Download International Master of Science in Environmental Technology and

Master’s dissertation submitted in partial fulfilment of the requirements for the joint degree of
International Master of Science
in Environmental Technology and Engineering
an Erasmus+: Erasmus Mundus Master Course jointly organized by
Ghent University, Belgium
University of Chemistry and Technology, Prague, Czech Republic
UNESCO-IHE Institute for Water Education, Delft, the Netherlands
Academic year 2015 – 2016
Enhanced Extraction Method for the Isolation of
Difficult-To-Culture Soil Bacteria
Host University:
University of Chemistry and Technology, Prague, Czech Republic
Marco Antonio Lopez Marin
Promotor: Assoc. Prof. Ondřej Uhlík, M.Sc., Ph.D.
This thesis was elaborated and defended at the University of Chemistry and Technology, Prague,
Czech Republic, within the framework of the European Erasmus Mundus Programme “Erasmus
Mundus International Master of Science in Environmental Technology and Engineering " (Course N°
2011-0172)
© 2016, Prague, Czech Republic, Marco Antonio Lopez
Marin, Ghent University, all rights reserved.
Declaration
This thesis was written in the Department of Water Technology and Environmental
Engineering of the University of Chemistry and Technology, Prague from February to August
of 2016
I hereby declare that this thesis is my own work. Where other sources of information
have been used, they have been acknowledged and referenced in the list of used literature and
other sources. I have been informed that the rights and obligation implied by Act No.
121/2000 Coll. On Copyright, Rights Related to Copyright and on the Amendment of Certain
Laws (Copyright Act) apply to my work. In particular I am aware of the fact that the
University of Chemistry and Technology, Prague has a right to sign a license agreement for
use of these work as schoolwork under § 60 paragraph 1of the Copyright Act. I have also
been informed that in the case this work will be used by myself or that a license will be
granted for its usage by another entity, the University of Chemistry and Technology, Prague is
entitled to require from me a reasonable contribution to cover the cost incurred in the creation
of the work, according to the circumstances up to the full amount. I agree to the publication of
my work in accordance with the Act No. 111/1998 Coll. On Higher Education and the
amendment of the related laws (Higher Education Act).
In Prague, 17th of August of 2016
Abstract
Only around 1% of all the estimated microorganisms that exist on Earth have been
cultured in the laboratory (Vartoukian et al 2010). The vast majority of Earth’s
microorganisms is still uncharacterized, and their roles in the environment may be not totally
known (Kell et al 1998). It is thought that the majority of these yet-uncultured organisms can
be dormant (Rittershaus et al 2013) and have thus escaped cultivation efforts throughout
decades. An enhanced extraction method for the isolation of difficult-to-culture soil bacteria
was developed using two growth factors of the bacterium Micrococcus luteus: the liquid
cultures’ supernatants of this bacterium containing the extracellular protein “resuscitation
promoting factor” (Rpf), and M. luteus cell walls.
Compared to a control soil extraction, the enhanced extraction with M. luteus
supernatant-Rpf (SRpf) increased the number of CFU formed on solid medium by ca. one
order
of
magnitude.
These
CFUs
were
analyzed
by
matrix-assisted
laser
desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) and their spectra
were analyzed and clustered to determine the resulting diversity in terms of different
operational taxonomical units (OTUs) present. The SRpf-enhanced extraction not only
increased the CFU number, but also the diversity in terms of different cultured OTUs. The
cell wall-enhanced extraction also increased the CFU number and the diversity, albeit not as
efficiently as the former extraction approach.
SRpf increases the culturability of soil bacteria. Because dormancy can be an universal
phenomenon (Rittershaus et al 2013) these enhanced extraction methods, specially the SRpfenhanced extraction, have a great potential in improving the culture of difficult-to culture soil
bacteria not only in soil, as it proved to be, but also in different environments. Yet-uncultured
microorganisms can be cultured for the first time following this enhanced extraction method.
i
ii
Acknowledgements
I would like to express my sincere gratitude to my promoter, Dr. Ondřej Uhlík and my
tutor, Ing. Michal Strejček, whose constant support and guidance have made this work
possible. I also owe a deep sense of gratitude to Ing. Jáchym Šuman and Petra Junková, PhD,
for their assistance with the laboratory work and for their invaluable counselling.
I would also like to thank the Mexican National Council for Science and Technology
(CONACYT) for the financial support received during the course of the master.
Finally, thanks to my parents for their unconditional support and for giving me the
opportunity to keep studying. This work is mainly the result of their endless support.
iii
iv
Content
Abstract ..................................................................................................................... i
Acknowledgements ................................................................................................. iii
List of abbreviations and acronyms ........................................................................ vii
Chapter 1.0. Rationale ............................................................................................ 9
Chapter 2.0. Literature Review. ............................................................................ 11
2.1 It is not dead. It is only sleeping................................................................... 11
2.1.1 Viable but non culturable bacteria: a mysterious bacterial state ........... 11
2.1.2 The VBNC state as an adaptive response for survival .......................... 14
2.1.3 Resuscitation of bacteria from the VBNC state ..................................... 17
2.1.4 An alternative model: VBNC cells as a result of cellular deterioration ... 19
2.1.5 Implications of the VBNC state ............................................................. 20
2.2 Culturing the unculturable ............................................................................ 20
2.2.1 Culturing the unculturable: breaking down a misnomer ........................ 20
2.2.2 Media and cultivation generalities ......................................................... 21
2.2.3 In situ culturing and recreating the environment in vitro ........................ 22
2.2.4 Co-culturing........................................................................................... 23
2.2.5 Improving cultivation using growth-promoting factors ........................... 24
2.2.6 Cultivation based on genomic data ....................................................... 25
2.2.7 Miscellaneous novel culturing techniques ............................................. 26
2.3 Peptidoglycan and Rpf, a bacterial cytokine ................................................ 26
2.3.1 Resuscitation promoting factor (Rpf) ..................................................... 26
2.3.2 Rpf’s mode of action ............................................................................. 28
2.3.3 Peptidoglycan-mediated resuscitation .................................................. 28
Chapter 3.0. Objectives......................................................................................... 31
Chapter 4.0. Materials and Methods ..................................................................... 33
4.1 Materials ...................................................................................................... 33
4.1.1 Media and buffers ................................................................................. 33
4.1.2 Soil ........................................................................................................ 33
4.2 Methods ....................................................................................................... 34
4.2.1 Bacterial Cultures .................................................................................. 34
4.2.2 Supernatant retrieval ............................................................................. 34
4.2.3 Micrococcus luteus cell wall preparation ............................................... 34
4.2.4 Supernatant activity determination by absorbance ............................... 35
4.2.5 Supernatant activity determination by fluorometry ................................ 35
4.2.6 Supernatant purification ........................................................................ 36
v
4.2.7 Rpf identification ................................................................................... 36
