encompasses two genes with
significant similarity to LJ_1254 (lp_1350 and lp_0146). All LJ_1254 homologs found
in LAB, apart from the copy in L. acidophilus NCFM have an N-terminal extension of
approximately 70-residues (total protein length 369 to 391 amino acid residues), which
is homologous with the LJ_1255 encoded protein (Figure 3.1 and Supplementary
materials, Table S3.2 an Figure S3.1).
79

Figure 3.1: Schematic representation of the nox-locus (panel A). Panel B shows the overlapping region of LJ_1255
and LJ_1254. Highlighted are the startcodon of LJ_1254, the stopcodon of LJ_1255 and the 13 bp-repeat region.
Irrespective of the peculiar genetic organization of the LJ_1254-1255 locus in L.
johnsonii NCC 533, we decided to consider both genes as part of the NADH oxidase
encoding locus (nox-locus) in further work, since both genes are expressed as a single
transcript and are homologous to NADH oxidoreductases in other lactic acid bacteria.
Consequently, we deleted both genes present in this locus to study its functionality (see
below).
H2O2 production by the NFR-deficient strain (NCC 9359) indicates the presence of a
secondary H2O2 source.
Previously, L. johnsonii has been shown to produce substantial amounts of H2O2 under
aerobic conditions (38). H2O2 production in L. johnsonii upon exposure to oxygen was
shown to predominantly depend on a novel, constitutively expressed NADH-dependent
flavin reductase (NFR) encoded by the LJ_0548-549 genes (nfr-locus, chapter 2).
Nevertheless, since in many other bacteria, the activity of NADH oxidases has been
identified as the main source of H2O2, a role of the oxygen-induced nox-locus in this
process cannot be excluded. To evaluate the role of the nfr- and nox-loci encoded
activities in the formation of H2O2 in L. johnsonii, NCC 533 (wild type), NCC 9337
(deletion of nox-locus) and NCC 9359 (deletion of nfr-locus), were cultivated under
aerobic growth conditions (aeration was elicited by vigorous shaking at 200 rpm) and
growth and H2O2 production were monitored. In agreement with earlier observations,
both the wild-type strain and its nox-locus deletion derivative produced H2O2 during
the 5-6 hours of aerobic growth (up to 0.36 mM), leading to stagnation of growth.
The nfr-locus deletion strain NCC 9359 did not produce any H2O2 during the initial
hours of aerobic growth, which is also in agreement with earlier observations. However,
prolonged aerobic incubation of the nfr-locus deletion strain resulted in recovery of
the H2O2 production capacity (Fig. 3.2B), eventually amounting up to 0.23 mM H2O2
80
after approximately 8-9 hours of aerobic growth. This delayed accumulation of H2O2 in
the medium, stagnated the growth of this strain (Figure 3.2A). The observed growth
stagnation could in all cases be prevented by addition of 0.5 mg/ml catalase to the
growth medium, underpinning that the accumulating H2O2 caused the observed
growth stagnation (chapter 2). These observations suggest that besides the main H2O2
producing enzyme NFR, a second system is present in L. johnsonii that is involved in
H2O2 production, which is postulated to involve the NOX-encoding genes.
To confirm the oxygen exposure mediated activation of the nox promoter in the nfrlocus deficient background pNZ5372 (Pnox-gusA) was introduced into the nfr-locus
deletion strain NCC 9359. The pNZ5372 derived GusA expression appeared to be 3.6
fold induced in this strain upon its exposure to oxygen (Table 3.3). This finding not only
confirms that the nox-locus is also induced by aeration in this strain, but indicates that
3
the oxygen-mediated regulation in this strain is even slightly enhanced as compared to
its parent strain NCC 533.
Figure 3.2: Growth and H2O2 production of wild type L. johnsonii NCC 533 (square closed symbols), its Δnoxlocus derivative (LJ_1254-LJ_1255; NCC 9337, circular closed symbols) and its Δnfr-locus (LJ_0548-LJ_0549; NCC
9359 square open symbols). Cultures were incubated at 37°C under continuous shaking at 200 rpm. Culture
densities were determined by optical density measurement at 600 nm (panel A) and H2O2 concentrations were
enzymatically determined by a phenol red assay (panel B). The data present biological triplicate experiments ±
standard deviation.
The candidate nox-locus of L. johnsonii contributes to H2O2 production and aerotolerance
The data presented above are in agreement with a role of the nox-locus in the recovery
of H2O2 production in the NFR-deficient background. To evaluate this, a strain lacking
both the nox- (LJ_1254-1255; Δnox-locus) and nfr- (LJ_0548-0549; Δnfr-locus) loci
was constructed and designated NCC 9360 (Δnox­-, Δnfr-locus; Table 3.1). Growth
rate, maximal biomass levels and H2O2 concentration during aerobic growth of this
Δnox­-, Δnfr-locus double deletion derivative was compared to the wild-type strain and
81

to the single Δnox-locus - and Δnfr -locus deletion derivatives. Notably, in contrast
to the wild-type strain or its Δnfr and Δnox single deletion derivatives, NCC 9360
(ΔnoxΔnfr) displayed a strongly reduced aerobic growth rate. Aerobic growth of this
strain stagnates at an OD600 of 0.3 (±0.01), whereas the other strains continue to grow
up to an OD600 of 2.1 (±0.01). Strikingly, the growth stagnation of NCC 9360 is not
accompanied by any detectable H2O2 formation (Table 3.4). Moreover, the premature
stagnation of growth of NCC 9360 cannot be prevented by the addition of 0.5 mg/
ml catalase (results not shown), confirming that it is not caused by accumulating H2O2.
On the basis of these results, it can be concluded that the combined activities encoded
by the nox-and nfr- loci plays an essential role during aerobic growth of L. johnsonii.
However, these experiments do not conclusively demonstrate that the nox-locus
contributes to H2O2 formation in the NFR deficient L. johnsonii strain, since the lack
of detectable H2O2 production in NCC 9360 (ΔnoxΔnfr) may be due its incapacity to
grow under aerobic conditions. To evaluate the role of the nox-locus in H2O2 formation
in the NFR deficient background, the NCC 9360 (ΔnoxΔnfr) was grown anaerobically
to mid-exponential phase (OD600 of 0.25 ± 0.04) and suddenly exposed to oxygen by
transferring the culture to shake flasks agitated at 200 rpm. Under these conditions, the
L. johnsonii wild-type and its single nox-locus deletion derivative initiate H2O2 production
immediately or after a delay (Δnfr strain), eventually leading to growth stagnation (see
also above). In contrast, although the NCC 9360 strain (Δnox, Δnfr) continued to grow
for at least two generations after the switch to aerobic conditions, it did not produce
detectable H2O2 levels (the estimated detection limit of H2O2 detection assay is 0.02
mM) (Figure 3.3). These results indicate that the H2O2 producing capacity detected in
the NFR-deficient strain upon prolonged oxygen exposure is depending on the LJ_12541255 nox-locus.
Table 3.4: Maximal growth rate, optical density (OD600) and H2O2 concentration determined during aerobic growth
of L. johnsonii NCC 533 (wild-type), NCC 9337 (Δnox), NCC 9359 (Δnfr) and NCC 9360 (Δnox, Δnfr) in LAPTg. H2O2
levels and OD600 levels were measured after 6 hours of aerobic growth. Data represent the average of triplicate
experiments ± standard deviation.
82
Strain
Genotype
OD600
µmax (h-1)
H2O­2 (mM)
NCC 533
wild type
2.0 (±0.1)
0.74 (±0.00)
0.36 (±0.01)
NCC 9337
Δnox
2.3 (±0.1)
0.85 (±0.08)
0.36 (±0.01)
NCC 9359
Δnfr
2.2 (±0.1)
0.72 (±0.02)
0.23 (±0.01)
NCC 9360
Δnox, Δnfr
0.3 (±0.03)
0.46 (±0.11)
<0.02
Figure 3.3: Anaerobic logarithmic cultures were transferred to aerobic shakeflask conditions at mid-logarithmic
stage of growth for L. johnsonii NCC 533 (closed square symbols), NCC 9359 (open square symbols) and NCC
9360 (open circular symbols). H2O2 was determined with an enzymatic phenol red method, with an estimated
detection limit of ~0.02 mM. Data represent the average of triplicate experiments ± standard deviation.
3
Discussion
H2O2 formation by members of the Lactobacillus acidophilus-group, including
L. johnsonii, is considered an important metabolic capacity which has been associated
with niche adaptation as well as probiotic functions (38, 50, 94). In chapter 2 we
presented experimented showing a novel NADH flavin reductase (NFR), that appears
to be conserved among the L. acidophilus-group of lactobacilli and is the dominant
enzymatic source of H2O2 synthesis in L. johnsonii upon its initial exposure to oxygen.
In the present study we show that a second H2O2-producing enzyme is induced in
L. johnsonii upon aerobic growth, which is homologous to the NADH oxidoreductase
family proteins (NOX). This oxygen-induced NOX allows the recovery of H2O2 production
in the NFR deficient L. johnsonii strain, and a double mutant strain that lacks both the
NFR and NOX encoding genes is completely defective in H2O2 production but is also
severely impaired in aerobic growth. Our results imply that the H2O2 producing capacity
of L. johnsonii is essential for its growth under aerobic conditions. This appears to be in
clear contradiction with the proposed toxicity of H2O2. Apparently, complete elimination
of the H2O2 producing capacity has detrimental consequences for the strain’s capacity
to deal with molecular oxygen exposure and to prevent the oxygen-derived molecular
damage, presumably by the formation of ROS.
The transcriptome comparison of aerobic and anaerobic cultures confirmed our
observations with respect to growth and metabolism of L. johnsonii in the presence
of oxygen, presented in chapter 2: apart from peroxide production (which induces
83

premature growth stagnation) and the alleviation of the CO2 and acetate dependencies,
the presence of molecular oxygen has little effect on metabolic end-products or maximal
growth rate of L. johnsonii (chapter 4). It is important to note that several functions
known in other bacteria to be induced in response to oxygen exposure are absent from
the L. johnsonii genome, including NADH peroxidase, alkyl hydroperoxidase, superoxide
dismutase, glutathione reductase and/or catalase. The thioredoxin / thioredoxin
reductase (Trx/TrxR) system is the only system known to play a key-role in oxidative
stress tolerance in other lactic acid bacteria that appears to be present in L. johnsonii
(113-115). Transcription responses to oxygen exposure in this bacterium included the
induction of expression of two copies of the thioredoxin gene. However, the potentially
enhanced thioredoxin-mediated ROS-scavenging capacity, is apparently not sufficient
to prevent premature growth stagnation elicited by the endogenous production
of H2O2. Many factors may play a role in the inability of the thioredoxin system to
sufficiently scavenge H2O2 or prevent aerobic growth stagnation. NADPH, which is the
electron donor for thioredoxin reductases (111), may be rapidly depleted in LAB that
are known to generally maintain low NADPH-pools (67). Furthermore, the discrepancy
between oxygen-mediated regulation of thioredoxin (induced) and its complementary
thioredoxin reductase (unaffected) may contribute to the limited ROS-scavenging
capacity in L. johnsonii. The limited responsiveness to oxygen of L. johnsonii and the
failure of the thioredoxin system to prevent oxygen induced growth stagnation may
reflect the adaptation of this species to its natural habitat of the mammalian intestinal
tract, where oxygen levels are very low and only transient exposure to significant
amounts of oxygen is expected to be encountered when bacteria reside closer to the
mucosal surface of the intestinal wall. Low level or transient oxygen exposure may
actually provide a benefit to L. johnsonii by relieving its requirement for exogenous C-1
and C-2 sources for growth (chapter 4).
The nox-locus in L. johnsonii consists of two overlapping ORF’s, i.e., a shorter gene
(LJ_1255) encoding the N-terminal region of typical NOX enzymes and a longer gene
(LJ_1254) encoding the C-terminal region. This could imply that the nox locus of L.
johnsonii encodes a pseudogene. However, the locus is expressed as a single transcript
and its transcription is elevated by oxygen exposure of the cells, through the activation
of its promoter region located upstream of LJ_1255. Moreover, the functionality and
H2O2 producing capacity encoded by the nox-locus is supported by the observed
H2O2-negative phenotype of the nox-locus deletion derivative of the L. johnsonii strain
that lacks the constitutively expressed nfr-locus, which encodes the predominant
84
peroxide producing enzyme in this species. Our results do not elucidate the exact
identity of the active NOX protein. Although a complementary role of the LJ_1255
encoded protein cannot be excluded, it seems likely that the activity is encoded by
the larger 3’-gene LJ_1254. This suggestion is supported by the observation that the L.
acidophilus NCFM genome encodes a full-length homologue of only LJ_1254, which is
not directly preceded by a LJ_1255 homologue.
NADH oxidoreductases are known to play a central role in aerobic metabolism of
facultative heterofermentative lactic acid bacteria: expression of an NADH oxidase
in Lactococcus lactis can induce a mixed acid fermentation, by dissipating the
cofactor (NADH) for lactate dehydrogenase activity (66). L. johnsonii, is an obligate
homofermentative lactic acid bacterium displaying homolactic pyruvate dissipation
(chapter 4). Analogously, L. johnsonii does not produce considerable amounts of
3
metabolic end-products other than lactate, irrespective of the presence of oxygen.
Nevertheless, NADH consuming enzymes like NFR and NOX could still influence aerobic
metabolism by draining the available NADH pool. This could restrict pyruvate dissipation
through the lactate forming, NADH consuming step catalyzed by lactate dehydrogenase.
Reduced pyruvate to lactate flux could lead to the accumulation of pyruvate, which
has previously been reported for L. lactis (48). The accumulation of pyruvate could
provide a chemical scavenger for environmental or endogenously produced H2O2,
because pyruvate reacts rapidly with this ROS to form acetate. However, in chapter 4,
we present experiments that show that aerobic growth of L. johnsonii does not lead to
substantial levels of acetate production, suggesting that the pyruvate accumulation and
its subsequent reaction with H2O2 is very limited in this species. Thereby the metabolic
effects of NADH oxidation in homofermentative LAB like L. johnsonii is limited and not
completely understood to date.
In this study we establish that the NFR and NOX enzymes are both involved in the capacity
of L. johnsonii to produce H2O2, which eventually leads to a stagnation of growth. In
contrast with this toxic effect of H2O2, the enzymes responsible for its production also
appear to contribute to aerotolerance in L. johnsonii. This apparent paradox resembles
the dualistic activity that has been described for alkyl hydroperoxide reductases (AHP),
which catalyzes NADH dependent reduction of H2O2 and organic hydroperoxides such
as cumene hydroperoxide. In E. coli this enzyme is part of the oxyR induced oxidative
stress response (238) and is the primary H2O2 scavenger (109). It consists of two subunits:
a larger flavoprotein (AhpF) and a smaller protein (AhpC) that displays homology
to thioredoxin reductases (74, 105). Remarkably, the flavoprotein subunit displayed
85

H2O2:NADH oxidase activity in Amphibacillus xylanus, Sporolactobacillus inulinus and
in Streptococcus mutans (72, 76, 239). In the latter species it was shown that addition
of the smaller AhpC subunit to the AhpF abolished its H2O2 production activity and
induced an alkyl peroxide and H2O2 scavenging activity. (74). Although neither the nox­locus nor nfr-locus encoded proteins display significant homology with these two AHPencoding genes, one could hypothesize that these genes function (or used to function)
together with a unidentified secondary protein in a comparable peroxidase complex.
This would explain the observation that these genes contribute to both H2O2 production
and to aerotolerance.
In this study we have identified NOX as a secondary H2O2 source in L. johnsonii. Our
results indicate that the mode of H2O2 production by L. johnsonii depends on the
environmental conditions. In predominantly anaerobic environments in which only
transient exposure to (low levels) of molecular oxygen occurs, the production of H2O2
will strictly depend on the NFR activity encoded by LJ_0548-0549. Such conditions may
be expected in the typical habitat in which L. johnsonii is encountered, the intestinal tract
of mammals. The intestinal lumen is considered anaerobic, but at closer proximity to
the strongly oxygenated intestinal mucosa the intestinal bacteria may become exposed
to (low levels of) oxygen (40, 41). Therefore, L. johnsonii would initiate the production
of H2O2 when residing in close proximity of the epithelial surfaces, which could play an
important role in its proposed effects on PPAR-mediated gene expression control (50).
Extended exposure of L. johnsonii to oxygen induces the expression of NOX, encoded
by LJ_1254-1255, which contributes to the H2O2 production by this species. Conditions
encompassing prolonged oxygen exposure may be encountered by industrially applied L.
johnsonii strains (e.g., as probiotic cultures) during production, processing and product
shelf-life conditions. This illustrates the relevance of understanding the NFR and NOX
dependent pathways for H2O2 production in L. johnsonii in relation to the lifestyle of
this microorganism. Such knowledge could enable the adaptation of production and
storage conditions for probiotic cultures to ensure higher survival of these bacteria,
while it can also be employed to enhance its in vivo health-promoting efficacy which
may very well depend on the capacity to produce H2O2 in situ close to the mucosa.
86
Supplementary materials
Table S3.1 L. johnsonii genes of which the expression was more than twofold lower in aerobic, as compared to
anaerobic batch cultures.
Gene ID
LJ_0058
LJ_0080
LJ_0081
LJ_0082
LJ­_0265
LJ_­0295
LJ_­0296b
LJ_­0298
LJ_­0299
LJ_­0300
LJ_­0301
LJ_­0302
LJ_­0326
LJ_0328
LJ_0388
LJ_0573
LJ_0718
LJ_0719
LJ_0720
LJ_0721
LJ_0849
LJ_0890
regulation
0,41
0,20
0,23
0,23
0,49
0,49
0,38
0,44
0,30
0,34
0,38
0,44
0,36
0,48
0,48
0,39
0,28
0,24
0,24
0,39
0,49
0,50
fdr
5,3E-04
9,8E-11
6,5E-11
6,3E-11
1,8E-03
4,8E-03
1,5E-04
1,4E-03
1,8E-04
8,9E-05
7,2E-03
2,4E-03
4,0E-03
2,7E-02
4,3E-04
4,9E-06
4,3E-05
2,5E-07
7,2E-06
3,4E-03
7,5E-04
7,1E-04
LJ_1117
LJ_1185
0,39
0,50
2,5E-06
2,1E-04
LJ_1187
LJ_1276
LJ_1429
LJ_1434
LJ_1443
LJ_1445
LJ_1446
LJ_1447
LJ_1448
LJ_1449
LJ_1450
LJ_1451
LJ_1452
LJ_1452b
LJ_1453
LJ_1455
LJ_1549
LJ_1649
LJ_1763
LJ_1764
0,45
0,45
0,42
0,48
0,44
0,39
0,40
0,45
0,38
0,28
0,40
0,42
0,39
0,31
0,38
0,34
0,48
0,46
0,37
0,32
3,5E-05
4,0E-03
2,3E-03
2,1E-03
9,2E-04
4,2E-05
9,1E-05
4,1E-03
1,8E-04
1,7E-06
3,6E-04
8,1E-04
4,1E-04
4,5E-05
5,4E-04
1,6E-05
9,2E-05
1,9E-04
4,5E-03
5,1E-07
LJ_1766
0,39
3,1E-08
annotation (or BLAST homologies)
Bile salt transporter
Uncharacterized
Glycosyltransferase
Endoglucanase Y
ABC transporter permease component
Lj965 prophage antirepressor
Lj965 prophage protein
Lj965 prophage protein
Lj965 prophage protein
Lj965 prophage replication protein
Lj965 prophage protein
Lj965 prophage antirepressor
LJ965 prophage protein
LJ965 prophage holin
Protein translocase subunit SecA 2
Pheromone response surface protein PrgC
Dipeptidase
Aminopeptidase C
Amino acid permease domain containing protein
Amino acid permease-associated protein
HPr kinase/phosphorylase
ABC transporter permease component, Spermidine/
putrescine
UPF0324 membrane protein
Carbamoyl-phosphate synthase, pyrimidine-specific,
small chain
Lipoprotein signal peptidase
Carbamoyl-phosphate synthase large chain
Lj928 prophage protein
Lj928 prophage protein
Lj928 prophage protein
Lj928 prophage protein
Lj928 prophage protein
Lj928 prophage protein
Lj928 prophage protein
Lj928 prophage protein
Lj928 prophage protein
Lj928 prophage protein
Lj928 prophage protein
Lj928 prophage protein
Lj928 prophage protein
Lj928 prophage protein
Exodeoxyribonuclease 7 large subunit
Dephospho-CoA kinase
Carboxymuconolactone decarboxylase
Carboxymuconolactone decarboxylase Related
Protein
Aldehyde-alcohol dehydrogenase
3
87