4.2.8 Soil extractions and cultivation .............................................................. 37
4.2.9 Diversity screening ................................................................................ 38
4.2.10 LC-ESI-Q-TOF mass spectrometry analyses ...................................... 39
Chapter 5.0. Results ............................................................................................. 41
5.1 Micrococcus luteus growth curve................................................................. 41
5.2 Micrococcus luteus supernatant activity to lyse peptidoglycan .................... 41
5.3 Micrococcus luteus supernatant activity against fluorogenic substrate ........ 44
5.4 Rpf detection ............................................................................................... 48
5.5 Soil extractions and cultivation: quantitative analyses ................................. 48
5.6 Soil extractions and cultivation: qualitative analyses (microbial diversity) ... 50
Chapter 6.0. Discussion ........................................................................................ 53
6.1 Supernatant activity ..................................................................................... 53
6.2 Enhanced extraction experiments ............................................................... 54
Chapter 7.0. Conclusion........................................................................................ 57
Bibliography .......................................................................................................... 59
Appendix. OTU Table............................................................................................ 69
vi
List of abbreviations and acronyms
AHL
(n-acyl homoserine lactone)
cAMP
(adenosine 3',5'-cyclic monophosphate)
CFU
(colony forming unit)
DVC
(direct viable counting)
DTT
(dithiotreitol)
IAA
(iodacetamide)
LB broth
(Luria-Bertani broth)
LC-ESI-Q-TOF-MS (liquid chromatography-electrospray ionization-quadrupole-time of
flight-mass spectrometry)
LMM
(lactate minimal medium)
MALDI-TOF MS
(matrix-assisted laser desorption/ionization-time of flight mass
spectrometry)
MPN
(most probable number)
MUF tri-NAG
(4-methylumbelliferyl- -D-N,N’,N’’-triacetylchitotrioside)
OUT
(operational taxonomical unit)
PBS
(phosphate buffer saline)
PCA
(plate count agar)
PUF
(polyurethane foam)
Rpf
(resuscitation promoting factor)
Rpf
(resuscitation promoting factor gene)
SDS-PAGE
(sodium dodecyl sulfate-polyacrylamide gel electrophoresis)
SPS
(stationary phase survival)
SRpf
(supernatant resuscitation promoting factor)
UHPLC
(ultra high pressure liquid chromatography)
VBNC
(viable but non-culturable)
vii
8
Chapter 1.0. Rationale
The planet we know today is largely the result of the microorganisms’ activity in the
biosphere. Earth’s smallest and simplest organisms created the conditions for the development
of more complex life forms; that is, all the present biodiversity of animals, plants, and other
organisms. Despite the central role of microorganisms in several biogeochemical processes,
and unlike plants and animals, whose importance to humanity has been acknowledged since
ancient times, the microbial world remained hidden from the human consciousness until the
seventeenth century. Antoine van Leeuwenhoek described microorganisms for the first time
using his self-built lenses, the first microscope.
Microbiology developed during the course of the succeeding centuries, always
constrained by the limitations of our own senses. Instruments and techniques had to be
developed to overcome these limitations and to start uncovering the immense diversity of
microorganisms. Despite technological advances that allowed microbiology to establish the
solid body of knowledge it has today, microbiologists realized that only a few organisms were
rewarding their endeavors: total microscopic counts greatly exceed viable counts in Petri
dishes. This inconsistency is known as the “great plate count anomaly”, a term coined in 1985
by Staley and Konopka (Staley and Konopka 1985).
In recent years, the improvement of molecular techniques for microbial analyses has
open new horizons, and has shed light upon some features of the uncultivable microbial
representatives. This implicates that today it is not necessary to culture bacteria to research
community structure and dynamics. Owing to the ever developing molecular technologies, it
would seem that the relevance of cultivation in today’s microbiology could be questioned.
In fact, only 1% of microorganisms can be cultured (Vartoukian et al 2010), but
metagenomic data can only be fully resolved based on knowledge derived from cultivation.
This means that cultivation is still necessary to ground the ever increasing data that is being
obtained through molecular techniques. Culture approaches give context to environmental
microbiology: a basis upon which to confirm the knowledge that metagenomics creates
(Nichols 2007). Only through the isolation of pure cultures can bacteria be fully characterized
(Vartoukian et al 2010), and solid knowledge on their actual behavior and function in the
environment can be acquired (Pham and Kim 2012). Moreover, the yet-uncultured bacteria
may also be a reservoir of bio-molecules, which could be useful for all kinds of industrial and
health applications (Alain and Querellou 2009).
9
10
Chapter 2.0. Literature Review.
2.1 It is not dead. It is only sleeping.
2.1.1 Viable but non culturable bacteria: a mysterious bacterial state
The physiological characterization of macroorganisms is easy to determine compared
to that of microorganisms (Bogosian and Bourneuf 2001). The “great plate count anomaly”
persuaded microbiologists to search for the existence of other states in which bacteria may
exist in nature, apart from being alive and dead. For a long time, it was considered that the
cells observed through a microscope but which at the same time were unable to grow in
laboratory media were dead cells; however, evidence has shown that they are not necessarily
dead, but instead they are waiting for environmental conditions to be favorable to revert to a
state in which they can grow and replicate normally (Kaprelyants et al 1994).
By combining the direct viable counting method (DVC) with direct plating on solid
medium, Xu et al. (1982) observed that Escherichia coli and Vibrio cholerae, when exposed
to stresses such as incubation in saltwater, maintained their numbers while measured with
direct microscopic examinations, but plate counts and most probable number (MPN) results
exhibited declines in the number of counts. The DVC method was proposed by Kogure et al.
in 1979. In this method, the number of viable bacterial cells is determined not by the
bacterium’s ability to divide, but rather by the elongation or growth of single cells once they
are exposed to substrate (Kogure et al 1979). Xu et al. (2012) resolved that the non-dividing
cells were not dead: although they did not divided, they remained substrate-responsive. They
were not dead; they were “only sleeping”.
This study was the turning point to sharpen the concepts of culturability and viability
(Pinto et al 2015). The definition of viability of a microorganism which stated as: “An
organism is viable if it is capable of multiplying to form two or more progeny [sic] in
conditions that are optimal for the species and strain of microbe concerned” (Postgate 1969),
was ultimately challenged. A cell is nowadays considered viable if it keeps its membrane
integrity and potential, enzymatic activity, respiration, responsiveness to nutritional stimuli
and mRNA production (Pinto et al 2015).
Assigning a name to this phenomenon of “sleeping cells” has been a problematic task
for microbiology. Several terms have been used to describe this bacterial dormant state, being
11
“viable but non-culturable” (VBNC) the most common name that appears in the literature.
Cells are not dead (are viable because they exhibit some of the characteristics previously
described), but they simply do not divide in cultures. This naming problem is mainly because
no large scale studies exist to compare the effects of diverse environmental conditions in the
induction of the VBNC state (Li et al 2014, Pinto et al 2015), and there has been no consensus
among different researchers on how to call the physiological states they observe. Some of the
terms commonly used in literature which can relate to “VBNC” include cryptobiotic,
dormant, latent, moribund, pseudosenescent, and somniscent cells (Kell et al 1998); injured,
and somnambulant cells (Colwell 2000). Other similar terms are “not immediately culturable”
(Kell et al 1998); “not readily culturable” (Bovill and Mackey 1997); “transiently non
culturable” (Mukamolova et al 2003); non culturable, active but non culturable
(Sachidanandham and Yew-Hoong Gin 2008); and “quiescence” (Rittershaus et al 2013). All
these terms are not necessarily synonyms.