Table S3.2: Homology of LJ_1254 and LJ_1255 in other lactic acid bacteria as determined by BlastP analysis.
Species-strain
Gene ID;
annotation
Length
(AA)
Homology with
LJ_1255
Homology with
LJ_1254
L. acidophilus
NCFM
NADH dependent
oxidoreductase
LBA_1418
306
No homology
Query coverage 84%,
Identical: 60%
residue 1-265
L. acidophilus
NCFM
Putative NADH
dependent
oxidoreductase
LBA_1421
57
Query coverage
65% Identical 61%
residue 1-56
No homology
L. gasseri K7
Uncharacterized
protein
LK7_09396
383
Query coverage
100%
Identical: 92%
residue 1-86
Query coverage 100%
Identical: 85%
residue 77-383
L. salivarius
NIAS840
NADH-dependent
oxidoreductase
NIAS840_00317
382
Query coverage
100%
Identical: 84%
residue 1-86
Query coverage 97%
Identical: 78%
residue 83-381
L. lactis subsp.
cremoris KW 2
NADH-dependent
oxidoreductase
KW2_1665
391
Query coverage
96%
Identical: 49%
residue 4-86
Query coverage 90%
Identical: 51%
residue 86-367
L. plantarum
WCFS1
NADH:flavin
oxidoreductase
LP_0146
369
Query coverage
100%
Identical: 30%
residue 1-81
Query coverage 89%
Identical: 35%
residue 77-354
L. plantarum
WCFS1
NADH:flavin
oxidoreductase
371
Query coverage
73%
Identical: 30%
residue 6-69
Query coverage 83%
Identical: 34%
residue 79-339
Figure S3.3: Genetic context of LJ_1254 and LJ_1255 in other lactic acid bacteria, using BlastP and Microbial
Genome Viewer.
88
3
89
Chapter 4
Oxygen relieves the CO2 and acetate
dependency of Lactobacillus johnsonii NCC 533
Rosanne Y. Hertzberger1,3,4, R. David Pridmore2,4, Christof Gysler2,4, Michiel
Kleerebezem3,5, M. Joost Teixeira de Mattos1
1) Molecular Microbial Physiology, Swammerdam Institute for Life Sciences, University
of Amsterdam, Science Park, Amsterdam, The Netherlands
2) Nestlé Research Centre, Vers-chez-les-Blanc, Switzerland
3) NIZO food research, Ede, The Netherlands
4) Kluyver centre for Genomics of Industrial Fermentation, The Netherlands
5) Host Microbe Interactomics Group, Wageningen University, Wageningen, The
Netherlands
Published in PlosONE (2013);8(2):e57235

Summary
The probiotic Lactobacillus johnsonii NCC 533 is relatively sensitive to oxidative stress;
the presence of oxygen causes a lower biomass yield due to early growth stagnation.
We show however that oxygen can also be beneficial to this organism as it relieves
the requirement for acetate and CO2 during growth. Both on agar- and liquid-media,
anaerobic growth of L. johnsonii NCC 533 requires CO2 supplementation of the gas
phase. Switching off the CO2 supply induces growth arrest and cell death. The presence
of molecular oxygen overcomes the CO2 dependency. Analogously, L. johnsonii
NCC 533 strictly requires media with acetate to sustain anaerobic growth, although
supplementation at a level that is 100-fold lower (120 microM) than the concentration
in regular growth medium for lactobacilli already suffices for normal growth. Analogous
to the CO2 requirement, oxygen supply relieves this acetate-dependency for growth.
The L. johnsonii NCC 533 genome indicates that this organism lacks genes coding for
pyruvate formate lyase (PFL) and pyruvate dehydrogenase (PDH), both CO2 and acetylCoA producing systems. Therefore, C1- and C2- compound production is predicted to
largely depend on pyruvate oxidase activity (POX). This proposed role of POX in C2/
C1-generation is corroborated by the observation that in a POX deficient mutant of
L. johnsonii NCC 533, oxygen is not able to overcome acetate dependency nor does it
relieve the CO2 dependency.
92
Introduction
The Lactobacillus acidophilus group was recognized early as the most prevalent
inhabitant of the vaginal microbiota (242, 243) and also as the pioneer bacteria in
the developing intestinal microbiota of neonates (16). Various strains and species of
the acidophilus group are marketed as functional ingredients in probiotic products,
associated with health benefits for the consumer. Therefore, understanding of the
physiology of members of this group of lactic acid bacteria is of importance both from
a medical and an economical point of view.
One of the probiotics belonging to this group is Lactobacillus johnsonii NCC 533,
whose genome sequence was published in 2004 (176). Its probiotic functionalities have
been explored in detail, including immuno-modulation (169, 182, 183) and pathogen
inhibition (171). Additionally, its ability to adhere to the epithelial cell was explored
(170, 195).
Analogous to many other members of the acidophilus group, L. johnsonii can be
considered as a highly auxotrophic species lacking the operons for a range of biosynthetic
pathways. The genome of L. johnsonii NCC 533 lacks genes for the synthesis of vitamins,
4
purines, fatty acids and all amino acids (except for the interconversion of L-asparagine
and L-aspartate and the interconversion of L-glutamate to L-glutamine) (176, 185). As
a consequence, L. johnsonii has fastidious growth requirements. Noteworthy in the
context of applicability, the organism does not grow autonomously on milk (244).
In addition to the above-mentioned auxotrophies, and analogous to many other closely
related species, L. johnsonii may require a source of acetate for growth. C2-compounds
are required in many anabolic reactions and acetate-mediated stimulation of growth has
been reported for lactic acid bacteria that exhibit a predominant homolactic metabolism
on hexose sugars, such as Lactobacillus sakei (245) and Lactobacillus delbrueckii (246).
Acetylation of the muramic and glucosamine residues of the peptidoglycan for instance,
involves O-acetylation for which a supply of C2 compounds like acetyl-CoA is essential
(247).
Heterofermentative lactic acid bacteria have the capacity for acetate production, and
are therefore assumed to be independent of exogenous acetate addition. However,
growth of a Δldh- Lactococcus lactis mutant was reported to be stimulated by acetate
which it uses for the conversion to ethanol as a means to regenerate NAD+ in order to
rescue its redox balance (248).
93

Another well-described growth requirement is CO2. L. johnsonii, is a so-called
capnophilic organism, i.e. it has a requirement for either gaseous CO2 or bicarbonate
supplementation for growth, which is a characteristic that is also observed in many
other lactic acid bacteria species (249-251). The C-1 source has been proposed to be
required for the synthesis of a common intermediate of the pyrimidine and arginine
production pathways, carbamoyl-phosphate. In L. plantarum carbamoyl-phosphate
can be synthesized from glutamine, ATP and bicarbonate involving two enzymes:
pyrimidine-regulated CPS-P (encoded by carAB) and arginine regulated CPS-A (encoded
by pyrAaAb) (252). Two regulators of this pathway, PyrR1 and PyrR2 control expression of
the pyr-operon in response to pyrimidine and inorganic carbon levels, respectively (253,
254). The genes of the pyr-operon are conserved amongst many lactobacilli, including
L, johnsonii NCC 533. Homologues of the argFGH genes for arginine biosynthesis are
absent, rendering this species auxotrophic for arginine.
The production and consumption of metabolites, like CO2 and acetate, are known to
stabilize microbial communities. For example, in yoghurt fermentation, Streptococcus
thermophilus and L. delbrueckii show close metabolic relations with the first species
providing the second with CO2, acetate, folate, and formate. In exchange, the
streptococcal species profits from the proteolytic activities of L. delbrueckii (255).
Analogously, it can be anticipated that specific nutritional requirements of microbes
play an important role in the composition of the human microbiota. In view of both its
industrial potential and its niche in the complex microbial environments where these
lactobacilli are generally found, such as the gut, understanding the mechanisms that
underlie these growth requirements are important.
Growth requirements may be strongly dependent on the growth conditions. For L.
johnsonii NCC 533 we observed major differences in growth and viability between
aerobic and anaerobic conditions, including a significantly higher viability in the
presence of molecular oxygen. This is surprising in view of the observation that
L. johnsonii is known to produce H2O2 under aerobic conditions, a compound that
is generally assumed to be toxic (38). The study presented here indicates that the
anaerobic dependency of L. johnsonii for carbon dioxide and acetate is related to its
limited flexibility in pyruvate dissipation pathways, which can be overcome by pyruvate
oxidase activity in the presence of oxygen, placing this enzyme in a pivotal position in
the central metabolism of L. johnsonii.
94
Materials & Methods Strains and culture conditions
Lactobacillus johnsonii NCC 533 was obtained from the Nestlé Culture Collection. NCC
533 plus NCC 9333, a pox-deletion derivative of NCC 533, were routinely cultured in
MRS medium at 37°C under anaerobic conditions. Erythromycin was supplemented at
5 µM/ml as required.
Anopore growth and micro-colony analyses
The AnoporeTM method (inoculation, incubation, imaging) was carried out as described
before by den Besten et al. (256). Multiple AnoporeTM inorganic membranes (AnodiscTM)
were placed on MRS-agar plates and four dilutions (102-105) of an overnight MRS
culture of L. johnsonii NCC 533 were spotted per slide (to ensure that colonies would
be physically separated to allow individual quantification). The plates were incubated at
37°C in Oxoid jars that were filled with a defined gas mixture by vacuuming, followed
by replacement with the gas mixture of choice, and repeated 3 times at the start of
the experiment as well as after every opening of the jar for sampling. Single AnoporeTM
slides were removed at different time points and transferred to a microscopic slide
4
with a pronarose layer that contained the cell permeable SYTO9 stain and the cell
impermeable propidium iodide stain (baclight live/dead staining, Molecular Probes,
Invitrogen). SYTO9 enters all cells but is replaced by propidium iodide whenever the
membrane integrity is compromised.
For every time point and condition between 20 and 147 microcolonies were randomly
selected from one slide and imaged directly without the use of a cover slip or immersion
oil. Photographs were taken through both red and green filters with a cooled chargecoupled device camera (Princeton Instruments, SARL, Utrecht, The Netherlands)
mounted on an Olympus BX-60 fluorescence microscope. A threshold was applied to
create a binary image of the image intensity plots and these were superimposed using
ImageJ. An example of such an image is shown in Supplementary materials, Figure
S4.1 in which images of several colonies are combined. The colony size and viability
were quantified using the ImageJ. The ImageJ plugin ObjectJ was employed to facilitate
colony selection.
Growth in liquid media and physiological characterization
Cells were inoculated at an OD600 of 0.01-0.05 in fresh medium. Growth was monitored
95

in continuously stirred vessels with 400 ml regular MRS medium (205) (for the CO2
dependency experiments) or a chemically defined medium (in case of the acetate
dependency experiments). The latter medium was described for Lactobacillus reuteri
(257) and has previously been shown to also support growth of L. johnsonii NCC 533.
Batches were sparged with specific gas mixtures varying in CO2 and O2 content. Cultures
were grown at 37ºC with constant mixing (ca. 200 rpm) and pH was maintained at
pH 6.5 by automatic 4M NaOH titration. Cell density was determined by measuring
the optical density at 600 nm. Growth rate was determined by fitting an exponential
trendline through the data points with a minimal R2 of 0.99. In cases of very slow
or no growth a trendline was fitted through the data points of the first five hours of
incubation (for instance where chemically defined medium is inoculated in the absence
of acetate and oxygen).
Construction of the pox-deletion derivative of L. johnsonii NCC533
The deletion of the gene LJ_1853 encoding a predicted pyruvate oxidase enzyme
was achieved as described previously (206) with the exception that the plasmid
pDP749 was used. In pDP749 the erythromycin resistance gene in pDP600-Ery has
been flanked by direct copies of the yeast 2-micron plasmid FLP recombination
target sites to facilitate excision of the erythromycin resistance marker by the
FLP recombinase. The 5’ region was amplified from L. johnsonii NCC 533 genomic
DNA with the primers A (ATATATGAGCTCAGCAAGAACGGCTTCTGC) and B
(ATATATGGATCCAGATGCTGCTTCTGGTGC) introducing SacI and BamHI restriction
sites, respectively. The amplicon was SacI-BamHI digested and cloned in similarly digested
pDP749, yielding an intermediate plasmid. The 3’ region was amplified using the primers C
(GTGAACGGCACCAGGACC) plus D (ATATATGGTACCGAAGCATATATTGGGGTC), the
amplicon obtained was PstI-KpnI digested and cloned in similarly digested intermediate
plasmid to yield the pox-deletion plasmid pDP887. Plasmid pDP887 isolated from
Lactococcus lactis was used to transform NCC 533 (185) and loop-in/loop-out gene
replacement was achieved as described previously (206). The deletion was confirmed
by PCR analysis.
Organic acids measurement by HPLC
Extracellular metabolite concentrations were determined as described previously (208)
using HPLC (LKB and Pharmacia, Oregon City, OR, USA) fitted with a REZEX organic
acid analysis column (Phenomenex, Torrance, CA, USA) at 45°C and a RI 1530 refractive
96
index detector (Jasco, Easton, MD, USA). The mobile phase consisted of a 7.2 mM
H2SO4 solution. Chromatograms were analysed using AZUR chromatography software
(St. Martin D’Heres, France).
Results
CO2 dependency of L. johnsonii NCC 533 during aerobic and anaerobic microcolony growth.
To study CO2 dependency of L. johnsonii, we used a high-resolution and quantitative
technique by using AnoporeTM slides to visualize growth on plates that were placed in
jars with a controlled atmosphere. This allowed for the rapid assessment of growth
requirements and in combination with time-resolved microscopic inspection at using
live/dead staining, enabled the generation of additional data related to the organisms’
physiological state, viability and population heterogeneity (256, 258, 259).
This set-up was employed to evaluate growth and viability during CO2 limitation under
aerobic and anaerobic conditions. To this end, AnoporeTM slides on MRS-agar plates
were inoculated with different dilutions of cells and incubated in jars filled with gasmixtures varying in CO2 and O2 content. At regular intervals the viability and size of the
4
colonies were determined using a live/dead baclight stain as described in Materials &
Methods. The sum of the propidium iodide stained pixels and the SYTO9 stained pixels
was used to estimate the size of the colony. The fraction of SYTO9 over all stained pixels
was used as a relative measure of viability.
CO2 supplementation to the gas phase (5%) was found to stimulate growth under
both aerobic (air) and anaerobic (N2) conditions. When plates were transferred to
a CO2 depleted environment, growth stagnated after 7 hours, both in aerobic and
anaerobic conditions. In the presence of supplemented CO2, microcolonies continued
growth with an estimated growth rate of 0.79 h-1 in the anaerobic, and 0.74 h-1 in the
aerobic environment, which is comparable to growth rate in liquid culture (data not
shown). This growth rate was estimated by fitting an exponential trend line through the
average colony size (Figure 4.1, panels A and B). Growth stagnation was accompanied
by loss of membrane integrity observed in microcolonies that are grown without CO2
supplementation, whereas microcolonies grown in CO2 supplemented environments
sustained viability above 90% throughout the experiment (Figure 4.1, panels C and D).
97

Figure 4.1. L. johnsonii NCC 533 is grown on AnoporeTM slides that are transferred from a 2 hour preincubation period in an N2+5% CO2 environment to environments that vary in CO2 and O2 content. Average
size of microcolonies grown aerobically (A) and anaerobically (B) and average viability of microcolonies grown
aerobically (C) and anaerobically (D). Growth after the pre-incubation was either in the presence (closed symbols)
or absence (open symbols) of 5% CO2. Data shown are the mean of all colonies counted for that time point and
condition ± standard deviation.
Notably, microcolonies grown in aerobic atmosphere displayed reduced loss of viability
albeit with a higher degree of heterogeneity, as compared to microcolonies grown in
a nitrogen atmosphere (Figure 4.1 C and D). This observation was remarkable since it
has been documented that L. johnsonii produces H2O2 in the presence of oxygen (38),
which was presumed to reduce growth rate and induce considerable cell death under
aerobic conditions. Taken together, these results suggest that CO2 depletion leads
to loss of membrane integrity and growth stagnation, while oxygenation appears to
support extended viability as compared to anaerobic conditions.
CO2 dependency of L. johnsonii NCC 533 during aerobic and anaerobic liquid growth.
To consolidate the results obtained with the Anopore system in the more routinely
employed liquid culture conditions, L. johnsonii NCC 533 was grown in a pH-controlled
stirred batch culture, sparged with predefined gas mixtures at a rate of 750 ml/min. When
L. johnsonii was grown in MRS medium in this experimental setup, a clear difference
between aerobic and anaerobic growth was observed. Anaerobic and aerobic cultures
reached an exponential growth rate of 0.85 h-1 and 0.69 h-1, respectively. After 6 hours
98
of incubation aerobic growth strongly slowed down and eventually the culture entered
stationary phase, whereas the anaerobic culture continued growth (Figure 4.2A). The
aerobic growth stagnation was related to the accumulation of H2O2 in the extracellular
growth medium, as is evidenced by the complete prevention of the growth stagnation
by the addition of 0.5 mg/ml catalase to the medium (Chapter 2).
Figure 4.2 Growth in stirred pH-controlled batch cultures sparged by N2 + 5% CO2 (closed symbols) or N2 + 20%
O2 + 5% CO2 (open symbols) as measured at OD600. In panel B, the gas regime was switched after 3 hours of
exponential growth from a CO2-rich gas to a CO2-free gas: N2 (closed symbols curve), N2 + 20% O2 (open symbols).
The switch is indicated by the dashed line. Growth curves are representative of at least triplicate experiments.
4
To assess the influence of CO2 on growth in these conditions, cultures were grown until
early-logarithmic phase of growth while sparging a defined gas composition, aerobic
(75% N2, 20% O2 and 5% CO2) or anaerobic (95% N2 and 5% CO2). Subsequently
sparging was switched to a CO2-free gas mixture. Depletion of CO2 in anaerobic cultures
resulted in growth stagnation and initiation of cell death within one hour (Figure 4.2B),
whereas in aerobic cultures this effect was not observed and growth continued until
it stagnated at a final OD of approximately 1.5, due to the accumulation of H2O2.
Overall, these data show that oxygen supplementation in the gas phase relieves the
CO2 requirement for growth, both on solid, as well as in liquid media.
Oxygen overcomes the acetate dependency of L. johnsonii NCC 533
In addition to CO2 dependency, growth of many lactobacilli also depends on the
presence of acetate in the growth medium (246). L. johnsonii was unable to grow in
chemically defined medium without acetate supplementation. Notably, the addition of
as little as 12 µM sodium acetate (1/1000 of the regular sodium acetate concentration
in the chemically defined medium) allowed for recovery of growth, albeit at a slower
rate and yielding lower final biomass concentrations. Acetate supplementation at a 100-
99

fold lower level as compared to its regular concentration in CDM (120 µM) completely
restored normal anaerobic growth (Figure 4.3). These results show that although there
is a strict acetate-requirement for growth, this requirement is already fulfilled with
concentrations that are substantially below the levels that are normally added to typical
Lactobacillus-laboratory media, such as MRS or CDM.
\
Figure 4.3. Growth of L. johnsonii NCC 533 in a chemically defined medium with varying concentrations of sodium
acetate: 12 mM as in standard CDM (closed square symbols) 120 µM (round symbols), 12 µM (triangular symbols)
and without any Na-acetate supplemented (open square symbols) in stirred pH controlled cultures sparged with
N2 + 5% CO2 at a rate of 500 ml/min. The growth curves are the average of duplicate experiments ± standard
error of the mean.
To assess whether the acetate requirement of L. johnsonii NCC 533 depended on the
growth conditions, the strain was grown in chemically defined medium with or without
acetate supplementation (12 mM), under aerobic or anaerobic conditions. Analogous
to what was observed with respect to the CO2 dependency, anaerobic growth of
L. johnsonii NCC 533 depended more strictly on acetate supplementation as compared
to aerobic growth, which could be sustained without an external acetate source, albeit
with a slower growth rate and a lower final biomass yield (Figure 4.4). This implies that
the endogenous production of acetate under these conditions may be expected to be in
the same range as the 12 µM that allowed similar growth restoration under anaerobic
conditions (see above).
Both aerobic and anaerobic growth of L. johnsonii in chemically defined medium
with 12 mM or 120 µM of acetate were analyzed with respect to acetate metabolism:
significant change in extracellular acetate were not detected by HPLC analysis nor by
a highly specific and sensitive acetate kinase/pyruvate kinase assay (results not shown).
This result is likely caused by analytical limitations that did not allow detection of the
minute amounts of acetate that are required to sustain growth under these conditions,
100
(estimated detection limit in spent medium is 200 µM).
Figure 4.4 Growth of L. johnsonii NCC 533 in a chemically defined medium with 12 mM Na-acetate (square
symbols) and without 12 mM Na-acetate (round symbols) in stirred pH controlled cultures sparged with N2 +
5% CO2 (closed symbols) or N2 + 20% O2 + 5% CO2 (open symbols) at a rate of 500 ml/min. Data are average of
independent triplicate experiments ± standard deviation.
In most organisms acetyl-CoA functions as the central C2-intermediate in several
biosynthetic pathways. This metabolite can be produced from pyruvate by reactions
4
catalyzed by pyruvate dehydrogenase (PDH) or pyruvate formate lyase (PFL). However,
apart from a homologue for one subunit of pyruvate dehydrogenase, the corresponding
genes appeared to be absent in the L. johnsonii NCC 533 genome (176). This genotype
is shared with the other members of the L. acidophilus-group (see Supplementary
materials, table S4.1A and S4.1B), indicating that these species lack the capacity for
autonomous acetyl-CoA production from their central energy metabolism (Figure 4.5).
The L. johnsonii genome does encode an enzyme that could provide the cell with both
CO2 and acetate, namely pyruvate oxidase (POX). POX catalyzes a reaction that requires
molecular oxygen as a co-substrate, and therefore its activity may directly explain the
observed physiological consequences of the presence of oxygen (aerobic growth is
independent of an external CO2 and acetate source). Therefore, we hypothesized that
oxygen availability relieves the CO2 and acetate dependency by the pyruvate oxidase
derived supply of both these metabolites.
101