Not only different terms have been used to describe the same physiological state, but it
is also possible that different states and conditions have been categorized under a single
VBNC state (Kell et al 1998). For instance, Kell et al (1998) define “dormancy” as a state of
metabolic inactivity: dormant cells are those which exhibit negligible metabolic activity, but
can further transit into a growing state; whereas VBNC (they call them active but non
culturable) cells are metabolically active but fail to grow to detectable levels. To determine
which bacteria are metabolically more inactive than others would imply setting a threshold at
which a bacterium can be considered to be in the VBNC state, what will indeed be
problematic. Oliver (2005) defines the VBNC state as a state of dormancy (dormant state).
The metabolic state and the cell’s ability to divide are not considered separately to name the
state in which bacteria are.
What is then the VBNC state? Kaprelyants et al (1993) proposed a simple definition:
VBNC cells are not alive in the sense of colony formation, but are not dead because they can
again be ‘alive’ if conditions become favorable again. The VBNC state is considered a
general survival strategy (Mukamolova et al 2003), since it is observed across several and
very diverse bacterial phyla (Pinto et al 2015). Resting stages can be mechanisms of microbial
adaptation to environmental changes to assure the dissemination of the population when
appropriate conditions return (Suzina et al 2006). The environmental phenomena which can
trigger stress in bacteria include changes in temperature, nutrient unavailability, salinity,
osmotic pressure, pH (Colwell 2000); and desiccation (Suzina et al 2006).
12
Other factors influencing the entry into the VBNC state include pollutants present,
redox conditions of the environment, and organic matter availability (Trevors 2011). Some of
these damaging environmental conditions may temporarily comprise the cell´s ability to
divide (Kell et al 1998), and trigger some responses that seem to be universal (Rittershaus et
al 2013). Owing to the possibility that different states have been described under one term, the
consideration of Ayrapetyan et al (2015) is interesting: they proposed the existence of a
“dormancy continuum”, in which the VBNC state is a deeper state of dormancy but part of a
common, shared phenomenon.
Two different physiological states are contained within this “dormancy continuum”:
the VBNC state, and a state called “bacterial persistency” (Ayrapetyan et al 2015). Both
persisters and VBNC cells are highly tolerant to antibiotics (Ayrapetyan et al 2015, Lewis
2010). Several environmental factors induce the formation of persisters, such as starvation,
carbon-source changes, pH, oxidative damage, etc (Ayrapetyan et al 2015). These factors are
also some of those responsible to induce the formation of VBNC cells (Mukamolova et al
2003). Persisters might also share with the VBNC cells the “shut down” mechanism of
cellular protection that appears when DNA-damaging agents are present (Lewis 2010).
Nevertheless, while persisters can appear in exponentially growing populations (Lewis 2010),
VBNC cells are the consequence of a “low metabolic cost existence” (Mukamolova et al
2003), which definitely is not the case during exponential growth. VBNC cells require a
longer resuscitation-promoting treatment than persisters, which can be readily cultivable e.g.
by removing the antibiotics (Ayrapetyan et al 2015).
It must be also noted that “starved” or “injured” cells have been described as a
different condition from the VBNC (Morita 1993, Mukamolova et al 2003). Injured cells have
the ability to grow on non-selective medium but will not grow on a selective medium (Kell et
al 1998) or in the presence of selective agents (Mukamolova et al 2003). Injured cells are
formed by means of cell degradation and are dying instead of adapting to adverse
environmental conditions (Pinto et al 2015). In contrast to injured cells, one of the
characteristics of the cells in the VBNC state is their cell integrity (Mukamolova et al 2003).
In laboratory conditions, entry into the stationary phase means that bacteria must also
start to adapt to an increasingly harsh environment, since many factors such as oxidative
stress, pH, and nutrient availability are changing (Mukamolova et al 2003). Even though the
stationary phase observed during laboratory culture is not the same as the low-nutrient
conditions observed in the environment (Mukamolova et al 2003), during this phase nutrients
can become limiting, so it is possible to conceive a stationary culture in the laboratory as an
13
occasion for the formation of VBNC cells. It must be also noted that the term “dormancy”
was coined for describing a physiological state of bacteria in nature (Stevenson 1977) before
the term VBNC was coined to describe an issue of non-culturability in the laboratory (Xu et al
1982). The fact notwithstanding, throughout this work the terms VBNC and dormant will be
considered as synonyms: VBNC cells are non culturable because they are physiologically
dormant.
The view that the VBNC state is a survival strategy (Mukamolova et al 2003) is not
the only explanation for the origin of VBNC cells. Three models have been proposed to
explain the origins of the VBNC phenomenon (Nyström 2003): (1) it is the culmination of an
adaptive, genetically programmed pathway generating dormant, survival forms; (2) it appears
due to cellular deterioration; and (3) it is the activation of genetic modules preceding cell
death. The two first models will be further analyzed in the present work.
2.1.2 The VBNC state as an adaptive response for survival
The view that the VBNC state is a general, genetically driven mechanism to generate
dormant and surviving cells is supported by a wide number of observations. Bacteria in the
VBNC state exhibit several types of morphological changes. Cells under these conditions can
predominate in cyst-like forms, have thickened cell walls, fine-grained cytoplasms, round and
reduced shape, condensed nucleoids, etc (Suzina et al 2006).
Of particular relevance is the cell wall state, since it is the structure which protects the
cell from the environment. Enterococcus faecalis cells in the VBNC state were twice as
resistant as measured for mechanical disruption with glass beads in a homogenizer and their
peptidoglycan appeared to be more crosslinked (Signoretto et al 2000). Other Enterococcus
species produce O-acetylated peptidoglycan (at the C-6 hydroxyl group of muramoyl
residues) when they enter the VBNC state, a modification that inhibits the activity of hen egg
white lysozyme (Pfeffer et al 2006) and regulates the sensitivity to hydrolytic enzymes (Pinto
et al 2015). The cell wall of Vibrio cholerae changes during its transition from the exponential
growth phase to the stationary phase, that is, when nutrients become limiting (Lam et al
2009). The content of D-amino acids, specially D-methionine and D-leucine increases, which
may influence the strength of the cell wall (Lam et al 2009). Staphylococcus aureus cell wall
also thickens when the cells enter the stationary phase (Zhou and Cegelski 2012). Membranes
of cells exposed to conditions that are thought to trigger the VBNC state also show a modified
14
structure, like changes in the lipid composition (Denich et al 2003). When exposed to
starvation, visible radiation or seawater, E. coli changes the protein composition of its outer
membrane (Muela et al 2008).
Bacteria better adapted to survive starvation conditions are small sized and have lower
metabolic activity (Colwell 2000). A size reduction when entering the stationary phase can
mean a reduction of the cell´s energy requirements (Mukamolova et al 2003). This reduction
can be as big as 15- to 300-fold (Colwell 2000). For Micrococcus luteus cells, a decrease in
the mean cell diameter (from 1.3 to 0.4 µm) was observed after the cells entered the stationary
phase (Kaprelyants et al 1993). Small bacteria, called ultramicrobacteria, predominate in the
environment (Bakken and Olsen 1987). Their smaller size can give them an advantage on
resource scavenging due to the increased area per unit volume (Morita 1988).
Bacteria respond to challenging conditions changing their morphology to a coccoid
form (Asakura et al 2007). Several bacteria have been observed to adopt a coccoid form once
entering adverse conditions, among them Campylobacter jejuni (Bovill and Mackey 1997),
Vibrio cholerae (Lam et al 2009), and Helicobacter pylori (Nilsson et al 2002). In chemostat
experiments at low dilution rates and in old bacterial batch cultures, bacteria exhibit also a
reduction in size (Kaprelyants et al 1993). For instance, Campylobacter jejuni grown under
different temperature and aereation conditions in sterile stream water microcosms has been
shown to form a continuum of morphological forms when entering the nonculturable phase
(Rollins and Colwell 1986). These shapes ranged from spiral to coccoid, and its predominance
depended upon their growth stage (Rollins and Colwell 1986).
When entering the VBNC state, bacteria still exhibit some detectable metabolic
activity (Oliver 2005). Gene expression occurs in Escherichia coli and Vibrio cholerae after
being exposed to low nutrient concentrations (Asakura et al 2007, Rozen et al 2002). In
Escherichia coli, enzymes essential for several catabolic pathways are upregulated (Rozen et
al 2002). The gene VCA0700 coding for chitodextrinase in Vibrio cholerae, important for the
chitin utilization by the bacterium, is also upregulated in dormancy (Asakura et al 2007). This
is relevant for the metabolism since the bacterium can rely on chitin as its sole carbon source
(Asakura et al 2007). VBNC cells preserve their membrane potential and ATP synthesis
despite their dormancy (Rittershaus et al 2013). ATP level in dormant Mycobacterium
tuberculosis cells under hypoxic conditions is five times lower, but they keep their
membranes energized to drive ATP production at the same level as replicating cells (Rao et al
2008).
15
Protein synthesis accelerates during the VBNC state (Rittershaus et al 2013). It was
found in Vibrio parahaemolyticus that, when incubated at 4 ºC in mineral medium, proteins
with functions associated with transcription, translation, ATP synthase, gluconeogenesis,
antioxidation and transmembrane solute uptake were enhanced (Lai et al 2009). In bacterial
cultures, protein production during the stationary phase can be achieved upon the degraded
proteins produced during the exponential phase (Shaikh et al 2010). The protein profiles of
Enterococcus faecalis cells in the VBNC state differ from its starved cell counterparts (Heim
et al 2002). While in VBNC cells, proteins homologous to the elongation factor Ts (EF-Ts) ,
fructose bisphosphate aldolase, and a catabolite regulator protein (CAA09491) were upregulated, in starved cells they were down-regulated (Heim et al 2002). EF-Ts plays a role in
the regulation of protein synthesis, and fructose bisphosphate aldolase is an enzyme needed
during growth in nitrogen limiting conditions (Heim et al 2002). The mannose-specific PTS
system component IIAB, a protein expressed under stress conditions, was present in starved
cells but not in VBNC cells (Heim et al 2002). This study supports the fact that the VBNC
state might not be only a product of cellular deterioration (Nyström 2003) because protein
expression patterns of both conditions differ and some up-regulated proteins, i.e. fructose
biphosphate aldolase, play an important role in responses against hostile conditions.
The regulation of mRNA stability and the preservation of DNA integrity are important
responses against stress in bacteria (Takayama and Kjelleberg 2000). An increase in mRNA
stability and half-life has been observed for Staphylococcus aureus (Anderson et al 2006) and
Mycobacterium tuberculosis (Rustad et al 2012) when these bacteria are exposed to
temperature shocks and hypoxic conditions, respectively. Stable mRNA transcripts were also
observed in the starved marine Vibrio sp. strain S14, which could be translated after a glucose
upshift (Marouga and Kjelleberg 1996).
For a cell to remain viable, it needs to keep its DNA protected from potential lesions.
Both spores and dormant cells alter their chromosomes to form more stable structures
(Rittershaus et al 2013). When E. coli cells starve for carbon, they induce a DNA binding
protein that renders the genome resistant to DNAses and hydrogen peroxide (Almiron et al
1992). This protein, Dps, prevents DNA damage by free radicals, yet does not interfere with
normal DNA metabolism, and can also be involved in nucleoid condensation (Martinez and
Kolter 1997). When Vibrio vulnificus enters the VBNC state, i.e. when cells are exposed to a
cold shock, they express several genes responsible for combating numerous environmental
stresses, such as rpoS and vvhA, the latter having a possible role in osmoprotection (Smith and
Oliver 2006). A condensed nucleoid can be an indication that the cell is in the VBNC state.
16
Ghost cells, bacteria without visible nucleoids, regain nucleoid visibility (and activity) once
they are placed under growth permissive conditions (Cole 1999). Choi et al (1996) described
that the proportion of starved marine bacteria with visible nucleoids, when resupplied with
nutrients, increased from 27% to 100%.
The entry into the VBNC state also implies storage of carbon reserves, in forms such
as glycogen, triglycerides, wax esters, polyhydroxyalcanoates, and poly-β-hydroxybutyric
acid, which are quickly mobilized for use once the conditions are restored (Rittershaus et al
2013). Acetyl-CoA that enters the Krebs cycle during active growth is redirected into lipid
synthesis pathways and stored in the mentioned chemical forms; this both reduces metabolic
rate and increases energy reserves for the cell to survive during dormancy (Rittershaus et al
2013).
2.1.3 Resuscitation of bacteria from the VBNC state
At some point during their dormant existence, bacteria must ‘wake up’ to start
dividing again or ultimately die, because the VBNC state is time-limited (Mukamolova et al
2003). Dormant bacteria can resume division once the appropriate conditions for their growth
reappear, a process called resuscitation (Mukamolova et al 2003). VBNC cells become
culturable again (Pinto et al 2015).
Determining whether a cell resuscitated or not can be a difficult task, since VBNC
cells may coexist with multiple phenotypic states, and interfering signals from other
physiological types within the same population may impede a correct result interpretation
(Sachidanandham and Yew-Hoong Gin 2008). Bacterial populations must likely contain a
mixture of VBNC/actively growing cells at the same developmental stage (Mukamolova et al
2003). If the state in which bacteria are found cannot be correctly determined, it is not
possible to determine whether a regrowth was due to resuscitation, or only because of the
growth of active cells (Kaprelyants et al 1994), or repair processes of injured cells (Kell et al
1998).
Kaprelyants et al (1994) determined the resuscitation of M. luteus cells starved for 3
months, after which they were transferred to fresh lactate minimal medium containing 0.5 µg
ml-1 penicillin G. They concluded that the posterior growth observed in the cell population
was caused only because of the resuscitation of dormant cells, since the antibiotic targeted
only dividing cells and left VBNC cells intact (Kaprelyants et al 1994). Campylobacter jejuni
17
cells subjected to dormancy by rendering their atmosphere microaerophilic, resuscitated when
they returned to aerobic conditions (Bovill and Mackey 1997). Wai et al (1996) researched the
resuscitation of Vibrio cholerae, after the application of a heat shock from 15 ºC (temperature
which triggered the VBNC formation) to 45 ºC for 1 minute. Cells that were not culturable in
liquid or solid media became culturable only after this heat shock (Wai et al 1996).
Bacteria can resuscitate when they are cultured together with other microorganisms,
either other bacteria or eukaryotes. Senoh et al (2010) demonstrated that resuscitation of V.
cholerae was possible when cultured with eukaryotic cells. Kaprelyants et al (1994) used the
autoclaved supernatant from centrifuged suspensions of starved cultures that may have
contained compounds excreted by some microbial species, which are necessary for others to
grow. When the cells were counted through the MPN method, it was observed that the use of
the autoclaved supernatant yielded better resuscitation results than the use of fresh liquid
growth medium (Kaprelyants et al 1994). Resuscitation is also possible in E. coli
supplementing dormant cells with amino acids, especially polar amino acids (Pinto et al
2011). The role that other microorganisms have in the resuscitation of dormant bacteria (or
culturing of VBNC bacteria in a technique called co-culture) and the use of growth factors for
the improvement of bacterial cultivation techniques will be further discussed in the following
section (2.2).