Figure 4.5: Simplified overview of pyruvate metabolism in L. johnsonii. LDH: Lactate dehydrogenase. POX:
pyruvate oxidase. ACK: Acetate kinase. PAT: Phosphate acetyltransferase.
Acetate and CO2 dependency of a pox-deletion mutant.
To test the proposed hypothesis, a pox deletion derivate that lacks the pyruvate
oxidase encoding gene was constructed. Under anaerobic condition in an atmosphere
supplemented with 5% CO2, the growth rate of the mutant in MRS was similar to that
observed for NCC 533. Moreover, under these conditions the wild-type and its poxdeletion derivative displayed a comparable growth arrest upon CO2 depletion. However,
under aerobic conditions, shutting down the 5% CO2 supply elicited rapid growth
stagnation of the pox mutant (Figure 4.6), which is in clear contrast to the wild-type
that continues to grow under these conditions. Clearly, the deletion of pox resulted in
a L. johnsonii mutant that depended on exogenous CO2 supplementation for aerobic
growth. This fully supports the proposed pivotal role of the pox-encoded pyruvate
oxidase enzyme in the generation of this essential C1-source under these conditions.
Analogous to the CO2 supply provided by the POX-pathway under aerobic conditions,
it would be expected that this pathway also provides an acetate supply when oxygen
is available. Consequently, the pox mutant would be expected to be more hampered
aerobically in media that lack exogenous acetate as compared to the wild-type strain.
Generally, the pyruvate oxidase deficient mutant displayed slower growth rates than
the wild type, independent of the presence of oxygen (Figure 4.7A). However, growth
of the pox mutant in the absence of acetate differed considerably, i.e., the typical
102
oxygen relief of the acetate dependency that was observed for the wild type was not
observed for the pox mutant (Figure 4.7B), which supports our hypothesized role of
pyruvate oxidase in generating C2-compounds.
Figure 4.6 Growth of the NCC 533 (closed symbols) and NCC 9333 (open symbols) as measured at OD600 in stirred
batch cultures sparged with N2 + 20% O2 + 5% CO2. The gas regime was switched after 3 hours of exponential
growth to N2 + 20% O2. Data are the average of quadruple independent experiments ± standard deviation.
4
Figure 4.7 Growth rate of L. johnsonii NCC 533 in the standard chemically defined medium with (panel A) and
without 12 mM Na-acetate (panel B) in stirred pH controlled aerobic batch cultures (open bars) or anaerobic batch
cultures (closed bars). Growth rates were determined as explained in Materials & Methods. Data are average of
triplicate experiments (panel A) and duplicate experiments (panel B) ± standard error of the mean.
103

Discussion
Lactobacillus johnsonii is generally described as an anaerobic fastidious lactic acid
bacterium. Fastidious because its growth is dependent on supplementation of various
nutrients to its growth medium, and anaerobic because oxygen cannot be used for
respiration. Moreover, L. johnsonii produces H2O2 when grown under aerobic conditions,
which inhibits growth. Here we present an example that auxotrophy can be dependent
on external conditions that seemingly are not related to the nutrient requirement: we
show that anaerobicity actually exacerbates the fastidious nature of L. johnsonii NCC
533 since the presence of oxygen is shown to relieve at least two of its anaerobic
growth requirements, i.e., the requirement for acetate and CO2.
Both on plates and in liquid culture, L. johnsonii showed clear CO2 dependent growth.
However, the oxygen relief of this dependency was more apparent in liquid culture
than on solid medium, as illustrated by the observation that aerobic growth on
plates without CO2 still resulted in smaller colonies and reduced viability. In contrast,
these CO2 dependent phenotypic differences were completely abolished by oxygen
supplementation in liquid culture. One explanation for the observed difference could be
found in the ambient pH, which is controlled at 6.5 in liquid culture and is uncontrolled
in the Anopore experiment. It should be noted in this context that pH influences the
equilibrium between the different dissolved carbonic species; CO2 dissolves in water as
H2CO3­ (pKa 6.1) and the latter species may be deprotonated in a pH dependent manner
to generate HCO3- and CO­32-, respectively. Thus, lower pH values shift the equilibrium
resulting in release of CO2 from the solution. It is to be expected that on solid media
especially the local pH within the direct environment of emerging microcolonies drops
substantially below 6.1 due to lactic acid production. These micro-scale differences in
environmental conditions experienced by bacteria grown in microcolonies versus liquid
cultures may explain the observed CO2 dependency differences observed.
Like the other species in the L. acidophilus-group (L. delbrueckii, L. gasseri, L. johnsonii, L.
crispatus, L. amylovorus, L. helveticus), the genome of L. johnsonii lacks two major systems
for the production of C2- and C1-compounds, namely the pyruvate dehydrogenase
complex (PDH) and pyruvate-formate lyase (PFL) producing acetyl –CoA (Supplementary
material, table S.1A and S4.1B). Instead, the genomes of these species all encode the
pyruvate oxidase gene that can provide a metabolic source of C2-compounds whenever
molecular oxygen is available for the POX reaction. The primary habitat of L. johnsonii
is considered to be the intestine, which is a predominantly anaerobic environment and
104
would therefore not support POX mediated C2-production. However, in close vicinity
to the mucosal tissues, local and a steep oxygen gradient may be encountered (41)
that may allow for the POX-mediated contribution to metabolism. Notably, preliminary
transcriptome studies of L. johnsonii grown under anaerobic, aerobic and CO2 depleted
conditions did not reveal regulation of the pox gene expression, suggesting that the
enzyme is constitutively expressed. Based on the physiological observations both on
plate and in liquid culture, combined with the absence of these genes, we hypothesized
that pyruvate oxidase activity would play a pivotal role in the acetate and CO2 supply for
the cell. Indeed, a pox-deletion derivative of L. johnsonii did not display a higher growth
rate under aerobic conditions in the absence of acetate, such as observed in the wild
type strain. Moreover, whereas the wild type strain continued to grow upon a switch
to CO2 depletion, growth of the mutant stagnated at a lower biomass concentration.
The observed time lapse between the onset of flushing with CO2 free gas and the
actual CO2 depletion of the system is most likely due to the slow removal of all carbonic
species at a pH higher than 6.1 (the pKa of carbonic acid). Both results show that, in
contrast to the wild type, the pox-mutant has lost the ability to aerobically generate CO2
and acetate. This corroborates the proposed role of pyruvate oxidase in the generation
4
of C1 and C2 metabolic intermediates.
It was observed that the pox mutant has a lower growth rate, both aerobically and
aerobically. Although it can be argued that under aerobic conditions the pox gene
might play a role in protection against its reaction product, H2O2 by allowing for a
faster production rate of ATP via the production of acetyl-phosphate and subsequent
generation of ATP by acetate kinase (91), this argument does not hold for anaerobic
growth conditions. So far, no specific role for POX under these conditions can be
brought forward and the cause of the effect of the deletion on growth remains to be
elucidated.
The major dependency of L. johnsonii on pyruvate oxidase for the supply of these
compounds was rather unforeseen since many other pathways are known and present
in L. johnsonii that can render CO­2 and acetate. Phosphoketolase, for instance, catalyzes
the deacetylation of xylulose-5-phosphate which yields acetyl-phosphate. Similarly,
CO2 can be produced through decarboxylation of amino acids, oxaloacetic acid and
phosphopantotenoyl. However, acetate and CO2 are both required for growth of L.
johnsonii in the absence of oxygen, even though very low concentrations of acetate
(<120µM) already suffice for growth. This suggests that the flux through these pathways
compared to pyruvate oxidase is marginal.
105

It is uncertain, however, that the lactobacilli that do possess PDH and PFL encoding genes
(Supplementary materials, Table S4.1A and B), can actually employ these pathways for
the synthesis of C1 and C2-compounds under aerobic conditions. Literature suggests
that L. plantarum does not possess a functional pyruvate dehydrogenase pathway, since
acetate production does not require CoA and is not hampered by PDH-inhibitors like
arsenate (260, 261). In addition, pyruvate formate lyase activity has been reported to be
highly oxygen sensitive and is only considered active under anaerobic conditions (262).
The presence of genes predicted to encode PFL or genes that resemble the PDH-genes
of other organisms does not preclude that a species still depends on pyruvate oxidase
under aerobic conditions for the production of C2 and C1 components, analogous to
what we concluded for L. johnsonii.
Clear data to support this hypothesis are lacking, although CO2 dependency of
L. plantarum was also reported to cause a characteristic growth stagnation under
aerobic conditions (250). In addition, another study showed that a pyruvate-oxidase
deficient mutant of L. plantarum is hampered in its acetate production capacity (83, 84),
supporting the role of this enzyme in aerobic acetate supply in lactobacilli that have a
broader genetic arsenal.
The effect of deletion of pox in L. johnsonii confirms the role of POX in the generation of
both C1 and C2 sources (CO2 and acetate) required for growth. However, a byproduct
of pyruvate oxidation by POX is H2O2, of which the accumulation induces oxidative
stress that leads to premature growth arrest under aerobic conditions (38). This brings
us to the conclusion that oxygen appears to both benefit and harm L. johnsonii. Under
aerobic conditions, clearly, a lower biomass yield is reached (chapter 2, Figure 2.1) on
the one hand, presumably as a consequence of H2O2 production. On the other, our
data also establish clearly that oxygen can increase the metabolic capacity of the strain,
relieving some of its fastidious growth requirements. These opposing consequences of
oxygen presence suggest that a micro-aerobic environment may be optimal for growth
of L. johnsonii NCC 533.
Here we have refined the metabolic requirements of L. johnsonii NCC 533 and
pinpointed the pivotal role of the pox gene in the requirement for C1 and C2 sources.
These findings can provide novel clues for the optimization of growth conditions of
these commercially relevant microbes, and may in more general terms facilitate a more
efficient regime for the production of probiotics belonging to this group of lactobacilli.
106
Acknowledgements
We would like to thank the Molecular Cytology group at SILS, University of Amsterdam
for letting us use the BX fluorescence microscope and in particular Norbert Vischer for
the assistance with ObjectJ. More information on ObjectJ can be found at http://simon.
bio.uva.nl/objectj
4
107

Supplementary materials
Figure S4.1 Superimposed image (Composite picture) of baclight-stained microcolonies in which images of
colonies after 7 hours of growth in environments that vary in oxygen and CO2 content are grouped. Images were
thresholded, colors were assigned artificially and superimposed as described in Materials & Methods.
108
lar_0608
lvis_1410
reuteri DSM 20016
brevis ATCC 367
lvis_1409
lar_0609
lsei_1306
lrhm_1267
lrhm_1266
lsei_1305
rhamnosus GG
lsl_0154
lca_1084
lsl_0153
lca_1085
salivarius UCC118
sakei subsp. Sakei 23K
casei ATCC 334
lp_2153
lp_2154
plantarum WCFS1
pdhB
pdhA
Lactobacillus
PDH complex
lvis_1408
lar_0610
lsei_1307
lrhm_1268
lca_1083
lsl_0155
lp_2152
pdhC
lvis_1407
lar_0611
lsei_1308
lrhm_1269
lca_1082
lsl_0156
lp_2151
pdhD
-
-
-
-
-
lhrm_1365
lsei_1410
lhrm_1366
lsei_1412
-
lca_0973
lsl_1873
lca_0974
lsl_1872
lca_0973
lp_2596
lp_3313
lp_3314
pflE
pflB
pflA
PFL
-
-
-
-
lca_0974
-
lp_2598
pflF
Table S4.1A: Presence of genes for pyruvate dehydrogenase or pyruvate formate lyase in lactobacilli. Overview of pyruvate dehydrogenase and pyruvate formate lyase
encoding gene prevalence in lactobacilli (Table A) and in species belonging to the L. acidophilus group (Table S4.1B). If no gene was found, a BLAST search was performed
using the protein sequence of the homologue in L. plantarum WCFS1.
4
109

Table S4.1B - Lactobacillus acidophilus group
PDH
complex
Lactobacillus
johnsonii NCC 533
pdhA
-
PFL
pdhB
pdhC pdhD
lj_1267
-
(88% 3e )
-
lba_1490
pflB
pflE
pflF
-
-
-
-
-
-
-
-
(95% 2e )
-17 1
acidophilus NCFM
lj_1757
pflA
-61 2
-
(87% 1e21)1
lba_1220
(96% 1e-65)2
helveticus DPC 4571
-
-
-
lhv_1961, (94%, 3e-66)2
-
-
-
-
delbrueckii ATCC 11842
-
-
-
ldb_0759
-
-
-
-
-
-
-
-
(95% 3e-66)2
gasseri ATCC 33323
-
-
-
lgas_1554 (95% 3e-61) 2
1 BLAST homology to a transketolase
2 BLAST homology to a pyrimidine-dinucleotide oxidoreductase
110
4
111
Chapter 5
Genome-wide transcriptome response to CO2
depletion in Lactobacillus johnsonii

Summary
Lactic acid bacteria belonging to the Lactobacillus acidophilus-group, including
Lactobacillus johnsonii, require a wide variety of nutrients for growth, such as amino
acids, vitamins, fatty acids, acetate and CO2. In chapter 4, we showed that CO2 depletion
during growth of L. johnsonii not only causes growth stagnation but also results in cell
death.
To study the molecular events following CO2 exhaustion, we followed the transcriptional
changes in anaerobically growing cultures that were exposed to a a CO2 depleted
condition. The transcriptome analysis revealed an extensive rearrangement of gene
expression profiles, with 343 genes that were more than twofold induced or repressed
after 120 minutes of CO2 depletion, including substantial rearrangements of the
expression of metabolic functions. These encompassed the strong upregulation of the
carbamoyl-phosphate synthesis pathway (up to 17-fold). This pathway utilizes CO2,
aspartate and glutamine as substrates in the biosynthesis of pyrimidines, suggesting
that intracellular depletion of the pyrimidine pool may be a direct consequence of
CO­2 depletion. However, supplementation of the MRS growth media with pyrimidines
could not alleviate CO2 induced growth stagnation. Moreover, addition of several other
metabolites of which the synthesis is associated with CO2 dependency in other lactic
acid bacteria (arginine and aspartate) could also not complement the CO2 requiring
phenotype, leaving the mechanism behind CO2 dependency to be determined.
114
Introduction
Lactic acid bacteria (LAB) are Gram-positive bacteria that produce lactic acid as their
main metabolic end-product. Several LAB have been recognized to be capnophilic,
which means that their growth is stimulated by supplementation with inorganic
carbon in the form of bicarbonate, gaseous CO2, formate or ureic acid (249-251). In L.
plantarum, the pyr-encoded uridine monophosphate biosynthesis pathway has been
shown to be related to its inorganic carbon requirement. This pathway incorporates
ammonia (from glutamine), phosphate and CO2 to synthesize carbamoyl-phosphate,
which is the precursor for arginine and pyrimidine nucleotides. The inorganic carbon
requiring isolates of L. plantarum depend on exogenous pyrimidine and arginine for
growth in environments that are low in inorganic carbon (252-254, 263).
Aspartate biosynthesis is another metabolic pathway that has been shown to contribute
to CO2 requirements in LAB. The enzyme pyruvate carboxylase uses CO2 to produce the
aspartate precursor, oxaloacetate (251). Correspondingly, supplementation with either
aspartate or citrate (which LAB can convert to oxaloacetate) could complement the
CO2 dependent phenotype (264). Additionally, an older report suggests that oleic acid
(Tween 80) could also be used to complement growth of certain lactobacilli in a CO2
deficient environment (265).
Along with the metabolic pathways that use inorganic carbon as a substrate, several
other biological processes may be affected by the level of environmental CO2.
Pathogenic bacteria are known to modulate virulence related genes in response to CO2
5
and bicarbonate (266-268). In L. plantarum protein levels other than those belonging
to the pyr-regulon undergo significant modulation by elevated CO2 levels, including
for example genes associated with purine synthesis (269). Although arginine and uracil
supplementation suffice to complement the CO2 requirement of L. plantarum, the
supplementation with inorganic carbon could still further stimulate its growth, even in
the presence of these compounds (270). These results indicate that a third unidentified
metabolic pathway requires CO2 as a substrate.
Lactobacillus johnsonii, a bacterium that is marketed as a probiotic, and is recognized
for its immunomodulatory properties (182, 183) and pathogen inhibition (38) was also
shown to require CO2 for growth. The removal of gaseous CO2, from an exponential
growing culture by flushing with dinitrogen gas resulted not only in rapid growth
stagnation but was accompanied by extensive cell death (chapter 4). Interestingly, low
level exposure to molecular oxygen could overcome this CO2 dependency, while it also
115

relieved the acetate-requirement for growth. These observations were explained by a
central role of the pyruvate oxidase pathway in the production of these compounds, as
was supported by the observation that a pox deletion derivative of this species remained
CO2 and acetate dependent, irrespective of the presence of molecular oxygen.
L. johnsonii has many auxotrophies, and is routinely grown in rich and complex
media like MRS (205), which are assumed to contain abundant levels of pyrimidines
and amino acids (176). Nevertheless, even under these rich conditions CO­2­ depletion
elicited growth stagnation and cell death, which we study here at the molecular level by
analyzing the transcriptome pattern changes over time following CO2 depletion. Despite
the rich media employed, CO2 depletion elicited a major adaptation of the expression
of the carbamoyl-phosphate pathway in L. johnsonii. However, unlike L. plantarum the
CO2 dependency of L. johnsonii could not be complemented by exogenous nucleotide
supplies, suggesting that CO2 may have an additional role in L. johnsonii.
Materials & Methods
Strain & cultivation
Lactobacillus johnsonii NCC 533 was obtained from the Nestlé Culture Collection and
was routinely cultured in MRS medium at 37°C under anaerobic conditions.
Growth in liquid and solid MRS media
Cells were inoculated in fresh MRS medium (205). Growth was monitored in continuously
stirred vessels with 400 ml working volume. Batches were sparged at 750 ml/min
with either a gas mix containing 5% CO2 and 95% nitrogen gas or pure nitrogen
gas. Cultures were grown at 37°C with constant mixing (ca. 200 rpm) and pH was
maintained at pH 6.5 by automatic 4M NaOH titration. Cell density was determined
by measuring the optical density at 600 nm (OD600). Viable counts were determined by
placing 2 µl drops of culture dilutions on MRS plates in triplicate and incubating these
plates overnight in a closed container in the presence of an AnaerogenTM bag.
Cultures of L. johnsonii were grown overnight in MRS medium (>16 hours) and serially
diluted in triplicate in fresh MRS medium. Drops of 2 µL from each dilution and each
replicate were placed on the agar plates. Plates were incubated at 37°C in air-tight jars
in CO2-rich or CO2-depleted conditions. Such conditions were achieved by applying
three consecutive cycles of vacuum (0.8 bar) and refilling with either 100% N2 gas (CO2
free) or 95% N2 + 5% CO2 gas (CO2 rich).
116
Transcriptome analysis
Cells were harvested at different time points: 0 minutes (prior to closing of the CO2
feed) and 30, 60 and 120 minutes after closing the CO2 feed. The first sample was
taken at OD600 0.13 ± 0.017 (inoculation level 0.025 ± 0.008, i.e. more than two
duplications after inoculation level), and cells were harvested from 50 ml of culture by
cold centrifugation (5’, 2600xg, 4°C). Sample volumes of the samples taken at later time
points were adjusted in order to obtain a similar amount of biomass for RNA isolation.
Cell pellets were resuspended in 0.5 ml ice-cold Tris-EDTA buffer and transferred to
screw-cap tubes with 0.5 gram zirconium beads (0.1 mm), 0.25 ml acidic phenol, 30 µl
10% SDS and 30 µl 3 M sodium acetate. After mixing, the samples were immediately
frozen in liquid nitrogen and stored at -80°C until further use.
Cells were disrupted by bead-beating in 3 rounds of 40 seconds in a Savant FastPrep
FP120, with in between cooling on ice. Cell debris was removed by centrifugation (20817
x g, 10’, 4°C) and residual phenol was extracted by addition of ice-cold chloroform
followed by centrifugation. RNA was isolated using a High Pure RNA Isolation Kit
(Roche Diagnostics, Mannheim, Germany). RNA purity and yield was determined by
comparison of absorption at 260 and 280 nanometer (Ultrospec 3000, Pharmacia
Biotech, Roosendaal, The Netherlands). RNA quality control was carried out using the
RNA 6000 Nano Assay in an Agilent 2100 Bioanalyzer (Agilent technologies, Palo Alto,
Ca, USA). The Cyscribe Post-labeling kit was used to synthesize cDNA using 5 µg of
total RNA, which was subsequently labeled according to the manufacturer’s protocol
5
(Amersham Biosciences, Amersham, UK). Samples in which the CyDye labeled cDNA
concentration was below 24 ng/µl were concentrated prior to cDNA synthesis using a
Hetovac VR-1 (Heto Lab Equipment A/S, Birkerod, Denmark).
A hybridization scheme was designed that allowed duplicate comparisons between
the transcriptome profiles of aerobic and anaerobic grown cultures. 60 Oligomer
microarrays (Agilent technologies) were used with 12 ± 2.5 probes per gene and 21841
probes in total (GEO accession number GPL18009). These arrays were employed as
previously described (173). In short, two differentially labeled cDNAs (300 ng) were
mixed (final-volume 25µl), incubated at 95° C for 3 minutes and subsequently cooled
to 68° C. To these mixed cDNAs 25 µl Slidehyb#1 hybridization buffer (Ambion, Austin,
USA) and 2X Hi-RPM hybridization buffer (Agilent Technologies) were added and 40
µl of the resulting solution was applied on a 8 * 15K slide preheated at 68°C. Slides
were hybridized at 65°C, rotating at 10 rpm for 16 hours in an Agilent hybridization
oven (Agilent technologies). Subsequently, slides were washed with wash buffer 1
117