Epstein (2009) proposed a different resuscitation approach, based on the fact that cells
can resuscitate stochastically instead of responding to environmental signals. If the conditions
found by a resuscitating bacterium are growth supportive, it will survive and start dividing;
otherwise the cell will simply die (Epstein 2009). Other cells will resuscitate later in the same
way to assess the environmental conditions; these cells are called ’scout cells‘ (Epstein 2009).
The sensing of extracellular signals can be impaired by dormancy (Sturm and Dworkin 2015).
Nevertheless, the scout theory and the resuscitation mediated by environmental cues are not
mutually exclusive, since scout cells which resuscitate when the conditions are growth
supportive can at the same time produce growth inducing signaling compounds that would
accumulate in the extracellular media, helping other bacteria in the population to resuscitate
(Epstein 2009, Pinto et al 2015).
18
2.1.4 An alternative model: VBNC cells as a result of cellular deterioration
Despite the fact that the VBNC state has recurrently appeared in the literature for more
than 30 years, its existence as a defined physiological phenomenon generating dormant,
resistant cells has been challenged by several researchers. VBNC cells, as previously
discussed, exhibit some changes that differentiate them from actively growing bacteria, but
these changes do not necessarily imply that bacteria entered the VBNC state (Pinto et al
2015). One of the main arguments against many of the studies that research on VBNC
bacteria, has been that they fail to adequately determine whether alleged resuscitation was
indeed due to resuscitation, or to the growth of some culturable cells initially present in the
population (Kell et al 1998).
Bacterial unculturability can also be a condition preceding cellular death
(Sachidanandham and Yew-Hoong Gin 2008). Clear degenerative changes can be observed in
cells that enter the stationary phase in laboratory cultures (Mukamolova et al 2003, Nyström
2003) . In a viability study of Vibrio vulnificus, CFU on medium supplemented with catalase
and sodium pyruvate to diminish H2O2 in the culture showed 1,000-fold higher counts than on
unsupplemented media (Bogosian et al 2000), supporting the idea that non-culturability is
really due to the damaging action of free radicals (Mukamolova et al 2003). Cells subjected to
nutrient starvation have an increased need for oxidation management (Nyström 2001), so they
express proteins aimed to protect themselves against oxidation damages. Upon starvation,
production of cytoplasmic superoxide dismutases, enzymes involved in protecting the cell
against oxidation, increase (Dukan and Nyström 1999). Depriving the starved cells from
oxygen makes them survive for longer periods (Dukan and Nyström 1999).
This close relationship between nonculturability and oxidation damages, especially
protein carbonylation, favors the stochastic deterioration theory (Nyström 2003). Specific
determinants to support the theory of VBNC phenomenon as adaptive, genetically
programmed pathway generating dormant cells are yet to be uncovered (Kell et al 1998, Pinto
et al 2015). Until now, not enough light has been shed upon the molecular mechanisms behind
the entry and exit from the VBNC state (Pinto et al 2015). If the genetic machinery behind the
VBNC state is neither characterized nor identified, the phenomenon will remain one of
microbiology’s biggest questions (Nyström 2003). But if there are several common features of
VBNC cells such as morphology, biochemistry and physiology alterations, there must
definitely be some basic genetic mechanisms orchestrating all these changes (Mukamolova et
al 2003).
19
2.1.5 Implications of the VBNC state
The VBNC state may have many implications in the environment and human health,
so unveiling the molecular basis of its perplexing nature is one of the pending milestones for
today’s microbiology. It is estimated that 60% of the Earth’s microbial biomass exist in a
dormant state (Lewis 2007, Rittershaus et al 2013). In oceanic ecosystems, the proportion of
dormant cells, which are inactive and can activate when conditions become suitable, ranges
from 20 to 90% (Cole 1999). Slow replication rates must be the norm in the environment:
organisms usually live in habitats which are not suitable for rapid growth, since the basic
nutrients for growth are not always abundant (Rittershaus et al 2013). If these dormant cells
maintain metabolic activity, as they have been shown to do, they might have a role in
environmentally significant processes such as nutrient cycling (Kell et al 1998).
Jones and Lennon (2010) proposed that bacteria, when dormant, become members of a
‘seedbank’ – a reservoir of inactive bacteria which can contribute to microbial diversity and
dynamics in future generations (or when pertinent environmental conditions for each of them
arise). The dynamics of this reservoir can be season-dependent (Mukamolova et al 2003).
VBNC cells must also play an important role for medicine. Infections could be able to enter a
dormant state, thus bacteria can become latent and resistant to antibiotics, since the
antibiotic’s action usually requires the organisms to be actively reproducing (Kell et al 1998).
When an environmental sample is inoculated on medium, the inability to observe
colonies of bacteria that can be otherwise observed under the microscope can be due to two
reasons: the suitable in vitro culture conditions have not been discovered, as is the case of the
majority of microorganisms, or the cells are temporarily present in the VBNC state and could
become culturable at a later time point (Kell et al 1998). Bacteria which have not been
cultured in vitro might be staying (or entering) the VBNC state once inoculated in laboratory
medium (Lewis 2007, Watve et al 2000).
2.2 Culturing the unculturable
2.2.1 Culturing the unculturable: breaking down a misnomer
Absolutely unculturable microorganisms are not likely to exist: since all microbes can
grow in nature, unculturability is only the microbiologists’ failure to mimic conditions
20
necessary for growth of many organisms (Stewart 2012, Watve et al 2000). The environment
in which bacteria live in nature is very different from the conditions we create for them in the
laboratory. In nature, the conditions are more likely oligotrophic (Kaprelyants et al 1993).
Bacteria live under what Koch (1971) called a “feast and famine existence”. Bacterial growth
observed under laboratory conditions, namely the lag phase, exponential phase, and stationary
phase, does not necessarily exist in nature, where bacteria are constantly experiencing
environmental changes (Pinto et al 2015). Oligotrophs are likely genetically adapted to low
nutrient environments, while copiotrophs are physiologically adapted to nutrient fluctuations
(Mukamolova et al 2003). Oligotrophs and copiotrophs can also be related to the strategies
which categorize higher organisms: R strategists for fast growing, copiotrophic bacteria, and
K strategists for oligotrophs, slow growers (Watve et al 2000). In natural environments, the
selective pressure favors cells with a low metabolic cost existence (Mukamolova et al 2003).
Laboratory conditions must mimic as close as possible natural environments of
bacteria (Mukamolova et al 2003). However, when isolating novel bacterial representatives,
highly specialized conditions or media can be disadvantageous, since as yet uncultured
bacteria are entities whose needs may not be totally characterized (Durbin 1961). This is why
most of the novel media for culturing difficult-to-culture bacteria are not designed but are
rather taken directly from nature, e.g. creating media based solely on water taken directly
from the environment and with a low inoculum of organisms (low concentrations) reflecting
that of the environment (Mukamolova et al 2003).