(Agilent technologies) at room temperature for 1 minute and wash buffer 2 (Agilent
technologies) at 37 °C. The slides were dried using nitrogen gas and scanned with
a ScanArray Express 4000 scanner (Perkin Elmer, Wellesley, MA). Image analysis and
processing were performed using the ImaGene Version 7.5 software (BioDiscovery Inc.,
Marina Del Rey, CA, USA). The microarrays were scanned at different intensities. For
each of the individual microarrays the best scan was selected on the basis of signal
distribution (combination of a low number of saturated spots and a low number of
low signal spots). The data were normalized using Lowess normalization as available
in MicroPrep (230). The data were corrected for inter-slide differences on the basis
of total signal intensity per slide using Postprep (230). The median intensity of the
different probes per gene was selected as the gene expression intensity. CyberT was
used to compare the different transcriptomes, taking into account the duplicates (dye
swaps) of each of the conditions (231). This analysis resulted in a gene expression ratio
and false discovery rate (FDR) for each gene. Differential gene expression values of
expression-ratios with FDR values <0.05 were considered to be statistically significant.
All microarray data is MIAME compliant and is available in GEO (accession number
GSE52876).
Data analysis
Time dependent gene expression during CO2 depletion was analyzed using Short
Time-series Expression Miner (STEM; http://www.cs.cmu.edu/~jernst/st/; (271)). Log
normalized expression data time series (with four data points obtained for every gene)
were assigned to model profiles. Model profiles are considered significantly enriched
when p<.05. Statistical significance of enriched model profiles was corrected for multiple
hypothesis testing using the Bonferroni method. Time-resolved gene expression of all
genes with significant changes in expression (minimally twofold induction or repression
between any of the four timepoints and p<0.05) can be found in Supplementary
material (Table S5.1A and S5.1B). Functional classes/categories were assigned to each
gene by using the GeneOntology (272) and KEGG platform (273). Categories were
considered statistically significant enriched when p<0.05.
Results and discussion
Inorganic carbon is one of the many growth requirements of Lactobacillus johnsonii.
In chapter 4, we have shown that anaerobic growth of this lactic acid bacterium is
dependent on supplementation of gaseous CO2. Upon termination of the CO2 supply
118
to an exponentially growing culture, growth quickly stagnates which is accompanied by
a considerable loss in cell-viability. Notably, exposure of this bacterium to low-levels of
molecular oxygen relieves this CO2 growth dependency in the wild type L. johnsonii but
not in a derivative that lacks a functional pyruvate oxidase encoding gene (pox deletion
derivative), indicating that endogenously produced CO2 through the pyruvate oxidase
pathway relieves the exogenous CO2 dependency (chapter 4).
To further explore the processes that play a role in CO2 dependency, the global
transcriptome pattern of logarithmic cultures grown under anaerobic conditions during
their gradual depletion for CO2 was analyzed. To this end, L. johnsonii was grown in
batches in which pH, temperature, mixing and gas flow were kept constant. The gas
supply was interrupted by switching from a 5% CO2/95% N2 gas mixture to 100% N2
gas. One hour after the onset of CO2 depletion growth stagnated, whereas growth
continued in the culture in which CO2 was continuously supplemented (Figure 5.1).
Along with growth stagnation, a 1-log drop in colony counts indicating cell death was
observed. These results were in good agreement with experiments presented in chapter
4.
5
Figure 5.1: Optical density (A) or colony forming units (B) of stirred pH-controlled batch cultures sparged by N2 + 5% CO2 (triangular
symbols) or switched from N2 + 5% CO2 to N2 (square symbols, dashed line indicates the switch to pure N2). RNA for transcriptome
analysis was isolated before the switch to CO2-depletion (sample 1), and 30 (sample 2), 60 (sample 3) and 120 minutes (sample 4)
after the intervention. Data shown are the mean of two independent experiments ± standard error of the mean.
The gas-intervention elicits a rapid stripping of the inorganic carbon from the system.
This CO2 removal is driven by the concentration difference between dissolved and
gaseous form of CO2. Many LAB express a carbonic anhydrase (CA), which is generally
assumed to convert CO2 to HCO3-­ (270), which could skew the equilibrium between CO2
and its protonated bicarbonate towards bicarbonate. Three types of CA are described
119

in LAB: the α-type (such as lp_2736 in L. plantarum WCFS1), the b-type (such as icfA/
bl_0616 in Bifidobacterium longum) and the γ-type (such as ef_2918 in Enterococcus
faecalis) (55). However, none of the three types of carbonic anhydrases has a homolog
in L. johnsonii, implying that L. johnsonii has no means to influence the HCO3-­ / CO2
equilibrium.
Samples were taken from the culture prior to closing of the CO2 supply (reference
sample) and at different time points after closing of the CO2 flow (30, 60, and 120
minutes). Notably, the sample taken two hours after closing the CO2 supply represents
a culture in which growth has completely halted and in which viability has decreased
approximately 10-fold (chapter 4).
Global transcriptome analysis
For the interpretation of the transcriptome data, the time-dependent pattern of gene
expression was analyzed using Short Time Series Expression Miner (STEM), which is a
pattern recognition suite that assigns each gene to a model profile based on its pattern
of expression over time. Gene expression data from duplicate independent time series
were compared and four model profiles were significantly enriched. A total of 384
genes passed the criterion of a minimal twofold induction or repression between any
of the four timepoints.
The model profile 40 and 42 that both fall in a cluster of model profiles showing a
general trend of induction of expression over time, comprised 259 genes. In addition,
34 genes were assigned to model profile 11 and 26 that both fall in a cluster of model
profiles that show a general trend of repression over time (see Figure S5.1 for general
trend of each model profile). These profiles were statistically significant enriched when
either a Bonferroni correction or an fdr-correction for multiple hypothesis testing was
applied (significance level p < 0.05). Table 5.1 provides an overview of the induced and
repressed model profiles and overrepresented GeneOntology and KEGG categories in
these model profiles (See Table S5.1A and B for expression values of the genes assigned
to the profile clusters).
Analysis of overrepresentation of genes associated with a specific function or pathway
amongst the induced and repressed groups showed that expression of a number of
predicted regulators and ABC-type transporters was induced. However, since the
predicted target genes of many of the regulators are unknown or have unknown
function and many of the transporters have unknown substrates, the regulation of
120
these particular genes does not readily provide biological insights in the adaptation that
the cell undergoes under these conditions.
Table 5.1: Overrepresented GeneOntology groups (p<0.05) and KEGG categories in the STEM model profiles.
Model
profile
# genes
assigned
GeneOntology group numbers
(p<0.05)
KEGG categories (p<0.05)
231
GO:0043565 sequence-specific
DNA binding
GO:0003700 sequence-specific
DNA binding transcription factor
activity
GO:0006355 regulation of
transcription, DNA-template
GO:0042626 ATPase activity,
coupled to transmembrane
movement of substances
M0051: Uridine
monophosphate
biosynthesis: glutamine
28
19
No categories enriched
(+ PRPP) ==>UMP
No categories enriched
15
Carbamoyl-phosphate pathway
To identify any metabolic patterns present in these data, a functional category
5
analysis was performed using a KEGG functional categories annotation. This led to
the observation that the genes belonging to the carbamoyl-phosphate pathway were
significantly overrepresented in the STEM model profiles with a general pattern of
induction (p<.0045). Previously, this pyrimidine and arginine synthesis pathway has
extensively been studied in L. plantarum. Two types of carbamoyl-phosphate synthase
(CPS) enzymes were identified that are involved in the first carboxylation reaction of
the synthesis pathway (252). In L. plantarum, expression of CPS-P (encoded by pyrAB)
is regulated by intracellular UMP-levels via a transcription attenuation mechanism (263),
whereas expression of CPS-A (encoded by carAB) is controlled by intracellular levels of
arginine. Moreover, two regulators control this pathway, PyrR1 acts as a repressor when
pyrimidine levels in the environment are high (252), while PyrR2 positively regulates
expression of the pyr-operon in response to low inorganic carbon levels (253).
The L. johnsonii genome encodes homologues of all the enzymes involved in the
pyrimidine synthesis pathway described in L. plantarum, including the two PyrR
121

regulators. Moreover, L. johnsonii also encodes two copies of the CPS encoding genes,
which are both present in an operon (lj_1276-1277 and lj_1185-1184). The entire
carbamoyl-phosphate and pyrimidine synthesis pathways were induced in L. johnsonii
120 minutes after the onset of CO2 depletion, with the highest fold-induction within
the entire datasets assigned to the aspartate carbamoyltransferase encoding pyrB and
dihydroorotase encoding pyrC genes that were 17- and 13-fold induced, respectively.
The CPS-A encoding genes (carAB) did not display any significant change in expression.
Moreover, the uracil permease (lj_0709; pyrP) of L. johnsonii was also strongly induced
(10-fold higher expression), indicating that similar to L. plantarum (274) the uracil
transport capacity is coregulated with the pyr-operon in L. johnsonii. However, the
uracil phosphoribosyltransferase enzyme (encoded by lj_0933; upp), which catalyzes the
conversion of imported uracil to UMP, was not significantly affected by CO2 depletion.
Notably, the absolute level of expression of upp was found to be among the 200 highest
expressed genes within the L. johnsonii genome, which may suggest that this function
may not be limiting for pyrimidine import. In a second potential uracil salvage pathway,
uridine monophosphate is formed via the ribonucleoside uridine. Genes encoding the
enzymes that catalyze the two reactions, uridine kinase (lj_0763; udk) and uridine
phosphorylase (lj_1217-lj1218; udp), are both present in the L. johnsonii genome but
their expression did not show significant induction or repression upon CO2 depletion.
A schematic overview of the carbamoyl-phosphate pathway is presented in Figure 5.2
including changes in gene expression.
The observed activation of expression of the carbamoyl-phosphate synthesis operon
and pyrimidine import strongly suggests that, analogous to what has been shown for
L. plantarum, depletion of the pyrimidine pool in L. johnsonii is the main cause of the
observed growth stagnation upon removal of inorganic carbon supplies. In L. plantarum,
approximately 30% of the strains displayed a high environmental-C1-carbon demand,
which could be complemented by supplementation of the growth media with either
arginine or uracil (249). L. johnsonii is auxotrophic for arginine, irrespective of the
availability of environmental CO2, suggesting that it is unlikely that arginine exhaustion
is the main cause of growth stagnation in this species. Moreover, the transcription of
the genes encoding the arginine-depletion responsive CPS-A (carAB) were unaffected
during CO2 depletion, supporting that intracellular arginine depletion is not occurring.
Analogously, supplementation of solid MRS medium with excessive concentrations of
arginine (10 mM), did not relieve the CO2 dependency of L. johnsonii (Figure 5.4).
Although the current experiments employed the rich laboratory medium MRS for
122
growth of L. johnsonii, the extracellular pyrimidine concentrations or their rate of
import may still be limiting to sustain normal growth under CO2 depleted conditions.
Moreover, it is well-established that the exhaustion of thymine, another product of
the carbamoyl-phosphate pathway, can cause cell death in auxotrophic bacteria, by
creating disbalanced endogenous dNTP-pools, leading to single and double strand DNA
breaks (275). This so-called thymineless death was also shown to occur in species of
the L. acidophilus group (276). Therefore, we evaluated whether pyrimidine exhaustion
could be the primary cause of cell death following CO2-depletion, by supplementing
MRS with pyrimidines and pyrimidine precursors at a very high level (100 mg/L).
Although the genome of L. johnsonii predicts that this bacterium can interconvert uracil
to the other pyrimidines (thymidine and cytosine), also thymidine, uracil and the uracil
precursor orotic acid were added to the medium. None of these supplementations was
able to overcome the growth stagnation and cell death induced by CO2 depletion. It
even appeared that the growth arrest of the pyrimidine supplemented culture was even
slightly accelerated as compared to the normal MRS media (Figure 5.3).
In addition to the experiment in liquid medium, growth of L. johnsonii on solid MRS
medium with and without addition of nucleotides was tested. To this end, overnight
cultures were spotted on MRS agarose, with or without 100 mg/l of uracil, thymidine,
cytosine and orotic acid and colonies were enumerated after growth with or without
5% gaseous CO2. The difference between colony forming units (cfu) in the presence
and absence of CO2 on regular MRS medium was approximately 2 log. Analogous
5
to liquid medium, growth in the absence of CO2 on solid media supplemented with
excess pyrimidines and pyrimidine precursors did not improve compared to regular
medium. We conclude that extracellular pyrimidine supplementation cannot overcome
the inorganic carbon requiring phenotype in L. johnsonii, which is in clear contrast
to previous observations made for L. plantarum. However, there is no experimental
evidence that the PyrP uracil transporter in L. johnsonii is functional, and ineffective
import could explain why supplementation of pyrimidines did not complement this
cell death. Alternatively, the upp- or the udk/udp-encoded pathway to convert uracil
into UMP may be defective in L. johnsonii, which could also explain why the uracil
supplementation is not effective. The transcriptome analyses imply clearly that drainage
of the intracellular pyrimidine pools may be the causal mechanism of CO2-related cell
death in L. johnsonii, but for as yet unknown reasons, extracellular supplementation
with the corresponding components failed to overcome such drainage and could thus
not overcome the observed cell death.
123
124
1,1 0,9 0,8 1,0 1,1 1,0 1,0 1,0 1,0 0,8 6,7 9,5 Arg argH argG argF Carba-­‐P pyrB Pi α-­‐KG Gln ATP CO2 + ADP 1,1 1,2 17,5 Asp 1,1 1,2 2,1 OAA 0,9 1,0 1,0 PEP PPi Carba-­‐Asp pyrC pyrD pyrE pyrF 1,0 1,2 1,2 1,1 1,1 1,9 1,7 1,5 13,1 4,4 4,8 6,0 UMP udk uridine 1,4 1,5 1,5 1,2 1,3 1,5 1,0 1,1 1,0 ATP PPi DNA synthesis OUT 0,9 1,9 10,9 D-­‐ribose-­‐1-­‐P pyrP ADP udp pyrimidines 0,9 0,9 0,6 H2O + NADH2 NAD++PRPP PPi + CO2 upp PRPP uracil IN uracil Figure 5.2: Aspartate and pyrimidine biosynthesis pathway in L. johnsonii and differences in their expression level 30 min, 60 min and 120 min after closing of the CO2
supply compared to CO2-rich conditions. Pathways were adjusted from Kegg-pathway (273) and (249, 274). PEP: phosphoenolpyruvate. OAA: oxaloacetate. Asp: aspartate.
Gln: glutamine. α-KG: α-ketoglutarate. Carba-P: carbamoyl-phosphate. Arg: arginine. Carba-Asp: carbamoyl-aspartate. PRPP: phosphoribosyl pyrophosphate UMP: uridine
monophosphate. carAB and pyrAB: carbamoyl-phosphate synthase. argF-H: ornithine carbamoyltransferase, argininosuccinate synthase, argininosuccinate lyase. pepCK:
phosphoenolpyruvate carboxykinase. aspC: aspartate transaminase pyrB: aspartate carbamoyltransferase, pyrC-F dihydroorotase, dihydroorotate dehydrogenase, orotate
phosphoribosyltransferase, orotidine 5’-phosphate decarboxylase. pyrP: uracil permease. upp: uracil phosphoribosyltransferase. udp: uridine phosphorylase. udk: uridine
kinase. Reactions/enzyme whose gene is absent in the L. johnsonii genome (argFGH) are depicted in grey. Green indicates induced expression (upper limit 5-fold induced), red
indicates repressed expression (lower limit at 5-fold repressed).
pyrAB carAB HCO3-­‐ + NH4+ (Gln) 2 ATP 2 ADP aspC pepCK glycolysis 
Figure 5.3: Optical density of L. johnsonii cultures grown in regular MRS (closed symbols) or MRS supplemented
with 100 mg/l of uracil, thymidine and orotic acid (open symbols). The dashed line indicates the time point where
gas regime is switched from sparging N2 + 5% CO2 to pure N2. Data shown are the mean of two independent
experiments ± standard error of the mean.
Aspartate biosynthesis
In other LAB, aspartate biosynthesis has been identified as one of the CO2 requiring
pathways (251, 277, 278). Aspartate is required in the carbamoyl-phosphate pathway
as a substrate for PyrB, the aspartate carbamoyl transferase. This amino acid is one of
the few amino acids that L. johnsonii is predicted to be able to synthesize autonomously
from intermediates of the glycolytic pathway. Aspartate transaminase (aspC, lj_1390)
(176) uses oxaloacetate and glutamine as substrates to produce aspartate and
α-ketoglutarate. The transcription of the aspC gene was 2.1-fold induced after 120
5
minutes of CO2 depletion, relative to CO2 rich conditions. Oxaloacetate is formed by
carboxylation of phospoenolpyruvate (PEP), catalyzed by a PEP-carboxylase (PEPC)
that uses primarily bicarbonate as a substrate and does not produce an additional
ATP, or by PEP carboxykinase (PEPCK) that uses CO2 and produces one ATP (279). The
PEPC function in L. johnsonii appears to be disrupted by a single point mutation that
introduces a premature stop-codon and thus is classified as a pseudogene (lj_1272 and
lj_1273). In contrast the PEPCK function appears intact and is encoded by the lj_0149
gene. Thus, PEPCK and ASPC could synthesize aspartate from central metabolism (PEP),
using glutamine and CO2 as co-substrates (Figure 5.2). Although transcription of aspC
is induced and transcription of pepCK is not significantly changed, we hypothesize that
aspartate may be synthesized through a CO2 requiring pathway. Although aspartate is
assumed to be presented in sufficient amounts in the MRS medium, we verified that
excess supplementation with this amino acid (30 mM) did not complement the CO2
requirement (Figure 5.4).
125

Figure 5.4: Enumeration of colony forming units of L. johnsonii on solid MRS agar supplemented with aspartate
(30 mM), arginine (10 mM) or pyrimidine nucleotides and precursors (100 mg/L) incubated under a N2 + 5% CO2
(open bars) or N2 (grey bars) atmosphere. Concentrations of the supplements were approximately 10-fold higher
than those normally present in chemically defined medium (see Materials & Methods section of chapter 4). Data
represent average of two independent experiments + standard error.
Concluding remarks
The experiments in chapter 4 show that L. johnsonii requires CO2 for growth. In
other LAB, the CO2 growth-dependency was shown to be related to the biosynthesis
pathways of pyrimidines (uracil) and/or amino acids (arginine and aspartate). Our results
clearly show that, analogous to L. plantarum, the carbamoyl-phosphate pathway is
regulated in response to CO2 levels. However, supplementation of uracil, thymidine
and the uracil-precursor orotic acid could not prevent the stagnation of growth
related to CO2 depletion, nor could it prevent the induction of cell death. This implies
that besides pyrimidine synthesis, an alternative pathway is responsible for the CO2
demand. However, it may also be that the pyrimidine uptake system is compromised
in L. johnsonii, which could explain why pyrimidine supplementation of the media did
not overcome the growth stagnation. The constitutively high expression of the uracil
phosphoribosyltransferase-encoding upp gene, may indicate that the catalytic function
of this enzyme is compromised. This could imply that it is part of a futile regulatory circuit,
in which the importer is transcriptionally upregulated but fails to increase intracellular
levels of its substrate and therefore provide negative feedback on its expression.
One of the possibilities to study the uracil transport capacity is to use the toxic uracil
analog 5-fluorouracil. If the uracil transport function in L. johnsonii is impaired, the cells
should be resistant to this cytotoxic molecule. Furthermore, uridine supplementation
should be able to bypass the postulated impairment of uracil import and conversion,
and could thus prevent the CO2-related cell death, provided that drainage of the
126
pyrimidine pool is indeed the prime cause of cell death.
An alternative pathway that could depend on CO2 is aspartate biosynthesis, where the
first step in the biosynthesis pathway involves the carboxylation of phosphoenolpyruvate,
leading to the formation of the aspartate-precursor oxaloacetate and ATP. However,
aspartate supplementation to the medium did also not complement the CO2 requirement.
Nevertheless, we cannot rule out that, besides aspartate, also other metabolites depend
on oxaloacetate as an intermediate and their synthesis may thereby be dependent on
an adequate CO2 supply.
Overall we conclude that L. johnsonii has limited decarboxylases that could generate
endogenous CO2. A comparison of the CO2 dependency of a wild type strain of
L. johnsonii and its pyruvate oxidase deficient derivative in the presence of oxygen
suggested that oxidation of pyruvate could provide an additional CO2 source (chapter 4).
L. johnsonii lacks most other genes encoding C1-generating enzymes, such as several
known amino acid decarboxylases, pyruvate dehydrogenase and pyruvate formate lyase.
Interestingly, L. johnsonii’s natural habitat is the intestinal tract of mammals, where
these bacteria may encounter substantial levels of CO2 and bicarbonate in certain areas
of the GI-tract, e.g., due to bicarbonate-rich pancreatic secretions (280) or as a result of
the metabolic activity of other microbiota members. We speculate that, analogous to
the loss of the capacity to synthesize many of the amino acids, or vitamins, evolutionary
adjustment to the CO2 rich environment of the GI-tract may include the loss of CO2
5
producing capacities.
Finally, it is noteworthy that at present many of the genes that were modulated by the
CO2 depletion lack a function prediction and/or do not contain recognizable domains,
including several genes of which the expression was quite drastically changed like
lj_1790 (10.2-fold induced) and lj_1300 (6.6-fold repressed). These genes may play a
significant role in the CO2 depletion response, but such a role remains to be established,
which illustrates the importance of continued elucidation of the biological functions of
such genes.
127

Acknowlegements
We thank Dr Anne de Jong from the Molecular Genetics Group of Groningen University
for the compilation of the gene assignments to functional categories used in our
analyses.
128
Supplementary material
Figure S5.1: General trend of expression of STEM model profiles.
120 min
30 min
STEM
locus
60 min
Table S5.1A: Expression ratio of all genes with minimal twofold induction between any of the four timepoints
(p<0.05). NCBI annotation is shown where available. Alternatively Gene Ontology classification of proteins or
conserved domains is shown (in grey). Where no Gene Ontology classification could be assigned, the closest BlastP
result is shown (displayed in grey, bold and italic). PTS: phosphotransferase system. PEP: phosphoenolpyruvate.
LJ_0020
LJ_0021
LJ_0025
LJ_0027
LJ_0030
LJ_0031
LJ_0034
LJ_0036
LJ_0037
LJ_0038
LJ_0039
LJ_0040
LJ_0041
LJ_0042
LJ_0047
LJ_0048
LJ_0050
LJ_0059
LJ_0062
LJ_0063
LJ_0072
LJ_0073
LJ_0074
LJ_0077
LJ_0083
LJ_0084
LJ_0089
LJ_0090
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
18
18
18
18
40
42
40
40
1,3
1,4
1,1
1
1,2
1,4
1,3
1,1
1,3
1,2
1,4
1,1
1
1,2
1,1
1,2
1,3
1,2
1,1
1,2
0,9
1
1
1
1,2
1,3
1,5
1,1
1,4
1,4
1,3
1,1
1,1
1,5
1,4
1,3
1,2
1
1,3
1
0,9
1,1
1,1
1,2
1,1
1,3
1
1,1
1,1
1,2
1,2
0,9
1,4
1,7
1,6
1,2
2,3
2,1
2,4
3,7
2,1
3,2
5,7
2,4
2,4
4,9
6
3,8
3,5
2,1
3,2
2,2
2
2,1
3
4,5
2,7
3,2
4,1
1,8
2,2
3,1
2,5
3
LJ_0091
18
1
1,1
2,9
LJ_0092
40
1
1,3
3,9
LJ_0093
LJ_0098
LJ_0099
LJ_0101
LJ_0107
LJ_0123
LJ_0128
LJ_0143
LJ_0154
LJ_0162
LJ_0163
40
42
40
40
40
40
42
40
40
40
40
1,3
1,4
1,1
1,2
1,1
1,3
1,6
1,1
1,2
1,1
1,2
1,5
1,6
1
1,3
1,1
1,3
2,2
1,7
1,5
1
1,1
2,9
2,3
2,3
2,2
3,2
2,4
2,7
3,7
2,7
2
3,7
NCBI annotation
Transcriptional regulator
Molybdenum cofactor biosynthesis, MoeB
EDD domain protein, DegV family
Transcriptional regulator
Endopeptidase O
Transcriptional regulator
Mg2+ transporter protein, CorA-like/Zinc transport protein ZntB
Exodeoxyribonuclease
Helicase, C-terminal
Cell surface hydrolase
Cell surface hydrolase
Uncharacterized protein
Predicted permease
FAD-dependent pyridine nucleotide-disulphide oxidoreductase
Adhesion exoprotein
Homeobox protein, antennapedia type, conserved site
LysR, substrate-binding
Phospholipid/glycerol acyltransferase
Glycosyl transferase, family 8
Phospholipid/glycerol acyltransferase
ABC transporter permease component
ABC transporter ATPase component
ABC transporter permease
LemA-like protein
HD containing hydrolase-like enzyme
HNH endonuclease
MarR family transcriptional regulator
Membrane protein
Methylated DNA-protein cysteine methyltransferase DNA binding
domain
Methylated DNA-protein cysteine methyltransferase ribonucleaselike domain
Sirtuin family
Ribonuclease H domain
Uncharacterized, extracellular protein
Alpha/beta superfamily hydrolase
SCP-like protein
Uncharacterized membrane protein
Universal stress protein
LPXTG-motif cell wall anchor domain protein
Uncharacterized small conserved protein
Putative secreted protein
Sensory box protein
5
129

130
LJ_0165
LJ_0172
LJ_0181
LJ_0185
LJ_0186
LJ_0187
LJ_0197
LJ_0201
LJ_0222
LJ_0227
LJ_0228
LJ_0234B
LJ_0243
LJ_0253
LJ_0254
LJ_0259
40
40
42
40
40
40
40
40
40
40
40
18
27
42
42
14
LJ_0260
40
1,2
1,2
2,9
LJ_0261
LJ_0262
LJ_0288
LJ_0292
LJ_0293
LJ_0294
LJ_0304
LJ_0305
LJ_0306
LJ_0309
LJ_0310
LJ_0324
LJ_0325
LJ_0328
LJ_0329
LJ_0376
LJ_0377
LJ_0378
LJ_0379
LJ_0383
LJ_0394
LJ_0396
LJ_0418
LJ_0420
LJ_0421
LJ_0422
LJ_0452
LJ_0456
LJ_0457
LJ_0458
LJ_0459
LJ_0459B
LJ_0462
LJ_0463
LJ_0464
LJ_0481
LJ_0482
LJ_0483
LJ_0486
LJ_0494
LJ_0495
LJ_0496
LJ_0499
LJ_0502
LJ_0522
LJ_0524
LJ_0525
LJ_0528
LJ_0529
40
40
40
40
18
40
40
40
18
14
40
14
40
40
40
40
40
40
40
14
40
40
18
40
42
40
40
40
40
40
40
40
40
40
40
42
40
40
40
40
40
40
40
18
40
40
18
40
40
1,1
1
1,2
1,1
1
1,2
1,1
1,2
1
0,9
1,2
0,9
1,2
1,1
1,2
1,1
1,2
1,1
1,3
0,9
1,2
1,1
0,8
1
1,3
1,1
1,2
1,3
1,3
1,4
1,3
1,2
1,1
1,2
1,2
1,1
1,1
1,2
1,3
1,1
1,2
1,1
1,1
1
1,1
1,2
1
1,1
1
1,1
1
1,2
1,1
1
1,2
1,3
1,4
1,3
0,7
1,1
0,8
1,3
1,1
1,2
1,1
1,4
1,2
1,4
0,8
1,3
1
1
1,1
1,9
1,2
1,4
1,4
1,5
1,7
1,6
1,6
1,5
1,4
1,2
1,8
1,1
1,2
1,5
1
1,3
1,1
1,2
1,4
1,4
1,3
1,1
1,2
1,3
2,4
2
2,2
2,6
3,1
4,5
3,1
2,4
2,3
2,3
2,2
3,3
4,7
4,4
5,9
2,9
5
2,2
2,1
2,4
3,5
2,5
1,8
2,3
3
3,7
2,1
3,4
3
3,5
3
3,8
3,7
2,3
3,6
2,2
2
2,5
2,2
2,3
3,5
2,2
2
2,6
2,3
3
2,7
2,6
2,1
1,2
1,2
1,2
1,4
1
1,3
1,2
1
1,2
1,3
1,2
0,8
1,1
1
1,2
1
1,6
1,1
1,6
1,5
1,2
1,4
1,3
1,3
1,3
1,5
1,4
1
1,9
1,6
1,8
0,9
3,2
2,1
2,1
2,6
2,3
2,3
3,8
8,2
2,3
2,5
2
1,8
0,8
2,5
2,1
1,9
Toxin-antitoxin system
Cell envelope-like function transcriptional regulator
Heat shock protein Hsp20
Transcription regulator, GntR
Transposase IS204/IS1001/IS1096/IS1165
Uncharacterized
Uncharacterized
Cation-transporting P-type ATPase
Uncharacterized
Xanthine phosphoribosyltransferase
Beta-carotene 15,15-monooxygenase
Uncharacterized
Uncharacterized
Uncharacterized
Uncharacterized
Raffinose operon transcriptional regulator
Raffinose permease containing PEP-dependent sugar PTS EIIA
domain
Alpha-galactosidase
Sucrose phosphorylase
Lj965 prophage integrase
Lj965 prophage protein
Lj965 prophage repressor
Lj965 prophage repressor
Lj965 prophage protein
Lj965 prophage protein
Lj965 prophage protein
Lj965 prophage portal protein
Lj965 prophage protein
Lj965 prophage protein
Lj965 prophage protein
Lj965 prophage holin
Lj965 prophage lysin
ArsR-type DNA-binding domain
Cation efflux protein
Uncharacterized
Uncharacterized
Amino acid/polyamine transporter I
Beta-1,6-galactofuranosyltransferase
LytR family transcriptional regulator
Uncharacterized
Putative flagellar protein FliS
Uncharacterized
tRNA-specific adenosine deaminase
Uncharacterized
Lysine decarboxylase
PEP-dependent sugar PTS EIIC cellobiose specific
LytTR DNA-binding domain
Uncharacterized
Uncharacterized
Transcriptional regulator
Putative transcriptional regulator
ABC transporter ATPase and permease components
uncharacterized hydrocarbon binding protein
Glutamate racemase
Non-canonical purine NTP pyrophosphatase
Small mechanosensitive ion channel protein
Glycerophosphoryl diester phosphodiesterase
Uncharacterized
5'-Nucleotidase, C-terminal
uncharacterized membrane protein
General stress protein 13
Glutamine amidotransferase
MFS transporter permease
Tat pathway signal sequence domain protein / cytochrome C5
Transcriptional regulator protein RpiR
Beta-lactamase class A
LJ_0536
LJ_0553
LJ_0573
LJ_0574
LJ_0589
LJ_0595
LJ_0599
LJ_0600
LJ_0613
LJ_0614
LJ_0615
LJ_0617
LJ_0618
LJ_0623
LJ_0637
LJ_0640
LJ_0644
LJ_0645
LJ_0646
LJ_0647
LJ_0648
LJ_0649
LJ_0652
LJ_0653
LJ_0669
LJ_0672
LJ_0673
LJ_0674
LJ_0675
LJ_0709
LJ_0712
LJ_0722
LJ_0723
LJ_0724
LJ_0725
LJ_0726
LJ_0730
LJ_0738
LJ_0741
LJ_0748
LJ_0749
LJ_0750
LJ_0751
LJ_0757
LJ_0758
LJ_0759
LJ_0760
LJ_0761
LJ_0762
LJ_0783
LJ_0784
LJ_0785
LJ_0788
LJ_0814
LJ_0815
LJ_0836
LJ_0864
LJ_0900
LJ_0902
LJ_0923
LJ_0965
LJ_1004
LJ_1005
LJ_1039
LJ_1042
LJ_1049
40
40
40
40
42
40
40
40
40
18
40
40
40
40
18
40
42
40
18
18
40
40
40
40
40
40
40
40
40
18
40
18
40
40
40
37
40
37
14
37
40
40
40
42
40
40
40
42
40
18
40
40
42
29
40
25
40
37
40
25
18
40
30
42
40
40
1,1
1,2
1,2
1,1
1,5
1,1
1,4
1,2
1,1
1
1,3
1,1
1,1
1,6
0,9
1,3
1,4
1
1
0,9
1
1
1,2
1
1,1
1,2
1,1
1,1
1,1
0,9
1,3
1
1,1
1,1
1,1
1,3
1,3
1
0,9
1,3
1,3
1
1
1,4
1,1
1,1
1,1
1,4
1,2
1
1
1,2
1,1
1,1
1,3
1
1,1
1,1
1
1
0,9
1,1
1
1,3
1,1
1,1
1,1
1,2
1,1
1,1
1,7
1,3
1,7
1,3
1
1
1,5
1,2
1,4
1,5
1
1,6
1,7
1,2
1,2
1,1
1,2
1,1
1,3
1
1,1
1,2
1,2
1
1,1
1,9
1,4
1
1,3
1,3
1,4
0,9
1,4
0,9
0,7
1
1,5
1,1
1
2,6
1,1
1
1,3
2,3
1,5
1
1,1
1,2
1,5
3,2
1,4
0,9
1,1
0,9
1,1
0,8
1,4
1,1
2
1,6
1,2
0,9
3,5
4,7
2,3
2,4
2,5
2
3,2
2,8
4,3
5,1
13,8
3,6
2,8
2,4
2
3,1
2,4
2,2
2,1
2,2
2,3
2,1
3,4
2,1
2,1
3,7
5,7
2,5
2,6
10,9
2,3
3,6
6,1
6,6
5
3,5
2,9
1,8
1,5
2,5
7,6
3,4
2,7
3,5
2,4
2,2
2
6,6
2,7
2,6
2,5
3,9
2,2
5,1
2,4
1,8
2,1
1,9
2,1
2,2
2,1
2,1
1,2
2,3
2,6
4,6
Alpha/beta hydrolase fold domain
Colicin V production, CvpA
YSIRK Gram-positive signal peptide
Gram-positive signal peptide protein
Plasmid maintenance toxin/Cell growth inhibitor
Uncharacterized
GTPase HflX
ABC transporter ATPase component
Cation-transporting ATPase
Cation-transporting ATPase
Metallopeptidase, catalytic domain
Cysteine protease YvpB
SGNH hydrolase-type esterase domain
CAT RNA-binding domain
Transcriptional regulator protein RpiR
Large-conductance mechanosensitive channel
Oxidoreductase
Peptidase T
Uncharacterized
Uncharacterized membrane protein
Sir2 family NAD-dependent protein
DNA polymerase III
DNA/RNA non-specific endonuclease
Dipeptidase
SNARE-like domain protein
Transcriptional regulator
Alpha/beta hydrolase fold-3
Uncharacterized membrane protein
Acyl-CoA N-acyltransferase
Uracil permease
Phosphoglycerate mutase
Major facilitator superfamily permease
Uncharacterized
Transcription regulator, MarR-type
Uncharacterized
Uncharacterized
Phophatidylserine decarboxylase
Beta-galactosidase
PEP-dependent sugar PTS EIID, probable fructose specific
Cell surface hydrolase
Transcriptional regulator, MarR family
ABC transporter ATPase and permease components
ABC transporter ATPase and permease components
Uncharacterized
Trehalose-6-phosphate hydrolase
Trehalose operon repressor
PEP-dependent sugar PTS EIIABC, probable trehalose specific
Uncharacterized
Uncharacterized
Cation-transporting P-type ATPase
Cation-transporting P-type ATPase
Acyl transferase/acyl hydrolase/lysophospholipase
Acyl-CoA N-acyltransferase
Uncharacterized
ATP-dependent clp protease ATP-binding subunit clpE
Glucose/ribitol dehydrogenase
UvrABC system protein A
Alpha/beta superfamily hydrolase
DNA/pantothenate metabolism flavoprotein
Glycopeptide antibiotics resistance protein
Uncharacterized
DNA polymerase III
30S ribosomal protein S20
Uncharacterized
Uncharacterized
dTDP-glucose 4,6-dehydratase
5
131

LJ_1050
LJ_1051
LJ_1052
LJ_1059
LJ_1060
LJ_1063
LJ_1064
LJ_1066
LJ_1070
LJ_1088
LJ_1107
LJ_1108
LJ_1118
LJ_1121
LJ_1122
LJ_1128
LJ_1136
LJ_1137
LJ_1146
LJ_1166
LJ_1194
LJ_1195
LJ_1202
LJ_1203
LJ_1204
LJ_1215
LJ_1229
LJ_1230
LJ_1231
LJ_1237
LJ_1238
LJ_1239
LJ_1247
LJ_1248
LJ_1249
LJ_1250
LJ_1252
LJ_1254
LJ_1255
LJ_1260
LJ_1266
LJ_1270
LJ_1271
LJ_1275
LJ_1276
LJ_1277
LJ_1278
LJ_1279
LJ_1280
LJ_1281
LJ_1282
LJ_1283
LJ_1284
LJ_1287
LJ_1325
LJ_1388
LJ_1390
LJ_1417
LJ_1422
LJ_1435
LJ_1436
LJ_1438
LJ_1441
LJ_1454
LJ_1457
LJ_1458
132
40
18
18
40
40
40
40
40
40
18
40
40
40
40
42
37
40
29
40
40
40
40
37
14
40
42
14
18
40
40
40
18
40
40
18
40
18
40
40
40
37
40
40
18
18
18
40
40
18
40
40
40
18
42
20
40
40
18
40
18
40
40
42
29
40
40
1
1
0,9
1,1
1,2
1,2
1,2
1,1
1,1
0,8
1,1
1,1
1,1
1,3
1,7
1,1
1,4
0,8
1,7
1,3
1,5
1,5
1
1
1
1,1
0,9
1
1,2
1,4
1,3
0,7
1,1
1,3
1
1,5
1
1
1,1
1,1
1,1
1,3
1,1
1
0,8
1
1
1,1
1
1,2
1,1
1,2
1
1,5
0,7
1,1
1
0,8
1,2
0,9
1,4
1,1
1,3
1,3
1,2
1
0,9
0,9
0,9
1,2
1,3
1,3
1,1
1,3
1,1
1,1
1
1
1,2
1,5
2,5
0,9
1,5
3,2
1,7
1,3
1,3
1,5
0,9
0,9
1
1,5
0,7
0,9
1,3
1,5
1,3
1,1
1,2
1,4
0,9
1,9
0,9
1
1
1
0,9
1,4
1,2
0,8
1
1
1,1
1,2
1,6
1,9
1,5
1,7
0,9
2,3
1,5
1,4
1,2
0,9
1,2
1,1
1,4
1,1
2
2,2
1,3
1,1
3,5
2,5
2,5
2,6
2,5
2,4
2,1
2,4
4,1
1,9
2,8
2,1
2,1
2,4
3,9
2,2
2,8
3
4,9
2,4
2,1
5,3
1,9
1,9
1,9
2,1
1,8
2
2,1
3,6
3,5
1,7
3
5,4
4,3
3,9
1,9
2,5
2,4
3,1
2,3
5,9
2,3
4,3
6,7
9,4
13,1
17,5
4,3
4,4
6
4,8
4
2,5
0,9
2,3
2,1
1,8
2,5
2,1
3,5
2,6
2,4
2
3,5
2,1
Glucose-1-phosphate thymidylyltransferase
dTDP-4-dehydrorhamnose 3,5-epimerase
dTDP-4-dehydrorhamnose reductase
Uncharacterized
NUDIX hydrolase domain
Uncharacterized
Rib/alpha-like repeat protein
Oxidoreductase
UvrABC system protein C
ECF transporter, Riboflavin transporter RibU
SMF protein
DNA topoisomerase 1
Transcription regulator, LysR
Uncharacterized
ABC transporter ATPase component
Domain of unknown function DUF1542
Uncharacterized
Branched-chain amino acid transport system carrier protein
Uncharacterized
ABC transporter ATPase component
Cell division protein GpsB
UPF0398 protein
ATP-dependent helicase, C-terminal
ATP-dependent helicase/nuclease subunit A
ATP-dependent helicase/deoxyribonuclease subunit B
Bifunctional protein FolD 1
Uncharacterized
Phospholipase D-like domain
Uncharacterized
Uncharacterized
Uncharacterized
Acyl-CoA N-acyltransferase
ABC transporter ATPase and permease components
ABC transporter ATPase component
ABC transporter ATPase and permease components
SPFH/Band 7/PHB domain protein
Sugar-phosphate isomerase
Aldolase-type TIM barrel
Aldolase-type TIM barrel
Lambda repressor-like, DNA-binding domain
Transketolase
Armadillo-type fold
Glycosyl transferase, family 1
Bifunctional protein PyrR
Carbamoyl-phosphate synthase large chain
Carbamoyl-phosphate synthase small chain
Dihydroorotase
Aspartate carbamoyltransferase
Bifunctional protein PyrR
Dihydroorotate dehydrogenase A (fumarate)
Orotidine 5'-phosphate decarboxylase
Orotate phosphoribosyltransferase
Beta-carotene 15,15-monooxygenase
UPF0145 protein
30S ribosomal protein S21
SGNH hydrolase-type esterase domain
Aspartate aminotransferase
Phosphatidic acid phosphatase type 2/halopero
Lj928 prophage protein
Lj928 prophage major head protein
Lj928 prophage minor head protein
Lj928 prophage protein
Lj928 prophage terminase large subunit
Lj928 prophage protein
Lj928 prophage repressor protein
Lj928 prophage protein
LJ_1465
LJ_1466
LJ_1501
LJ_1502
LJ_1504
LJ_1505
LJ_1509
LJ_1510
LJ_1511
LJ_1523
LJ_1544
LJ_1556B
LJ_1557
LJ_1565
LJ_1566
LJ_1567
LJ_1568
LJ_1571
LJ_1574
LJ_1575
LJ_1576
LJ_1577
LJ_1588
LJ_1590
LJ_1610
LJ_1615B
LJ_1621
LJ_1657
LJ_1658
LJ_1659
LJ_1660
LJ_1666
LJ_1679
LJ_1686
LJ_1687
LJ_1688
LJ_1689
LJ_1690
LJ_1702
LJ_1724
LJ_1725
LJ_1727
LJ_1728
LJ_1729
LJ_1730
LJ_1738
LJ_1740
LJ_1747B
LJ_1751
LJ_1752
LJ_1753
LJ_1754
LJ_1765
LJ_1774
LJ_1778
LJ_1779
LJ_1785
LJ_1786
LJ_1787
LJ_1788
LJ_1789
LJ_1790
LJ_1791
LJ_1794
LJ_1796
LJ_1802
40
40
40
40
42
40
40
40
42
42
42
40
40
40
40
40
18
40
42
40
40
42
40
40
40
30
40
40
40
40
40
40
40
18
40
40
40
42
40
18
40
14
14
40
40
42
40
40
40
40
40
40
49
18
42
18
18
40
40
40
40
40
40
40
40
40
1,3
1,3
1,2
1,1
1,1
1,1
1,4
1,5
1,5
1,5
1,3
1,1
1,2
1,1
1,2
1,2
1
1,2
1,3
1,1
1,3
1,5
1,1
1,2
1,1
0,9
1
1,1
1
1,1
1,2
1,4
1,2
1
1,3
1
1
1,4
1,3
1
1,1
0,9
0,9
1,3
1,1
1,3
1,2
1,2
1,2
1,3
1,1
1,5
1,4
1
1,3
0,9
1
1
1
1,2
1,2
1,2
1,1
1,1
1
1,2
1,4
1,3
1,2
1,3
1,4
1,3
1,4
1,5
1,6
1,7
1,7
1,5
1,1
1,1
1,6
1,3
1
1,4
2
1,3
1,6
2,3
1
1,2
1,4
2,2
1,1
1,3
1,1
1,2
1,3
1,4
1,4
1,3
1,3
1,1
1,1
1,7
1,5
1
1
0,7
0,8
1,6
1,2
1,7
1,2
1,4
1,1
1,2
1,1
1,6
2,1
1,3
1,8
1,2
1,2
1,2
1,2
1,5
1,4
1,9
1,1
1,6
1,2
1,4
2,1
2,7
2,1
2,8
2,1
2,2
2,3
2,3
2,2
2,3
2,7
3,5
2,6
2,7
2,8
3,3
3,2
4,1
2,1
2,2
4,1
5
2,1
2
3
1,6
2
4,4
2,4
2,1
2,5
3,2
2,8
2,4
2,8
2,7
4,1
2,4
4,2
2
2
2,3
2,4
3,4
2,1
2,1
2,3
2,1
2,2
2,5
2,6
2,7
1,3
2
3,6
2,2
2,7
4,6
5,6
4,6
4,1
10,2
2,6
3,1
4,4
2,7
Lj928 prophage integrase
LysR, substrate-binding
DNA methylase, N-6 adenine-specific, conserved protein
Phospholipid/glycerol acyltransferase
ABC transporter ATPase and permease components
ABC transporter ATPase and permease components
Uracil-DNA glycosylase
NUDIX hydrolase domain
Signal peptidase I
Oligopeptide ABC transporter solute-binding component
Uncharacterized
Uncharacterized
Uncharacterized membrane protein
Uncharacterized membrane protein
Alpha/beta hydrolase fold-3
DNA binding domain, AraC-type
RmlC-like cupin domain
Uncharacterized
ABC transporter solute-binding component
Transcriptional regulator
Major facilitator superfamily permease
Uncharacterized
RelA/SpoT
K homology domain-like, alpha/beta
Alpha/beta hydrolase
Uncharacterized
ABC transporter permease
GntR family transcriptional regulator
Two-component system histidine kinase
Two-component system response regulator
Bacterial extracellular solute-binding, family 1
Uncharacterized
Pseudouridine synthase
Uncharacterized
Uncharacterized
Aminoglycoside 3-phosphotransferase
Beta-lactamase
Uncharacterized
DNA-directed RNA polymerase subunit delta
Zinc-metalloprotease
Glycosyl transferase
Polysaccharide biosynthesis protein
CDP-glycerol glycerophosphotransferase
Transcriptional regulator
ABC transporter ATPase and permease components
Glycerol-3-phosphate cytidylyltransferase
Uncharacterized
Phosphate-starvation-induced PsiE
Replication initiation factor
ATP transporter
Uncharacterized
Uncharacterized
Uncharacterized
Amino acid transporter
Uncharacterized
Major facilitator superfamily permease
Uncharacterized
Quinonprotein alcohol dehydrogenase-like superfamily
Uncharacterized
Uncharacterized
Uncharacterized
Uncharacterized
Cystathionine beta-synthase, core
Uncharacterized
Transcriptional regulator
Uncharacterized
5
133