Pham and Kim (2012) mention some common mistakes when attempting to culture
soil bacteria: (1) addition of inhibitors, (2) high concentration of nutrients, and (3) no
consideration for density dependent cell/signaling mechanisms. In the environment, bacterial
populations are very heterogeneous, with some individuals actively growing, some other
injured, and some dormant. In vitro, opportunities for these bacteria to grow should be
equalized. The overgrowth of oligotrophs by copiotrophs should be avoided (Giovannoni and
Stingl 2007). Several novel culturing strategies will be described in the following sections.
2.2.2 Media and cultivation generalities
Indeed, one of the successful modifications that led to the culture of previously
unculturable bacteria is the use of dilute nutrient media, since slow growers can be inhibited
when inoculated in substrate rich conventional media (Vartoukian et al 2010). High nutrient
21
media might work well for human-residing bacteria and pathogens, since the interior of a
human being must be very high in carbon resources and nutrients (Dewi Puspita et al 2012)
and has a stable temperature. If most of natural environments in which microorganisms live
are oligotrophic, then laboratory media aimed to their recovery must also have a low nutrient
concentration. Inhibitors found in media such as oxidative agents (e.g. H2O2) can cause
oxidative stress (Dewi Puspita et al 2012), and could hinder the growth of injured cells.
Other relevant variables when culturing microorganisms are cultivation time and
inoculum size. When incubation time is increased, some rarely isolated groups appear in the
culture medium (Davis et al 2005). Song et al (2009) attempted the isolation of SAR11 cells,
abundant members of bacterioplankton in the ocean. The cells did not appear under laboratory
conditions until 8 weeks of culturing. Since oligotrophs are slow growers, they likely require
higher cultivation times. At the same time, if the scout cell awakening theory proposed by
Epstein (2009) is the case for resuscitation of VBNC cells, a longer cultivation time (and also
larger inocula) would allow a higher formation rate of scouts and thus increase the chance of
observing the growth of colonies (Epstein 2013).
Choosing the right inoculum size is relevant when culturing difficult-to-culture
bacteria, since some bacteria appear to be culturable only when they are inoculated above a
certain cell density (Stewart 2012). Also, some of the signaling mechanisms among bacteria
are density dependent, so the lack of consideration for initial density may have a direct
relation with the VBNC phenomenon. If bacteria do not detect the environmental signals
necessary for their resuscitation, they will not start dividing in vitro. Dilution of the inoculum
can be beneficial, because in this way the inoculum would contain the most abundant cells
rather than the most nutrient tolerant, fast grower organisms (Button et al 1993), which may
exist in lower numbers but adapt and grow quickly. Low inoculum sizes can also prevent
bacteria from being affected by antimicrobials produced by other community members (Dewi
Puspita et al 2012).
2.2.3 In situ culturing and recreating the environment in vitro
If laboratory conditions are unable to mimic the natural environment, then culturing in
situ can be an option (Alain and Querellou 2009). Yasumoto-Hirose et al (2006) brought their
cultivation chambers, polyurethane foam (PUF) supplemented with agar medium, directly to
the ocean to culture marine bacteria. They targeted different bacteria by supplying the PUF
22
with different culture media, and in the laboratory, the PUFs where homogenized with sterile
seawater, diluted and bacteria were inoculated into agar medium (Yasumoto-Hirose et al
2006). For bacteria which grow in biofilms, colonization carriers can be used (Alain and
Querellou 2009). Titanium rings pierced with numerous apertures were used as colonization
carriers for bacteria in deep-sea vent worms, which were further isolated through a series of
enrichment cultures in media enriched with electron donors and acceptors found in situ such
as iron, nitrate and sulphate (Alain et al 2004).
Natural environments can also be constructed in the laboratory (Stewart 2012), or the
environment where bacteria live can be mimicked using directly natural matrices as media,
such as water or soil (Vartoukian et al 2010). Ferrari et al (2008) developed a protocol for
cultivating previously uncultured soil bacteria employing, what they call, a soil substrate
membrane system. The medium of this system consists of natural non-sterilized wet soil and a
polycarbonate membrane where colonies grow. Some methane oxidizing bacteria were also
cultured for the first time on a polycarbonate membrane, using non sterile soil slurry as a
medium while incubated in a methane-air atmosphere (Svenning et al 2003).
Bacteria inoculated in the polycarbonate membranes profit from the nutrients and
chemicals from their own environment (the natural medium used), since these substances can
pass through the membrane but bacteria cannot. The basic reasoning behind diffusion
chambers is to provide bacteria with their natural environment while in culture by artificially
creating this environment, such as in an aquarium (Bollmann et al 2007, Kaeberlein et al
2002). After bacterial colonies appear on the membrane, they can be selected and propagated
with successive reinoculations (Stewart 2012). Sterile natural lake water has been used as a
culture medium for low nucleic acid bacteria (Stingl et al 2008, Wang et al 2009).
2.2.4 Co-culturing
Because microbes do not live isolated in their environment and establish relationships
with other microorganisms in the community, co-culture approaches have been established
(Pham and Kim 2012). It must be remembered that resuscitation of some bacteria was
possible in the presence of other living cells, either with growing cells of the same species or
with different cells (Kaprelyants and Kell 1993, Senoh et al 2010). So, in a petri dish, cells
might be releasing signaling molecules which indicate dormant cells that they are in a suitable
environment for their growth (Lewis 2007).
23
The isolation of novel microorganisms from a given environment can be done by coculture with cultured specimens of that environment, so called helper cells (D'Onofrio et al
2010). Kaeberlein et al (2002) were able to culture previously uncultured marine bacteria in
nutrient medium only in the presence of other marine microorganisms. Pagnier et al (2008)
isolated novel bacteria in co-culture with amoebae, since they live in symbiosis with the
eukaryote. Marine bacteria isolated via the diffusion chamber might be difficult to be further
cultured in a pure culture, but they can grow in co-culture with culturable marine bacteria on
an agar medium (Nichols et al 2008).
Taking advantage of symbiotic relationships with higher organisms such as plants and
insects might help to bring some of the most challenging as-yet-uncultured bacteria to culture
(Stewart 2012). The genomes of symbiotic bacteria can be reduced due to close host
association and can lack critical genes essential for microbial reproduction (Kikuchi 2009,
Stewart 2012). Bacteria which live in symbiosis with insects have been mainly cultured using
insect cell lines (Kikuchi 2009).
Associations of bacteria in communities require them to have an effective cell-to-cell
communication system, which is called ‘quorum sensing’ (Bassler 1999). Quorum sensing
occurs when bacteria are at high population densities (Bassler 1999) and this system involves
several substances for cell-cell communication. These substances include growth factors,
which can also be incorporated in the cultivation strategies and thus increase the efficiency for
culturing environmental bacteria (Dewi Puspita et al 2012).
2.2.5 Improving cultivation using growth-promoting factors
Media can be also supplied with metabolites that microbes need in their natural
environment for growth. A lot of different substances can be mentioned that have helped to
culture as-yet-uncultured microorganisms in vitro: different carbon sources, electron donors
and acceptors, inorganic ions, aminoacids (Pham and Kim 2012, Vartoukian et al 2010), and
the number of these substances might be as vast as the number of as-yet-uncultured
microorganisms. Two exemplatory growth promoting factors are the adenosine 3',5'-cyclic
monophosphate (cAMP) and n-acyl homoserine lactone (AHL).