LJ_1803
LJ_1804
LJ_1805
LJ_1806
LJ_1807
LJ_1816
LJ_1817
LJ_1818
LJ_1819
LJ_1822
LJ_1823
LJ_1828
LJ_1831
LJ_1833
LJ_1834
LJ_1839
LJ_1841
LJ_1843
LJ_1845
LJ_1848
LJ_1850
134
40
18
40
40
40
40
40
40
40
40
40
40
40
40
40
40
40
18
42
40
40
1,4
0,9
1,1
1,1
1,1
1,1
1,1
1,4
1,2
1,4
1,3
1,4
1,2
1,3
1,3
1
1,7
1
1,6
1
1,3
1,6
1
1,3
1,3
1,2
1,5
1,3
1,3
1,4
1,5
1,7
1,6
1,3
1,4
1,6
1
2,1
0,9
2
1,1
1,4
3,2
2,5
2,3
2,3
3,1
3,5
3,3
2,3
2,2
3,9
3,6
2,5
2,3
2,3
3,6
2
4,5
1,9
2,8
2,1
3,1
Uncharacterized
PEP-dependent sugar PTS EIIC, probable galactitol specific
Uncharacterized membrane protein
CAAX amino terminal protease
Uncharacterized membrane protein
Basic membrane lipoprotein
Transcriptional regulator protein RpiR
Aldolase-type TIM barrel
N-acetylmuramic acid 6-phosphate etherase
Aldo/keto reductase
Glucosamine-6-phosphate deaminase
DNA binding domain, putative
Transcriptional regulator
Uncharacterized
Beta-carotene 15,15-monooxygenase
Protein of unknown function DUF1085
Foldase protein PrsA 2
Ornithine decarboxylase
Acyl-CoA N-acyltransferase
ABC transporter ATPase component
DNA mismatch repair protein MutS, C-terminal
60 min
120 min
LJ_0085
LJ_0086
LJ_0087
LJ_0129
LJ_0132
LJ_0240
LJ_0268
LJ_0302
LJ_0312
LJ_0313
LJ_0318
LJ_0319
LJ_0320
LJ_0326
LJ_0416
LJ_0490
LJ_0506
LJ_0512
LJ_0604
LJ_0605
LJ_0627
LJ_0628
LJ_0629
LJ_0630
LJ_0700
LJ_0745
LJ_0803
LJ_1012
LJ_1027
LJ_1028
LJ_1029
LJ_1030
LJ_1031
LJ_1035
LJ_1132
LJ_1156
LJ_1174
LJ_1211
LJ_1262
LJ_1299
LJ_1300
LJ_1301
LJ_1302
LJ_1320
LJ_1392
LJ_1415
LJ_1426
LJ_1429
LJ_1448
LJ_1451
LJ_1473
LJ_1476
LJ_1533
LJ_1625
LJ_1745
LJ_1746
LJ_1763
LJ_1827
30 min
locus
model
profile
Table S5.1B: Expression ratio of all genes with minimal twofold repression between any of the four timepoints
(p<0.05). NCBI annotation is shown where available. Alternatively Gene Ontology classification of proteins
or conserved domains is shown (in grey). Where no Gene Ontology classification could be assigned, the
closest BlastP result is shown (displayed in grey, bold and italic). PTS: phosphotransferase system. PEP:
phosphoenolpyruvate.
NCBI annotation
15
27
11
9
26
23
11
9
34
9
22
9
11
38
34
11
26
22
11
26
15
11
25
11
11
26
26
26
15
11
11
11
11
22
26
9
26
26
9
26
26
11
34
11
11
9
20
11
26
26
11
11
26
34
27
11
26
14
0,8
1
0,7
0,8
0,8
0,8
0,7
0,6
1,2
1
0,8
0,8
0,8
1,3
1,1
0,9
1
0,8
0,8
0,9
0,8
0,7
0,9
0,7
0,8
0,9
0,9
0,8
0,7
0,9
0,8
0,8
0,8
0,8
1
1,1
1
1,1
1
1
1
0,6
1
0,8
0,7
1
0,5
0,8
1,1
1,1
0,8
0,8
0,9
1,1
0,9
0,8
1
0,7
1,1
1,3
0,9
0,5
0,9
0,5
0,4
0,3
1
0,7
0,5
0,5
0,8
1,5
0,9
0,7
0,8
0,4
0,9
1,1
1
0,8
0,4
0,8
0,9
0,9
0,8
0,9
0,9
0,6
0,7
0,8
0,7
0,5
0,9
0,6
1
0,9
0,7
0,8
0,8
0,5
0,7
0,5
0,5
0,7
1,5
0,8
1,2
1,1
0,6
0,8
0,9
0,9
1,3
0,9
0,8
0,5
0,5
0,6
0,4
0,2
0,4
0,5
0,1
0,1
0,6
0,4
0,6
0,5
0,5
0,7
0,6
0,4
0,4
0,5
0,4
0,5
0,5
0,5
1,9
0,5
0,4
0,4
0,4
0,4
0,5
0,4
0,3
0,4
0,5
0,7
0,5
0,3
0,5
0,5
0,5
0,5
0,5
0,2
0,5
0,2
0,2
0,4
1
0,4
0,5
0,5
0,2
0,5
0,5
0,5
0,5
0,4
0,5
1,6
Uncharacterized
ABC transporter ATPase component
ABC transporter permease component
Phosphonate-binding periplasmic protein
Phosphate/phosphonate ABC transporter permease component
Transcriptional regulator
50S ribosomal protein L31 type B
Lj965 prophage antirepressor
Lj965 prophage scaffold protein
Lj965 prophage protein
Lj965 prophage protein
Lj965 prophage major tail protein
Lj965 prophage protein
Lj965 prophage protein
50S ribosomal protein L7/L12
Xaa-Pro dipeptidase
Seryl-tRNA synthetase
Drug/metabolite transporter
ABC transporter ATPase component
ABC transporter permease protein BceB-type
PEP-dependent sugar PTS EIIAB, probable mannose specific
PEP-dependent sugar PTS EIIC, probable N-acetylgalactosamine specific
PEP-dependent sugar PTS EIID, probable N-acetylgalactosamine specific
Uncharacterized
Glycerol uptake facilitator protein
Dipeptide/tripeptide permease
Probable xylulose-5-phosphate/fructose-6-phosphate phosphoketolase
Probable GTP-binding protein EngB
Glycosyl transferase, family 1
Glycosyl transferase, family 1
Glycosyl transferase, family 1
Glycosyltransferase
Oligosaccharide repeat unit polymerase
Acyltransferase 3
Two-component system response regulator
Transcription regulator, MarR-type
ATP-dependent DNA helicase RecQ
Major facilitator superfamily permease
CAT RNA-binding domain
Uncharacterized
ABC transporter permease
ABC transporter ATPase component
Transcription regulator, GntR
Glycine--tRNA ligase alpha subunit
Histidine--tRNA ligase
SWEET sugar transporter
Lj928 prophage protein
Lj928 prophage protein
Lj928 prophage protein
Lj928 prophage recombination protein
Amino acid transporter
Sortase
50S ribosomal protein L28
Phenylalanine--tRNA ligase alpha subunit
Surface protein, aggregation promoting factor
Surface protein, aggregation promoting factor
Carboxymuconolactone decarboxylase
Purine permease PbuG-like
5
135
Chapter 6
General discussion and outlook

This thesis addresses the consequences of oxygen and CO2 exposure on the metabolism
and transcriptome profile of the lactic acid bacterium Lactobacillus johnsonii. These two
gaseous components were found to have a major influence on metabolism, growth,
gene expression, yield, and viability. Here we discuss several questions that logically
follow from our findings and we suggest future experiments that could bring us closer
to answering these questions. Furthermore, we discuss the relevance of the research
presented in this thesis for the interactions between L. johnsonii and the mucosal
surfaces of host organisms.
CO2 growth dependency and cell death
The growth requirement of L. johnsonii for CO2 in the absence of oxygen is one of
the clear novel findings in this thesis. Previously, other LAB, including S. thermophilus
and a subset of L. plantarum and L. lactis strains (251, 252) were shown to depend
on an exogenous C-1 source under certain conditions. Given the similarities in LAB
metabolism, we expect that CO2 dependency is a characteristic that is conserved among
a variety of LAB.
One of the transcriptional responses to CO2 depletion was the induction of the
carbamoyl-phosphate pathway genes, reported in chapter 5. Regulation of this
pathway was previously shown to play a role in governing CO2 and pyrimidine
metabolism in L. plantarum (252, 263, 269). Expression of the pyr-operon was also
found to be controlled in several other LAB by a variety of conditions. For example, the
pyr-operon was repressed during bile and acid stress of L. rhamnosus GG (281, 282),
coculturing of L. lactis with S. cerevisiae led to substantial repression of the pyr-operon
in L. lactis (283), and growth of L. casei in ‘soy milk’ compared to bovine milk induced
the expression of this operon (284). We consider that many of these environmental
changes also include a change in the availability of an environmental C-1 supply; bile
solutions may contain bicarbonate, and low pH influences the CO2/HCO3- equilibrium,
while cocultivation with S. cerevisiae is expected to increase environmental CO2 levels.
Taken together, we consider it likely that many of the environmental conditions known
to control the expression of the pyr-operon in LAB, may in fact be responses to the
availability to an appropriate C-1 source for growth, which thereby plays an important
role in metabolic control in these bacteria.
There are three distinctive characteristics of CO2 dependency that we report here, that
were not described before. Firstly, in contrast to other LAB, the CO2 dependency of L.
138
johnsonii cannot be relieved by addition of pyrimidine nucleotides, arginine or aspartate.
Even with addition of excessive amounts of these nucleotides and amino acids, the
typical 2-fold difference in viability of L. johnsonii grown under pure nitrogen versus
CO2-rich conditions could not be complemented. One of the possible causes may be a
dysfunctional pathway for incorporation of exogenous uracil. An indication for this is the
high level of expression (amongst the 200 most highly expressed genes in L. johnsonii)
of the upp-encoded uracil phosphoribosyltransferase in normal, CO2-rich, conditions.
This enzyme catalyzes the conversion of imported uracil to uracil monophosphate. Its
high-level transcription would be expected in case L. johnsonii would depend on the
continuous import of uracil for regular growth in MRS. However, preliminary studies
with chemically defined medium from which uracil was omitted, did not reveal uracil
auxotrophy in L. johnsonii in medium in which CO2 is not actively removed. These
findings imply that the carbamoyl-phosphate pathway is functional in vivo. The
metabolic mechanism underlying CO2 dependency in L. johnsonii deserves further
exploration, since no supplementation strategy could be identified to complement this
dependency by specific nutrients, which is clearly different from what has been found
in various other LAB (see also chapter 5).
A second characteristic that was not previously identified in LAB, is the substantial
induction of cell death that was associated with CO2 depletion. Both in microcolonies
(Anopore studies) and in liquid cultures, we observed that CO2 depletion led to loss of
membrane integrity (as demonstrated by propidium iodide staining) and abolished the
capacity to grow (as demonstrated by viability plating). This is a remarkable observation,
since the absence of components required for growth is normally bacteriostatic rather
than bactericidal. Following our hypothesis on the relation between CO2 and pyrimidine
biosynthesis, we speculate that a depletion of the intracellular pyrimidine pool is
6
the mechanism underlying the lethality of CO2 depletion. Fatal DNA breaks due to
disbalanced nucleotide pools is a well-known consequence of thymidine exhaustion in
both prokaryotes and eukaryotes (275, 276), and such a mechanism may explain the
CO2-related cell death in our experiments.
This cell death is especially of relevance for the application of L. johnsonii in the food
industry, in particular in its application as a probiotic. Survival of probiotic bacteria in
industrial processes as well as in the final products can be considered as a prerequisite
for the health beneficial effect they elicit in the consumer. It is an important notion that
not only oxidative stress may lead to viability loss, but depletion of the available C-1
nutrients in the industrial and product environments may also be an important cause
139

for loss of bacterial (probiotic) viability.
A third surprising characteristic of CO2 depletion was the relation to environmental
oxygen, reported in chapter 4. In contrast to the expectations, bubbling a fermenter
with N2 + O2 resulted in higher growth rates and biomass yields than bubbling with
pure N2. Similarly, L. johnsonii displayed significantly higher growth rates in the absence
of acetate under aerobic conditions as compared to anaerobic conditions. This growth
stimulatory effect of oxygen was shown to be dependent on pyruvate oxidase-mediated
CO2 and acetate production, since it could be abolished in a pox deletion derivative of
L. johnsonii. These findings imply that oxygen, besides its deleterious consequences
in terms of hydrogen peroxide production (see below), also has beneficial effects by
reducing the fastidious growth requirements of L. johnsonii.
The role of NFR and NOX in aerotolerance of Lactobacillus
johnsonii.
One of the most prominent differences between aerobic and anaerobic growth of L.
johnsonii is the production of substantial amounts of H2O2. Accumulation of this H2O2
results in an approximately 10-fold lower biomass yield in the presence of oxygen, due
to premature H2O2-induced growth stagnation. Continuous removal of H2O2 through
the addition of exogenous catalase prevents aerobic growth stagnation.
In chapter 1 and 2 we discuss the role of two enzymes, NOX and NFR, in H2O2
production of L. johnsonii. Based on our findings we propose that NFR is constitutively
expressed, whereas NOX is expected to complement NFR after longer-term oxygen
exposure. This redundancy for H2O2 production capacity, in combination with the
strongly increased oxygen sensitivity observed in a L. johnsonii derivative that lacks both
NFR and NOX, implies that NADH oxidation, oxygen consumption and/or hydrogen
peroxide production are important to sustain aerotolerance and to allow aerobic
growth of L. johnsonii. However, our experimental approaches did not directly enable
the identification of the precise role of H2O2 production in the overall aerobic physiology
of L. johnsonii, and below we discuss some of the possible mechanistic explanations.
Can H2O2:NADH oxidases confer aerotolerance by scavenging oxygen?
One of the most striking observations on the role of the H2O2 producing enzymes
NOX and NFR, is that they appear to be essential for aerotolerance of L. johnsonii:
a deletion of the genetic loci that encode these enzymes resulted in a strain that
140
displayed significantly lower growth rate and final OD under aerobic conditions. This
finding provides an interesting paradox; H2O2 production is the main source of growth
inhibiting stress under aerobic conditions, but at the same time appears to be essential
for growth under these conditions.
Preliminary experiments comparing oxygen consumption rates in wild type L. johnsonii
and its Δnfr and Δnfr Δnox derivatives, establish that nfr and nox encode the main
oxygen scavenging capacity in L. johnsonii, since the deletion of both genes led to a
complete elimination of oxygen consumption. Based on these results and the other
findings presented in this thesis, we postulate that NFR and NOX may contribute to
aerotolerance by scavenging oxygen. In this proposition, we consider the possibility that
molecular oxygen and possibly superoxide derivatives resulting from the spontaneous
autoxidation of cellular components, may under certain conditions be more damaging
than H2O2. The most predominant damaging reaction in bacteria due to H2O2 is the
result of Fenton chemistry, demonstrated by the effectivity of iron chelators to reduce
H2O2-induced cell death (131, 157, 285). L. johnsonii has a remarkably low number
of enzymes that are predicted to contain an iron-sulfur cluster (3 vs 145 in E. coli).
If L. johnsonii maintains very low intracellular iron pools, similar to other LAB (151,
286), hydroxyl formation through Fenton chemistry would be rare. Thereby, the cellular
make-up of L. johnsonii would render it intrinsically tolerant against low-levels of H2O2.
Moreover, we assume that in the natural habitat in which this type of lactobacilli are
encountered, they are only transiently exposed to limited amounts of oxygen. Oxygen
is thought to freely diffuse over the bacterial membrane, driven by the intra- and
extra-cellular concentration difference. Once inside the cell, oxygen could react with
cellular components such as flavins, resulting in superoxide production and launching
a cascade of damage inducing reactions. However, superoxide formation and its
6
detrimental consequences could be prevented by the effective scavenging of oxygen
by NADH oxidases that convert it into H2O2. The small, uncharged H2O2 molecule can
freely diffuse out of the cell as long as the extracellular H2O2 levels remain low (287). In
a crowded environment such as the intestinal microbiota, where L. johnsonii co-occurs
with numerous catalase and peroxidase producing bacteria, one could plausibly assume
that the released H2O2 would be quickly scavenged and is unlikely to accumulate to
substantial levels.
The argumentation raised above could explain how in certain environments H2O2producing NADH oxidation could contribute to aerotolerance, by the prevention of
141