A possible link between cAMP and the VBNC state has been proposed, since cAMP
can be a signal for the regulation of starvation survival proteins (Schultz et al 1988). In
Mycobacterium tuberculosis, gene Rv3676 codes for a transcriptional factor belonging to the
24
cAMP receptor family, which is a direct regulator of the gene rpfA, one of the M. tuberculosis
genes that encodes for a resuscitation promoting factor (Rickman et al 2005). Cyclic AMP is
widely present dissolved in aquatic ecosystems; it is released from other organisms, including
multicellular eukaryotes (Ammerman and Azam 1981).
AHL is a quorum sensing molecule; its extracellular concentration increases when the
cell density increases (Dewi Puspita et al 2012). When attempting to recover Acidobacteria
and Verrucomicrobia uncultured representatives, Stevenson et al (2004) determined that the
supplementation of medium with catalase or AHL resulted in one of the best culture
modifications among several for a greater occurrence of Acidobacteria in culture. Similarly to
cAMP, AHL could also be related to the VBNC state. Gorschkov et al (2010) suggested that,
when starved, cell sensitivity toward AHL increases, which allows the quorum-dependent
stress response to start at lower population densities. Other molecules that could act as growth
factors are resuscitation promoting factor (Rpf) of Micrococcus luteus and peptidoglycan/cell
wall fragments. Their role in the VBNC phenomenon and their potential as signaling
compounds will be discussed in section 2.3.
2.2.6 Cultivation based on genomic data
When designing new culturing techniques, there is no need for trial and error
approaches: finding out which growth factors do bacteria need can be found within their
genomes (Alain and Querellou 2009). For instance, genome analysis of three members of the
SAR11 alphaproteobacterial clade showed a deficiency in sulphate reduction genes (Tripp et
al 2008). In nature, members of this clade take reduced sulphur compounds exclusively from
other organisms; therefore supplementing reduced sulphur species to their growth medium
could bring them to culture (Tripp et al 2008).
Bomar et al (2011) cultured for the first time a leech symbiont based on the
information provided by sequencing the transcriptome of the leech’s gut. The information
suggested that the metabolism of the bacterium relied on the conversion of fermented
sialylated-mucin glycans to acetate, so addition of these glycans to the designed medium
brought the bacterium into culture.
25
2.2.7 Miscellaneous novel culturing techniques
Other approaches for optimizing culturing techniques involve looking after individual
cells. Zengler et al (2002) developed a technique for encapsulating cells individually in gel
microdroplets, sorting the droplets through flow cytometry, and inoculating each cell in the
wells of microtiter plates. A similar approach using agar spheres to encapsulate bacteria
directly from a simulated environment was developed by Ben-Dov et al (2009). Bacteria
inside the spheres can be cultured in their own natural medium, from which they can be
isolated and enriched in agar plates (Ben-Dov et al 2009).
The use of microbial culture chips is another high-throughput culture technology that
can be used to segregate and grow microorganisms (Ingham et al 2007). As an analogy, these
devices resemble microscopic egg carton boxes, with a porous aluminium oxide base so that
they allow the exchange of nutrients between the medium and the sorted cells. An advantage
of microbial culture chips is their high throughput screening capacity (Ingham et al 2007).
Individual cells can be physically manipulated using optical tweezers in the form of infrared
lasers to isolate them and transfer them to a growing medium (Vartoukian et al 2010).
2.3 Peptidoglycan and Rpf, a bacterial cytokine
2.3.1 Resuscitation promoting factor (Rpf)
Bacterial growth factors, or bacterial cytokines, are compounds whose name is derived
from substances present in higher organisms that control cell growth and division, called
growth factors (Kell and Young 2000). In eukaryotic cells, cytokines activate receptor
tyrosine kinases, which communicate with the nucleus through phosphorylation cascades
(Kell and Young 2000).
It was observed that, when supplementing dormant Micrococcus luteus cells with
sterile supernatant from their actively growing counterparts, dormant cells were resuscitated
and the viable count as estimated by MPN raised several orders of magnitude (Mukamolova
et al 1998). M. luteus is a gram positive bacterium with a high G+C content that can be found
in diverse environments such as soil, water and human skin (Mukamolova et al 2002).
Mukamolova et al (1998) isolated a protein from the Micrococcus luteus culture supernatant:
a small protein called resuscitation promoting factor, Rpf, essential for M. luteus growth.
26
The lytic activity of Rpf was hypothesized by Cohen-Gonsaud et al (2004), since the
enzyme revealed a high structural similarity with lysozyme. Mukamolova et al (2006)
confirmed the muralytic activity of Rpf. When expressed in E. coli and secreted into its
periplasm, Rpf lysed the bacterium; it also lysed fluorescamine labelled cell walls of M.
luteus, the artificial substrate 4-methylumbelliferyl β-D-N,N′,N′′-triacetylchitotrioside (MUF
tri-NAG) (Mukamolova et al 2006), and crude preparations of M. luteus cell wall (Telkov et
al 2006). It cleaves the
-glycosidic bond found between N-acetyl-muramic acid and N-
acetylglucosamine in the peptidoglycan (Mukamolova et al 2006), while releasing its
fragments.
Rpf is a member of a broad protein group found throughout high G+C gram positive
bacteria (Mukamolova et al 2003). Firmicutes possess a protein family related to the
actinobacterial Rpf, which also improves their culturability, the SPS (stationary phase
survival) protein family (Ravagnani et al 2005). Rpf is encoded for by the M. luteus gene rpf
(Koltunov et al 2010). The protein is formed by a 70-residue lysozyme like domain and a
LysM domain found in cell-wall-associated proteins, which is thought to facilitate binding to
peptidoglycan (Koltunov et al 2010). A linker region which connects both domains has a
variable size in different strains; this determines whether Rpf remains bound to the surface of
the cell, or is released to the medium (Koltunov et al 2010). Rpf production reaches maximum
before the late exponential phase of M. luteus growth, and rapidly declines before entering the
stationary phase (Mukamolova et al 2002). Optimum activity of Rpf is at pH 6 (Telkov et al
2006).
Rpf proteins control culturability of one of the largest taxonomic units within the
bacterial domain, Actinobacteria (Mukamolova et al 2006, Ventura et al 2007). This phylum
is of extreme relevance because it includes some of the main antibiotic-producing organisms
for the pharmaceutical industry (Ventura et al 2007). Rpf stimulates also the growth of other
high G+C gram positive organisms like Mycobacterium spp. Rpf genes are not exclusive to
Micrococcus luteus: Mycobacterium tuberculosis has 5 analogous proteins with similar
properties to those of Micrococcus Rpf (Mukamolova et al 2002), and these genes can be
widely distributed among high G+C gram positive bacteria (Kaprelyants et al 1999). Much
research has been done to understand the role of each of the 5 rpf genes of M. tuberculosis
(Kana and Mizrahi 2010), but their structure and specific roles in Mycobacterium spp.
dormancy will not be further discussed in this work.