superoxide mediated damage, but at the same time preventing the excessive accumulation
of H2O2 by exploiting the ecosystem’s H2O2 defusing capacity. Consequently, the absence
of elaborate oxidative stress defense mechanisms in L. johnsonii may be a result of its
adaptation to its natural habitat, where it expresses only a minimal defense against the
limited oxygen exposure, and exploits its environment for effective detoxification of the
H2O2 it produces, which is relatively nontoxic to L. johnsonii anyway.
The argumentation raised above could explain how in certain environments H2O2producing NADH oxidation could contribute to aerotolerance, by the prevention
of superoxide mediated damage, but at the same time preventing the excessive
accumulation of H2O2 by exploiting the ecosystem’s H2O2 defusing capacity. Consequently,
the absence of elaborate oxidative stress defense mechanisms in L. johnsonii may
be a result of its adaptation to its natural habitat, where it expresses only a minimal
defense against the limited oxygen exposure, and exploits its environment for effective
detoxification of the H2O2 it produces, which is relatively undamaging to L. johnsonii
anyway.
The notion that LAB use oxidases to scavenge oxygen from its environment is supported
by two studies. Rezaiki et al. showed that L. lactis grown under respiratory-permissive
condition in Erlenmeyer-flasks can effectively create an anaerobic environment,
indicated by low GFP fluorescence and ceased H2O2 production (53). In addition, Gibson
et al. demonstrate that the deletion of a protein identified as a water-forming NOX in
S. pyogenes resulted in a H2O2 producing strain that displayed a severe aerobic growth
defect. One of the possible explanations of this observation is that the protein that
was annotated as NOX is in fact an NADH peroxidase, since it is difficult to distinguish
between these enzymes (see above; Table S1.1). However, if the enzyme was correctly
identified as an NADH oxidase, it could scavenge oxygen to a level that is sufficient to
prevent H2O2 production via the aerobic lactate utilization pathway (288).
We propose to explore this hypothesis by the following set of experiments:
•
a careful assessment of the toxicity of the different reactive oxygen species, for
instance by engineering superoxide dismutase and catalase expressing variants
of L. johnsonii.
•
an analysis of the intracellular iron pools, or the total iron content compared to
species that do express ROS-scavenging enzymes. This could provide support
for the hypothesis that L. johnsonii can withstand low H2O2 levels due to its
intrinsic resistance against H2O2 damage.
142
•
quantification of superoxide production in cultures and cell free extracts of
L. johnsonii that lack both NOX and NFR (nox,nfr deletion derivative) could
provide support for the theory that oxygen scavenging by NOX and NFR
prevents superoxide production.
Interestingly, preliminary experiments with the introduction of plasmid borne expression
of a SOD gene from L. paracasei in wildtype L. johnsoni and its Δnfr and Δnfr Δnox
derivatives, indicated that SOD expression recovers H2O2 production in the Δnfr Δnox
background, indicating that these cells contain superoxide radicals that act as substrate
in the SOD catalized reaction leading to H2O2 production. Further experiments should
include a comparison of aerotolerance levels in these strains. In case SOD expressions
indeed improves aerotolerance in the Δnfr Δnox mutant, this would substantiate the
proposed primary role of the H2O2 forming NADH oxydases in oxygen scavenging to
prevent the formation of the more detrimental superoxide.
Perspectives: the relevance of ROS in host/microbiota interactions
The experiments that are presented in this thesis observe L. johnsonii in a relatively
unnatural single-species environment which provides useful insights in the metabolism
and physiology of L. johnsonii, but neglects all metabolic and physiological effects in
complex microbial communities associated with mucosal surfaces that can be considered
as its natural habitat. Here, we would like to take a broader perspective on bacterial
H2O2 production and discuss how it may affect host/microbe interactions.
Role of ROS in host defense and signaling
Reactive oxygen species have a central role in the non-specific innate immune response.
6
Pathogenic bacteria are engulfed in phagocytic vesicles of dedicated immune cells
such as macrophages and neutrophils. Activity of phagocytic-NADPH oxidases (Nox2)
generates high levels of superoxide, referred to as respiratory or oxidative burst. The
high levels of superoxide results in the formation of secondary ROS such as H2O2 and
hypochlorous acid (HOCL) collectively leading to the death of the phagocytized bacterial
cell.
Besides this dedicated anti-bacterial use of ROS, H2O­2 produced in non-phagocytic tissue
by NOX (1,3,5) and DOUX (1,2) plays a role as signaling molecules in a variety of pathways.
In many of these processes H2O2 does not transduce the signal directly but oxidizes thiol
143

peroxidases, thioredoxin or glutathione that function as secondary messengers. The
cysteine residues of many regulatory enzymes, such as tyrosine phosphatases, protein
kinase phosphatases, NF-κB and ubiquitins are sensitive to oxidation by these thiols
and have been reported to be modulated by such redox signaling. Additionally, H2O2
produced by epithelial NOX can also interfere with signaling processes in pathogenic
bacteria such as Campylobacter jejuni, where the oxidation of a tyrosine kinase prevents
capsule formation, which is an important virulence factor (289).
NOX-related ROS production serves a variety of roles: it can act directly antibacterial,
and simultaneously recruit an immune response by activating inflammasome, cytokines
and prostaglandins (290). Dysregulation of this process can result in excessively
high levels of ROS production and can induce hyperinflammation, which is a typical
characteristic encountered in Inflammatory bowel disease (IBD). Hyperinflammation
cascades induce mucosal tissue morphological destruction, including the formation of
lesions, ulcerations and fibrosis (291). Interestingly, the oral administration of LAB that
overexpress superoxide dismutase or catalase has been shown to attenuate disease
symptoms in experimental animal models for IBD, like the chemically induced rodent
(mouse or rat) colitis models (138, 292, 293).
At the same time, ROS-producing NOX, provides negative feedback on this sytem
and plays an important role in suppressing the inflammatory cascades in inflamed
tissue to recover its homeostatic, non-inflamed status. A defect in the Nox2 causes
chronic granulomatous disease in humans, which is not only characterised by recurrent
infections, but also by hyperinflammation. Similarly, Nox2 expression was found to play
an essential role in the prevention of insulin resistance and diet-induced obesity in mice.
These apparently dualistic roles of ROS indicate that its effect is highly dependent on its
temporal, spatial and quantitative production pattern (294).
Role of ROS in host/microbiota interactions
The epithelial cells that line the digestive tract are continuously exposed to enormous
amounts of bacteria: ranging from ~103/g in the upper parts of the small intestine to
~1012/gram in the colon. It is of utmost importance that immune responses are balanced
and appropriately eradicate pathogenic and invading bacteria, whereas they should not
respond excessively to the bulk of commensal bacteria in the gut.
An intriguing question is how H2O2 produced by bacteria may impact gut homeostasis
and host signal transduction. Essential to bacterial H2O2 to emerge and affect host
144
immune responses is the presence of sufficient oxygen in the GI-tract. Studies on the
oxygen content demonstrate that the majority of the gut volume is anaerobic, due to
continuous scavenging of oxygen by the microbiota. Oxygen is continuously leaking
in from epithelial tissue, creating a very steep gradient from the mucosal surface to
the lumen (40, 41). Another gradient appears to be present in which the proximal
regions of the small intestine (duodenum and jejunum) are considered to contain higher
levels of oxygen compared to the strictly anaerobic colon (196). Oxygen availability in
the micro-environments of the intestinal tract may be one of the dominant drivers of
the spatial distribution of microbial specialists, in which microaerophilic microbes are
generally found in samples taken close to the mucosal surfaces whereas the obligate
anaerobic species are mostly found in the more anaerobic environments of the lumen
and the colon (41, 295, 296). There are several studies detailing how intestinal oxygen
levels impact bacterial metabolism, including the modulation of pathogenicity factors
of Shigella (41), respiration of E. coli (297) and thiol/flavin export by Faecalibacterium
prauznitsi (298). We consider that aerotolerance is an important factor for bacteria to
reside in close proximity of epithelial surfaces, which can be considered as an important
driver for direct modulation of host immune responses.
In such environments with fluctuating oxygen levels, ROS produced by LAB, could
potentially have an impact on health and disease in the host. In in vitro cell culture
systems it was shown that superoxide produced by E. faecalis leads to host-cell lipid
oxidation generating the reactive compound 4-hydroxy-2-nonenal which induces DNA
damage (62, 299). Also, H2O2-producing streptococci, such as S. pyogenes, S. mutans
and S. pneumoniae are associated with disease in humans. S. pneumoniae is regularly
found amongst the upper-respiratory tract microbiota of healthy individuals, but can
cause bacteremia, meningitis and pneumonia in immunocompromised individuals.
6
S. pyogenes can show a similar transformation from harmless commensal to lethal
pathogen, whereas S. mutans and S. gordoni are mostly associated with dental caries.
As we described in chapter 1, H2O2 production in these species is mostly catalyzed by
the lactate and pyruvate oxidation pathways (LOX, POX, ACK). Importantly, expression
of the pyruvate oxidase encoding spxB gene has been identified as an important factor
for virulence of S. pneumoniae (88, 90, 300-302). Similarly, SpxB and H2O2 production
was also correlated with the ability of oral streptococci to adhere to the tooth surface
and release DNA, which is an important factor for biofilm formation and natural
competence (85, 303-306), although the exact mechanism behind these correlations
remains unresolved. Bacterial H2O2 production could play a role in pathogenesis on basis
145

of its cytotoxic effect on epithelial tissue, but at the same time the bacterial production
pathways are of importance for their aerotolerance and oxidative stress resistance. Other
studies indicate that modulation of characteristics like aerobic respiration or expression
of ROS-scavenging enzymes are also considered as virulence factors, while they also
contribute to survival of the bacterial cell (57, 116, 117). Thereby, it becomes difficult
to distinguish between a direct role in the virulence cascade and the ability to persist in
the host environment. Obviously, the latter capacity is a prerequisite to allow bacteria
to exert their detrimental and pathogenic effects, creating a intrinsic dilemma in the
design of adequate experiments to discriminate between direct, and persistence-driven
effects on pathogenesis.
In many of the aforementioned cases, bacterial ROS production is associated with
adverse health outcomes. However, there are several reports indicating that bacterial
H2O2 may also have a beneficial, anti-inflammatory effect. One study, by Voltan et
al., shows that L. crispatus derived H2O2 induces PPAR-g expression in epithelial cell
cultures. Such increased PPAR-g expression was previously associated with improved
gut homeostasis and reduced severity of colitis (201), through its capacity to decrease
NF-κB expression and thereby suppress inflammatory cascades. Administration of
H2O2-producing L. crispatus in mice, could substantially reduce severity of (chemically
induced) colitis, whereas a low hydrogen-peroxide producing (spontaneous) mutant
of L. crispatus did not show such an effect. This study illustrated in multiple ways that
H2O2 is essential for PPAR-regulation. However, since the spontaneous low-level-H2O2
producing mutants failed to adhere to the epithelial cells while the wild-type strain
adhered effectively, the prerequisite of close proximity of the bacteria and the epithelia
may have been a strong confounder in this study. Thereby, secondary mechanisms to
explain the differences between the two bacterial strain that are independent of their
H2O2 production capacity can not be excluded to play a role in the observed protection
against colitis (50).
Another study described the effect of L. johnsonii on rats that are genetically predisposed
to develop type 1 diabetes (T1D), which to a certain extent could also be linked to the
bacterial capacity to produce H2O2. The L. johnsonii strain used in this study, strain
N6.2 was previously found to be overrepresented in the microbiota of T1D-susceptible
rats that did not develop diabetes (307). Administration of this isolate to the rats was
shown to increase a variety of mucosal proteins associated with gut barrier function
and decrease the inflammatory cytokine IFNγ. Furthermore, it decreased the oxidative
stress response and the overall incidence of T1D in this rat model, while these effects
146
were not seen with another Lactobacillus strain (of the species L. reuteri) that also
negatively correlated with type 1 diabetes symptoms (308). Interestingly, L. johnsonii
administration was shown to inhibit the expression of indoleamine 2,3-dioxygenase
(IDO) in host ileal mucosa. IDO is primarily involved in tryptohan catabolism, but also
plays a central role in inflammation cascades, and its activity has been associated with
several adverse health outcomes (309). L. johnsonii administration was shown to lead
to higher ileal H2O2 levels, and in in vitro cell cultures it could be shown that H2O2
(produced by L. johnsonii) abolished IDO activity. Taken together these results create
a tentative connection between the IDO inhibition and the H2O2 producing capacity
of this bacterium in vivo, although it can not be excluded that the elevated ileal H2O2
levels were (in part) host-derived, and thus may not have depended (exclusively) on the
production by L. johnsonii (203). Nevertheless, this study provides a strong indication
that bacterially produced H2O2 could modulate IDO expression in vivo, and it would be
very interesting to evaluate the effect of the L. johnsonii mutants described in this thesis
that are unable to produce H2O2 in this rat model.
Collectively, these studies indicate that H2O2 may act as a bacterial effector molecule that
can evoke immune modulations in the host. We hypothesize that analogous to hostrelated H2O2 production, the effect of bacterial H2O2 depends on its temporal, spatial
and quantitative release in the GI-tract. Intestinal conditions that stimulate high level
production of ROS by bacteria could potentially induce epithelial and mucosal damage,
whereas low-level production of ROS by bacteria like L. johnsonii may successfully
suppress excessive host immune responses by modulating the expression of central
metabolic regulatory nodes, such as PPAR-g and IDO that have established roles in
metabolism-immune cross talk.
6
Relevance of bacterial H2O2 in the vaginal microbiota
Compared to the oral and intestinal microbiota, where ROS-producing bacteria form
only a small subset of the overall community, the vaginal microbiota stands out by
its high abundance of such bacteria. The vaginal microbiota of the vast majority of
women in the reproductive age is dominated by one (or more) of four different species
(L. crispatus, L. gasseri, L. jensenii and L. iners) belonging to the L. acidophilus group,
of which at least three (L. gasseri, jensenii and crispatus) can produce substantial
amounts of H2O2. Although colonization patterns vary as a function of the menstrual
cycle, health and disease and are influenced by sexual intercourse and pregnancy, all
major studies that have analyzed the constituents of the vaginal microbiota identify this
147

lactobacilli dominated ecosystem in healthy women (310-313). In addition, the absence
of lactobacilli in this ecosystem is often accompanied by overgrowth of a collection
of opportunistic species such as Gardnerella vaginalis and Atopobium vaginae (314316). This microbial disbalance is referred to as bacterial vaginosis (BV), which affects
approximately 10-20% of European women (317). Women with BV-type microbiota are
also at a significant higher risk to acquire sexually transmitted disease, and increased
risk of womb infections that can elicit preterm births (199, 200, 318, 319).
The lactobacilli-dominated microbiota of the vagina provide an intriguing contrast to
the mucosal surfaces of the digestive tract that are colonized by tens to hundreds of
different species, none of which is really dominating the ecosystem. Conversely, the
vaginal environment is colonized by only a handful of dominant species with very
similar features, including their capacity for H2O2 production (94, 187, 320). A longstanding hypothesis states that bacterial H2O2 production in the vaginal niche is the
molecular trait by which lactobacilli prevent the colonization of other organisms (319,
321). However, this hypothesis is debated, and as an alternative explanation it has been
proposed that the high levels of lactic acid in combination with a low pH may be more
effective in creating colonization resistance. In addition, the oxygen availability, as well
as the stability of H2O2 in the vaginal environment have been contested (312, 322). The
precise role of bacterial H2O2 in vaginal homeostasis remains to be determined. The
identification and mutation of the H2O2 producing enzymes in a species closely related
to vaginal lactobacilli, that we presented in this thesis, may advance our understanding
of this topic. We envision that a comparison between the ability of wildtype and nonH2O2 producing lactobacilli to prevent bacterial vaginosis in animal models could help
understand the influence of this physiology on stability of the vaginal microbiota.
Relevance of bacterial H2O2 in the neonatal gut
The composition of the vaginal microbiota is not only related to health of women, but
may also contribute to the health of an infant after vaginal delivery. Although it has
been proposed that fetuses already acquire microbes prenatally (323), the predominant
colonization of the infant gut takes place after birth. The vaginal bacteria of the
birth canal may present an important part of microbial transfer between mother and
infant. Comparison of microbial constituents of the neonatal gut following vaginal
delivery with those found after caesarian delivery, revealed prominent differences and
supported the idea that the vaginal (and/or fecal) microbes of the mother are amongst
the pioneer colonizers of the neonatal gut after birth (16, 324, 325). Interestingly, these
148
lactobacilli containing early microbial colonization patterns are inversely correlated with
the prevalence of necrotizing enterocolitis (NEC) in preterm infants (326, 327). The
administration of probiotic lactobacilli has been shown to be effective in lowering the
incidence of NEC (25).
Oxygen levels are generally assumed to show a significant drop during subsequent
phases of colonization of the infant gut, which is reflected by the aerotolerance levels
of the colonizing species. The first microbial groups that are found in the neonatal gut
in the first 48h after birth are mostly aerotolerant, facultative or obligate aerobic species
(16). Outgrowth of the strictly anaerobic groups, such as clostridiae, faecalibacteria and
sulfate-reducing bacteria, is typically found at later stages in the development of the
microbiota (328). Although this dynamic colonization is influenced by many parameters,
the establishment of the aforementioned oxygen gradients in the developing intestinal
environment may play an important role in suppressing outgrowth of the more
aerosensitive members of the microbiota. Some authors even suggest that oxygen
consumption by the first aerobic colonizers is essential to pave the way for colonization
of their anaerobic successors (40).
The observations described above raise the question whether H2O2 produced in the
neonatal gut by the vaginal lactobacilli that were transferred during passage through
the birth canal, may play a role in initiating or shaping the early immune system. This
hypothesis is based on three assumptions: (1) that the absence of high numbers of
bacteria lead to high oxygen levels in the GI-tract during the first hours after birth, (2)
that mother-to-child microbial transfer leads to prominent colonization of the infant gut
with H2O2-producing lactobacilli and (3) that bacterial H2O2 can modulate host-immune
reactions. The third assumption is supported by observations described above that
highlight the potential role of bacterially produced H2O2 in signaling to the mucosal via
6
PPAR and IDO. Moreover, the capacity to scavenge oxygen could help the colonization
of less aerotolerant species such as the bifidobacteria that are typically overrepresented
in the intestinal microbiota of breastfed infants. However, one can also not rule out
that the bacterially produced H2O2 may in certain cases also have adverse effects, since
the presence of high intestinal oxygen levels and the absence of catalase expressing
bacteria in the infant intestine may also cause ROS induced mucosal damage. Also here,
small amounts may go a long way. The balance between ROS-production and ROSscavenging are key in maintaining a healthy intestinal environment.
149
Perspectives: how the findings in this thesis could help unravel the role of bacterial
H2O2 in host/microbe interactions
An important bottleneck in studying the effect of ROS in immune responses is the
difficulty in teasing apart host and bacterially derived H2O2. Adequate negative controls
that exclusively eliminate bacterial H2O2 from the interplay are currently not available.
Especially the effects of ecosystem-derived catalase and SOD may scavenge both host
and bacterially-derived ROS and may influence the colonization by specific microbial
groups including the lactobacilli. A promising approach to resolve this issue was followed
by Voltan et al., who used a spontaneous non-H2O2 producing isolate of L. crispatus
(strain MU5) as a negative control. Unfortunately, this isolate was affected in various
phenotypic traits besides its H2O2-producing capacity, including a non-aggregating
phenotype and the inability to adhere to epithelial tissue cells (329). These deviations
may be of critical importance for the in vivo effect that these bacteria could elicit in the
intestinal tract of a host model, which was also supported by the finding that epithelial
adherence was essential for the immunomodulatory capacity of L. crispatus.
The identification of the H2O2 producing enzymes in L. johnsonii and the availability of
deficient mutants, opens novel avenues to further study the relevance of bacterial H2O2
in various host/microbiota interactions. The isogenic strains that are no longer able to
produce H2O2, developed and studied in this thesis, NCC 9359 (Δnfr) and NCC 9360
(Δnfr, Δnox) could be instrumental in studies with epithelial tissue cultures and/or animal
models. Moreover, similar mutants may more readily be constructed in other members
of the L. acidophilus group, now that key-enzymes involved in H2O2 production (NFR
and NOX) are identified in this group of bacteria. Inversely, H2O2-overproducing variants
of L. johnsonii or other members of the L. acidophilus group could also be of interest.
Although transformation with NFR encoding multi-copy plasmids (Chapter 2) did not
lead to elevated H2O2 production by L. johnsonii in our experiments, it can not be
excluded that alternative expression systems or specific (micro-aerobic) environments
may allow the construction or exploitation of H2O2 overproducing strains in the same
cell-based or animal models.
150
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Summary (for a scientific audience)
Lactic acid bacteria (LAB) are intimately entwined with human life. They ferment several
key food products in our diets and they reside on the mucosal surfaces of the mouth,
intestine and vagina. Administration of adequate amounts of certain LAB species has
been shown to provide health benefits, such as attenuation of antibiotic-associated
diarrhoea. Lactobacillus johnsonii is a LAB that is used in the food industry for such
health-benefit, or probiotic effects. The functionality of L. johnsonii and other related
LAB in the food industry and in the host-related environment are strongly dependent
on environmental factors. Especially oxygen and carbon dioxide were found to have
a major influence on metabolism, growth, gene expression, yield, and viability. This
thesis addresses the consequences of exposure to these two gaseous components on
L. johnsonii.
Chapter 1 contains a general overview of research literature on aerobic metabolism
and oxidative stress of lactic acid bacteria. LAB are classified as aerotolerant anaerobes;
although they commonly grow well in the presence of oxygen they do not show aerobic
respiration unless a hemin source is added. Instead, oxygen is used for direct oxidation of
various metabolic intermediates. Facultative heterolactic LAB produce acetate and CO2
aerobically through the pyruvate oxidase and lactate oxidase pathway. These products
are more oxidized than lactate and therefore additional oxidation of the reducing
equivalent NADH is required. When exogenous hemin is added to the medium, NADH
can be channelled through the electron transport chain. This respiratory growth is
associated with higher yield, robustness and lower oxidative stress. Alternatively, water
or H2O2-forming NADH oxidases can regenerate NAD+. NADH oxidase is a central switch
in the metabolism of heterolactic LAB. Activity of these oxidases and autoxidation of
other cellular components results in the generation of reactive oxygen species, such
as hydrogen peroxide and superoxide, which cause oxidative stress. We provide an
overview of the anti-oxidative enzymes (ROS-scavengers) and physiological adaptation
found in LAB to reduce oxidative stress.
A prominent characteristic that L. johnsonii shares with several other lactobacilli
and streptococci is the accumulation of substantial amounts (>1 mM) of hydrogen
peroxide in its environment. L. johnsonii lacks the key ROS scavenging enzymes, such
as catalase, alkyl hydroperoxide reductase and superoxide dismutase. Hydrogen
peroxide accumulation is the primary cause of oxidative stress in L. johnsonii. It induces
a premature stationary phase and a ~10-fold lower biomass yield in the presence of
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oxygen. Addition of catalase abolishes this growth stagnation. In chapter 2 and 3
we report on the identification and characterization of two proteins that contribute
to hydrogen peroxide production of L. johnsonii. In chapter 2 we showed that H2O2
production is unrelated to expression of pyruvate, lactate and NADH oxidase. In cell
extracts of L. johnsonii an enzymatic hydrogen peroxide forming activity could be
detected upon addition of flavin and NADH (not NADPH). Partial purification of the
unidentified enzyme displaying this activity showed that two small flavoproteins,
LJ_0548 and LJ_0549, were overrepresented in the hydrogen peroxide forming fraction.
Genetic disruption and overexpression confirmed that these proteins constitute an
NADH flavin reductase (NFR). A deletion derivative of these two genes did not produce
any hydrogen peroxide when exposed to oxygen, indicating that these enzymes catalyze
the reaction that produces hydrogen peroxide in L. johnsonii.
However, after prolonged cultivation in the presence of oxygen, the NFR deletion
derivative regained partial H2O2 producing capacity. In chapter 3 we report on the
identification of a second H2O2 producing enzyme, an NADH oxidase (NOX), encoded by
the LJ_1254-1255 locus. Expression of this locus was 2.1-fold induced in the wildtype
under aerobic growth conditions and 3.7-fold in the NFR-deletion derivative. Deficiency
of this NOX activity did not impact H2O2 production of the wildtype, but completely
abolished all H2O2 production in its NFR deficient derivative. Intriguingly, this mutant
also showed hampered growth and lower biomass yield in the presence of oxygen,
despite its H2O2-negative phenotype. We conclude that the oxygen-induced NADH
oxidase produces hydrogen peroxide in the absence of NADH flavin reductase, and may
also contribute to hydrogen peroxide production during longer exposure to oxygen.
Oxygen is not in all cases detrimental for growth and viability of L. johnsonii. In the
second part of this thesis we focus on the growth stagnation observed during growth
under a N2 atmosphere, observed both in liquid (sparged batch cultures) as on solid
media (AnoporeTM slides). The cause of this growth stagnation was shown to reflect
a lack of CO2 which L. johnsonii requires for growth. Two aspects of this observation
were unexpected. Firstly, oxygen could fully relieve this CO2 growth dependency, and
secondly, lack of CO2 apparently led to cell death of L. johnsonii. Especially the latter
7
effect is unusual, since removal of essential nutrients generally only halts bacterial
growth. We further study these two factors in chapter 4 and 5. In chapter 4, we
showed that this oxygen relieve of CO2 dependency also accounted for the acetate
growth dependency: L. johnsonii cannot grow in an environment without acetate
unless the culture is aerated. Both these effects of oxygen could be traced back to one
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common denominator, which is the pyruvate oxidase reaction leading to both CO2 and
acetate production. Pyruvate oxidase could therefore allow L. johnsonii to overcome its
acetate and CO2 dependency in conditions that include oxygen exposure. A pyruvate
oxidase deletion derivative confirmed this hypothesis as it rendered the organism both
aerobically and anaerobically dependent on acetate and CO2 supplementation. Our
results demonstrate that certain growth requirements of L. johnsonii are not hardwired
but depend on environmental factors.
In chapter 5, we attempted to further understand the metabolic requirement for CO2
by analyzing genome-wide transcriptome changes in an anaerobically growing culture
upon CO2 depletion. We detected an extensive rearrangement of gene expression,
including many transporters and regulators. Additionally, expression of the pyr-operon
encoded pyrimidine biosynthesis pathway, also referred to as the carbamoyl-phosphate
pathway, was strongly upregulated (up to 17-fold). In other LAB, this pathway has been
associated with CO2 dependency, which is required in the first enzymatic step of the
pathway and generally growth stagnation due to CO2 depletion could in these LAB be
prevented by addition of sufficient levels of pyrimidines. However, in L. johnsonii such a
relation between pyrimidine supplementation and CO2 dependency was not observed.
Supplementation of other compounds that have previously been associated with CO2
growth dependency in LAB, such as arginine and aspartate also could not prevent CO2
induced growth stagnation. We speculate that L. johnsonii is unable to incorporate
exogenous pyrimidines and propose further experiments to test this preposition.
In chapter 6, we present a general discussion of the results in this thesis and place
them in the environmental context of the intestinal and vaginal microbiota. One of
the most prominent questions that remains is why NOX and NFR activity is required
for aerotolerance. We propose that H2O2 production of L. johnsonii may be a means to
scavenge oxygen in an physiologically attractive way, i.e. without spending much NADH
which is ultimately required for lactate production. High levels of intracellular molecular
oxygen and possible resulting superoxide formation could be more hazardous than
hydrogen peroxide, which quickly diffuses out of the cell and is potentially scavenged by
neighboring catalase-expressing bacteria or other environmental factors. We propose
several experiments to study this hypothesis.
Lastly, we discuss how hydrogen peroxide production and aerotolerance could play a
role in the interactions of L. johnsonii with a host organism. Reactive oxygen species
play a central role in a wide variety of immune reactions. We discuss how bacterially-
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derived superoxide and hydrogen peroxide could contribute to gut homeostasis through
immune signaling. Especially in the neonatal intestine, which generally contains higher
levels of oxygen, H2O2-producing lactobacilli, transferred from the mother to the
infant during birth, could produce substantial levels of hydrogen peroxide, which may
contribute to shaping the neonatal immune system. We propose how the NOX and/
or NFR deletion derivatives that were constructed in the scope of this thesis, can be
employed to testing the role of bacterial H2O­­­2 in host/microbiome interactions.
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Samenvatting (voor breed publiek)
Melkzuurbacteriën zijn belangrijke organismen in het leven van de mens. Niet alleen
zijn ze essentieel in de bereiding van gefermenteerde producten in ons voedselpatroon,
zoals yoghurt, kaas, olijven, zuurdesem en vleesproducten, maar ze zijn ook prominente
bewoners van ons lichaam, bijvoorbeeld in de mond, darm en vagina. Verder hebben
sommige melkzuurbacteriën zogenaamde probiotische eigenschappen: als je er
voldoende van consumeert kan dat een gezondheidseffect teweeg brengen, zoals het
verminderen van de ernst van diarree. In dit proefschrift presenteren we onderzoek
over één van deze probiotische melkzuurbacteriën, Lactobacillus johnsonii en we
concentreren ons specifiek op de invloed van zuurstof (O2) en koolstofdioxide (CO2) op
metabolisme en fysiologie van deze bacterie. Met deze gassen komt L. johnsonii veel in
aanraking, zowel in de voedingsmiddelenindustrie als in het menselijk lichaam. Het is
daarom belangrijk om de nogal ingrijpende rol van deze twee gassen beter te begrijpen.
In hoofdstuk 1, geven we een overzicht van de huidige kennis over de invloed van
zuurstof op melkzuurbacteriën (LAB). Zuurstof is voor veel levensvormen schadelijk, zelfs
voor soorten die geen vijf minuten zonder zuurstof kunnen overleven zoals de mens.
Tijdens de ademhaling wordt zuurstof omgezet in water (H2O), maar als bijproduct
worden giftige zuurstofradicalen geproduceerd, zoals waterstof peroxide (H2O2) en
superoxide (O2-), die continue uit de weg moeten worden geruimd. Melkzuurbacteriën
kunnen niet ademhalen, maar ze gebruiken zuurstof wel op andere manieren,
bijvoorbeeld in de omzetting van melkzuur naar azijnzuur. In dat proces worden veel
zuurstofradicalen geproduceerd, met name H2O2. Ook L. johnsonii produceert veel H2O2
maar heeft geen adequate mogelijkheid om dit op te ruimen. Ophoping van die H2O2
zorgt ervoor dat deze bacterie niet meer verder kan groeien na een paar uren beluchting.
Uit ons onderzoek blijkt dat, in tegenstelling tot veel andere melkzuurbacteriën, de
azijnzuurproductie geen prominente bron is van H2O2. In hoofdstuk 2 presenteren
we een aantal experimenten waarin we op zoek gaan naar de oorsprong van die H2O2
in L. johnsonii. In cellen worden chemische reacties gefaciliteerd (gekatalyseerd) door
eiwitten, en eiwitten worden gemaakt door het kopiëren van erfelijk materiaal (het DNA,
de genen). We hebben het eiwit dat verantwoordelijk is voor de H2O2 productie kunnen
identificeren, door alle eiwitten van L. johnsonii te fractioneren en de H2O2 productie
in elk van die fracties te meten. Door de fracties telkens kleiner te maken konden we
uiteindelijk de chemische reactie toeschrijven aan een handjevol eiwitten. Na analyse
van die eiwitten, met zogeheten massaspectrometrie, konden we twee kleine eiwitten
detecteren met een aantal eigenschappen die erop wezen dat ze inderdaad de H2O2
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productie in L. johnsonii zouden kunnen faciliteren. Die suggestie werd bevestigd: als we
de genen die voor die specifieke eiwitten coderen, uit de bacterie verwijderen, maakt
de resulterende gemuteerde bacterie ook geen H2O2 meer. Alhoewel, na langdurige
blootstelling aan zuurstof, blijkt ook in deze mutant bacterie de H­2O2 productie weer
terug te komen. Er is blijkbaar dus nog een tweede eiwit dat H2O2 vorming faciliteert.
In hoofdstuk 3 beschrijven we de zoektocht naar dat tweede eiwit. Door te meten
welke genen L. johnsonii aan en uitzet bij zuurstof blootstelling, kwamen we erachter
dat het gen dat het sterkst wordt aangeschakeld door zuurstof, voor een eiwit codeert
dat ook eigenschappen heeft die erop wijzen dat het een rol kan spelen in H2O2 productie.
Als we het gen van dit eiwit verwijderen, verandert er niets aan de bacterie, maar het
verwijderen van beide genen (ook het gen dat we hadden gevonden in hoofdstuk
2) levert een bacterie op die helemaal geen H2O2 meer maakt. Deze bacterie bleek
alleen ook niet meer goed te kunnen groeien bij beluchting. Dat was een opvallende
karakteristiek, omdat juist die H2O2 vorming zo schadelijk was. Kennelijk spelen deze
twee eiwitten L. johnsonii een dubbelrol, waarbij ze zowel het schadelijke H2O2 vormen,
maar tegelijkertijd op een tot nu toe onbekende manier de bacterie beschermen tegen
andere schadelijke bijproducten van zuurstof.
In het tweede deel van dit proefschrift beschrijven we het effect van kooldioxide, CO2.
L. johnsonii heeft CO2 nodig om te kunnen groeien en om in leven te blijven. Het
is opvallend dat als we zuurstof toevoegen aan de atmosfeer waarin de bacteriën
groeien, er ineens wél gegroeid kan worden zonder CO2. In hoofdstuk 4 zijn we op
zoek gegaan naar de oorzaak van dit zuurstof-effect. Ten eerste constateerden we dat
wat geldt voor CO2 afhankelijkheid, ook blijkt te gelden voor azijnzuur-afhankelijkheid.
L. johnsonii heeft azijnzuur nodig om te kunnen groeien, maar in de aanwezigheid
van zuurstof is die azijnzuur afhankelijkheid opgeheven. Eén reactie blijkt hierin een
centrale rol te spelen. Pyruvaat is een metaboliet dat kan reageren met zuurstof waarbij
CO2 en azijnzuur worden gevormd. Deze reactie wordt gefaciliteerd door het eiwit
pyruvaat oxidase. De hypothese was dat L. johnsonii zonder azijnzuur en CO2 kan
groeien als zuurstof aanwezig is, omdat onder die condities beide stoffen gemaakt
kunnen worden door de reactie tussen pyruvaat en zuurstof. Dit bleek te kloppen,
7
omdat na uitschakeling van het gen voor pyruvaat oxidase L. johnsonii ook niet meer
zonder azijnzuur en CO2 kon groeien als er zuurstof aanwezig was. We concluderen dat
zuurstof niet alleen maar schadelijk is voor deze bacterie, maar dat zuurstof er ook voor
kan zorgen dat de bacterie kan groeien zonder azijnzuur en CO2.
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In hoofdstuk 5 concentreren we ons verder op de vraag waarom L. johnsonii afhankelijk
is van CO2. Zulk soort afhankelijkheid wordt in andere melkzuurbacteriën veroorzaakt
doordat CO2 nodig is als grondstof voor het maken van pyrimidine, een bouwsteen van
het DNA. We analyseren welke genen in L. johnsonii aan en uit worden gezet wanneer
CO2 wordt verwijderd uit de omgeving. De expressie (aan/uit) van zeer veel genen blijkt
te reageren op de aan of afwezigheid van CO2, voornamelijk genen die betrokken zijn
bij membraantransport en genregulatie. Daarnaast zien we dat genen die betrokken
zijn bij de eerder genoemde productie van DNA-bouwstenen (uit CO2) zeer sterk
worden aangeschakeld bij verwijdering van CO2, wat erop kan duiden dat de productie
van DNA bouwstenen inderdaad de belangrijkste oorzaak is van de CO2 behoefte van
L. johnsonii. In andere melkzuurbacteriën kon het toevoegen van deze bouwstenen aan
de groeimedia de afhankelijkheid voor CO2 opheffen. In onze experimenten bleek dit
niet op te gaan voor L. johnsonii. Onze hypothese is dat L. johnsonii de toegevoegde
DNA-bouwstenen niet goed kan inbouwen en we stellen een aantal experimenten voor
waarmee die hypothese zou kunnen worden getest.
In hoofdstuk 6 presenteren we een algemene analyse van al onze resultaten. Een
prominente vraag die overblijft is waarom L. johnsonii de H2O2-producerende eiwitten
nodig heeft om in de aanwezigheid van zuurstof te kunnen groeien. Onze hypothese
is dat deze bacterie zuurstof liever omzet in H2O2 dat snel naar buiten verdwijnt en
daar uit de weg wordt geruimd door omliggende bacteriën. Misschien kan L. johnsonii
daarmee voorkomen dat er nog schadelijkere zuurstof radicalen zoals superoxide
worden gevormd.
Verder bespreken we hoe H2O2 productie door bacteriën een rol kan spelen in de
interacties met de gastheer. Het is welbekend dat zuurstofradicalen, geproduceerd door
de cellen van de darmwand, een regulatie effect uitoefenen op ons immuunsysteem.
De vraag is of zuurstofradicalen die door darmbacteriën worden geproduceerd ook
kunnen bijdragen aan deze immuunregulatie. Dit zou vooral belangrijk kunnen zijn
in de darm van pasgeboren baby’s, waar vermoedelijk veel zuurstof aanwezig is om
zuurstofradicalen te produceren. De vaginale bacteriën van de moeder, die tijdens
de geboorte worden overgedragen aan het kind, zijn typisch H2O2-producerende
lactobacillen, sterk verwant aan L. johnsonii. We stellen voor dat dit een manier is
waarop bacteriën een rol kunnen spelen bij de vroege vormgeving en regulatie van
het immuunsysteem. De kennis die is ontwikkeld in dit promotieonderzoek, en de
mutanten die we hebben geconstrueerd, zouden een bijdrage kunnen leveren aan het
bevestigen van de voorgestelde rol die bacteriën zoals L. johnsonii zouden kunnen
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spelen in de interacties met de gastheer.
List of Abbreviations
ACK
AHP / AHPR
ATP / ADP
CCP-A
COX
DLD
FAD
FMN
GSH(R)
HemCat
LAB
LDH
LOX
MnCat
MnSOD
NADH / NAD
NOX
NPR
PAT
PDH
PFL
POX
ROS
SOD
spx
TRX(R)
acetate kinase
alkyl hydroperoxide (reductase)
adenosine tri/diphosphate
catabolite control protein A
cytochrome oxidase
dihydrolipoamide dehydrogenase
flavin adenine dinucleotide
flavin mononucleotide
Glutathione (reductase)
hemin-dependent catalase
lactic acid bacteria
lactate dehydrogenase
lactate oxidase
manganese dependent catalase
manganese-dependent SOD
nicotinamide adenine dinucleotide
NADH oxidase
NADH peroxidase
phosphate acetyltransferase
pyruvate dehydrogenase
pyruvate formate lyase
pyruvate oxidase
reactive oxygen species
superoxide dismutase
pyruvate oxidase encoding gene
Thioredoxin (reductase)
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Dankwoord
Dit proefschrift is het stoffelijke eindproduct van vijf jaar promotieonderzoek. Vijf
jaren van verrassende inzichten en harde lessen, fundamentele en toegepaste vragen,
inspirerende ontmoetingen en goede gesprekken, Eureka-momenten en grote
teleurstellingen, anoniem zwoegen en publiek koketteren. Het is in vele opzichten een
verrijkende tijd geweest. Daarvoor ben ik veel mensen dankbaar. Te beginnen bij mijn
promotores:
Joost Teixeira de Mattos. Nadat Jeroen Hugenholtz dit project helaas vroegtijdig
moesten verlaten, was jij bereid om de supervisie namens de UvA over te nemen. Je was
de tweede die ik tegenkwam op de UvA en dat was een hele prettige kennismaking
die ervoor zorgde dat ik graag bij SILS kwam werken. Ik bewonder je bevlogenheid,
je liefde voor het vak en voor het onderwijs. Je had een onverwoestbaar vertrouwen
in mij als wetenschapper en in de goede afloop van dit onderzoek. Dat heb ik als
heel waardevol ervaren. Je zorgde regelmatig voor licht en lucht in dit project, met je
aanstekelijke positivisme en humor.
Michiel Kleerebezem. Nadat Eddy Smit dit project helaas vroegtijdig moest verlaten,
was jij bereid om de supervisie namens NIZO over te nemen. Een enorme aanwinst!
Ondanks je overvolle schema, nam je met regelmaat uitgebreid de tijd om van
gedachten te wisselen. Onvermoeibaar tilde je mijn schrijfsels naar een hoger niveau.
Je voortdurende nieuwsgierigheid en je aanmoediging voor nieuwe experimenten,
betere teksten, consistentere notaties waren een zegen voor dit proefschrift en voor
mijn ontwikkeling. Ik heb ontzettend veel van je geleerd, het was een privilege om met
je samen te werken.
Christof Gysler and David Pridmore. It was a pleasure to have worked together with
you. This project gained a lot from the continuous interest from Nestlé’s side and your
input during our monthly updates and the Kluyver meetings. The mutant strains were
an essential element in this thesis, which we owe to the excellent cloning skills of David
and his perseverance.
Ik wil de Leescommissie bedanken voor het kritisch lezen van mijn manuscript.
Het was een genoegen om onderdeel uit te maken van de MMP groep, met zijn vele
bijzondere persoonlijkheden. Zowel in kantoor C3.258 als in het 12:00 lunchgroepje,
werd er veel gediscussieerd, geroddeld, gescholden, gesnoept, gewerkt, gelanterfant,
en ook veel en hard gelachen. Dit waren voor mij ook vooral hele gezellige jaren. Dankzij
178
jullie, lieve collega’s! In het bijzonder: Martijn, jij was een baken van enthousiasme,
uitbundigheid en onredelijk positivisme in het overheersend cynische academische
landschap. Gertien, ik heb veel van je geleerd en ik bewonder je ambitie, je kracht,
je toewijding en je onvermoeibare streven naar meer en beter wetenschappelijk
onderzoek en onderwijs. Jos, ik heb jouw creatieve projecten en prachtige foto’s zeer
gewaardeerd! Jeroen, Andreas, Pascal, Aniek, jullie hebben me geholpen met de
meest uiteenlopende zaken zoals boekjes drukken, figuren maken, wetenschapper
worden en hoe je moet omgaan met de nieuwste Android-versie op je telefoon. Milou,
Poonam dank voor de vriendschap en alle mooie tijden die we samen beleefden.
Felipe, you arrived a bit late but in those few months you added a lot of energy, new
ideas and a lot of fun. Johan, in lab, kantoor en kantine ben jij de afgelopen jaren een
dierbare vriend geworden en ik ben dankbaar dat je mijn paranimf wilt zijn. Sarah, jij
bent een representant van de vele intelligente, ambitieuze en lieve vrienden die ik de
afgelopen jaren in Amsterdam heb gemaakt. Dank dat je mijn paranimf wilt zijn. All
other colleagues and friends, although I will not list all your names, you know who you
are, thanks for everything!
Annereinou, Iris, Marjo en vele andere NIZO’ers, ondanks dat ik geen échte NIZO’er
was, voelde ik me altijd welkom en was het niet alleen nuttig, maar ook ontzettend
gezellig om naar Ede te komen. Dank!
Dr. Rebecca ten Cate, mama, jij leerde mij dat een promotieonderzoek een voorrecht
is. Je bracht me liefde voor de wetenschap bij, doorzettingsvermogen en geduld. Je
leerde me hoe je zeven levens kan hebben en elke tegenslag te boven kan komen. Je
bent een blijvend voorbeeld voor mij.
Papa, een aantal eigenschappen die essentieel waren voor dit project en andere
activiteiten, heb ik aan jou te denken. Schaamteloos ambitieus zijn, groot denken,
hoffelijkheid en positivisme. Ik ben gezegend met een inspirerende familie.
Liefste Arjen, zonder jou was dit boekje er ook gekomen, maar door jou was ik een
hele gelukkige, vrolijke en verliefde promotiestudent. Dank je wel, dat je geduldig was,
dat je me altijd steunt en dat je me telkens weer aanmoedigt om hoger te mikken. Ik
7
heb ontzettend veel zin in ons volgende hoofdstuk in St. Louis.
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About the author
Rosanne Y. Hertzberger (1984, Rotterdam) completed
a BSc and MSc in Life science & Technology from Delft
University of Technology and Leiden University, including
courses at the École nationale du genie rural, des eaux et
des fôrets in Paris. During her master thesis she studied
micronutrient accumulation in Saccharomyces cerevisiae
at the Nestlé Research Centre in Switzerland. In April 2009
she started her PhD project as a collaboration between
the Nestlé Research Centre and NIZO Food Research,
carried out as a part of the Molecular Microbial Physiology lab of the Swammerdam
Institute for Life Sciences, University of Amsterdam. During her PhD studies she wrote a
weekly column for the Dutch newspapers NRC Handelsblad and nrc.next. From July 8th
2014, she will start a postdoctoral research project on the role of vaginal lactobacilli in
women’s health at the Centre for Women’s Infectious Disease Research at Washington
University in St Louis, Missouri, USA.
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