27
2.3.2 Rpf’s mode of action
Although the resuscitating mechanism of Rpf in dormant bacteria is not clear, several
models have been proposed (Pinto et al 2015): (1) Rpf acts as a cytokine, binding to specific
receptors in dormant cells and triggering resuscitation; (2) Rpf hydrolyzes bacterial cell wall
of dormant cells, remodeling it and thus starting resuscitation; (3) Rpf hydrolyzes the cell
wall, resulting in peptidoglycan fragments release, which bind to specific cell receptors that
trigger resuscitation; and (4) Rpf mediated hydrolysis of the cell wall releases muropeptides,
which then bind to specific receptors and trigger resuscitation. Rpf is active at picomolar
concentrations, so its mode of action can be more similar to a cytokine (1) rather than by
restructuring the cell wall (2) (Mukamolova et al 2003). Nevertheless, a receptor for the Rpf
has not been identified yet (Pinto et al 2015, Telkov et al 2006).
As previously discussed, one of the characteristics of VBNC cells is their thickened
cell wall, and cells might need a specialized enzyme for cleaving that reinforced structure
(Keep et al 2006, Pinto et al 2015). When bacteria divide, they must cleave peptidoglycan to
separate the new daughter cell attached at the septum (Hett et al 2007). The lytic activity of
Rpf against peptidoglycan can aid the cell during the divisions after resuscitation occurs (Hett
et al 2007). Telkov et al (2006) determined by directed mutagenesis that an alteration in the
catalytic site of Rpf, namely the replacement of the glutamate residue at position 54 with
glutamine, alanine and lysine, and cysteine residues 53 and 114 with threonine and lysine,
respectively, reduced its activity and at the same time minimized its resuscitation potential.
This finding strongly favors the hypothesis that activity of Rpf is directly related to its
resuscitation potential (Telkov et al 2006).
2.3.3 Peptidoglycan-mediated resuscitation
The third Rpf’s mode of action described previously (3), implies catalytic activity of
Rpf and the possibility that the peptidoglycan fragments resulting from hydrolysis are
detected by receptors in other cells (Pinto et al 2015), possibly promoting growth and, in the
case of dormant bacteria, triggering resuscitation (Dworkin and Shah 2010). Peptidoglycan is
constantly released in the environment when bacteria replicate (Dworkin and Shah 2010), and
this polymer comprises a large fraction of the organic material found in aquatic environments
(Dewi Puspita et al 2012). Peptidoglycan has the potential of activating bacterial spores and
28
non-spore forming dormant bacteria (Dewi Puspita et al 2012). Nikitushkin et al (2013)
observed that peptidoglycan fragments obtained either by lysis with Rpfs or by sonication had
stimulatory effects on the resuscitation of dormant Mycobacterium smegmatis and
Mycobacterium tuberculosis.
29
30
Chapter 3.0. Objectives
The present work proposes a method to increase the yield of the extraction and
cultivation approaches for soil bacteria, employing growth promoting factors. These include
purified supernatants from the liquid culture of the bacterium Micrococcus luteus, which
contains the Rpf protein, and cell wall fragments from the same bacterium. The main goal is
to ‘resuscitate’, or promote the culturability of VBNC bacteria from soil during the soil
extraction step employing these growth factors, and then culture these bacteria on low nutrient
solid media. The results will be analyzed in terms of (1) differences in counts of total colony
forming units (CFU) between the extraction with versus without purified supernatant and cell
wall fragments; and (2) the difference in the microbial diversity observed in both approaches.
The hypothesis is that difficult-to-culture soil bacteria will be isolated with this approach,
with some being cultured for the first time.
Prior to the extraction experiments, the lytic activity of the M. luteus supernatant will
be proved. The presence of Rpf in the purified supernatant of M. luteus will also be addressed.
31
32
Chapter 4.0. Materials and Methods
4.1 Materials
4.1.1 Media and buffers
1. Lactate minimal medium (LMM), prepared according to Kaprelyants and Kell (1992):
4.0 g l-1 NH4Cl; 1.4 g l-1 KH2PO4; 0.005 g l-1 biotin*; 0.02 g l-1 L-methionin*; 0.04 g l1
thiamin*; 1.0 g l-1 inosin*; 0.07 g l-1 MgSO4*; 0.000024 g l-1 CuSO4*; 0.0005 g l-1
MnCl2*; 0.001 g l-1 FeSO4*; 0.000025 g l-1 Na2MoO4*; 0.0005 g l-1 ZnSO4*; 10 g l-1
lithium lactate. The pH was adjusted to 7.5 and the medium was autoclaved.
2. PCA: Tryptone glucose yeast agar (17.50 g l-1) was mixed with 7 g l-1 agar
bacteriological (agar no. 1) and the medium was autoclaved.
3. Half-strength LB broth: 12.50 g l-1 LB, autoclaved.
4. Sodium pyruvate (0.5%) mineral medium: 10.955 g l-1 Na2HPO4·12H2O; 2.7 g l-1
KH2PO4; 1.0 g l-1 (NH4)2SO4; 0.03 g l-1 Ca(NO3)2*; 0.2 g l-1 MgSO4*; 0.01 g l-1
FeSO4*; 5.0 g l-1 sodium pyruvate . The pH was adjusted to 7.5, and the medium was
autoclaved.
5. Modified R2 broth: 0.50 g l-1 casein acid hydrolysate; 0.50 g l-1 yeast extract; 0.50 g l-1
special peptone; 0.50 g l-1 starch; 0.03 g l-1 K2HPO4; 0.024 g l-1 MgSO4; 0.30 g l-1
sodium pyruvate. 15 g l-1 agar bacteriological (agar no.1) were used for the modified
R2 agar. The pH was adjusted to 7 and the medium was autoclaved
6. Phosphate buffer saline (PBS)*: 8.0 g l-1 NaCl; 0.2 g l-1 KCl; 1.42 g l-1 Na2HPO4; 0.24
g l-1 KH2PO4.
7. Physiological solution: 0.85% NaCl, autoclaved.
Marked (*) components were filter-sterilized (0.22 m) and added to media after autoclaving.
4.1.2 Soil
The soil used for the extraction experiments was retrieved from Prague-Suchdol,
Czech Republic. It is a chernozem soil with a clay-loamy texture (Casova et al 2009).
33
4.2 Methods
4.2.1 Bacterial Cultures
Micrococcus luteus NCTC2665 (“Fleming strain”) was grown in 200 ml LMM at
28°C and 120 rpm. Its growth curve was determined during 250 hours by absorbance at 600
nm using an IMPLEN Nanophotometer® spectrophotometer, model P300. The purity of the
M. luteus liquid cultures was assessed by plating on PCA at 28°C. Colonies formed were
analyzed by MALDI-TOF MS (4.2.9).
Escherichia coli was grown overnight in 150 ml half-strength LB broth at 200 rpm
and 28°C. The culture was autoclaved to use it in the supernatant activity determination
experiments (4.2.4).
4.2.2 Supernatant retrieval
M. luteus cultures in LMM at different growth stages were centrifuged at 5000 × g for
10 minutes, after which the pellet was collected to prepare cell walls (4.2.3). The supernatant
was sterilized using a 0.22 m filter and further used for its activity determination. As activity
control in all the experiments, a supernatant aliquot boiled for 10 minutes was used.
4.2.3 Micrococcus luteus cell wall preparation
M. luteus cell wall for the supernatant activity determination was prepared according
to the procedure described by Mukamolova et al (2006): collected M. luteus cells (exponential
phase) after centrifugation (10 min, 5000 × g, described in 4.2.2) were washed with deionized
water, and then resuspended in 40 ml SDS (5% w/v). This was boiled for 20 min and
centrifuged again. The pellet was resuspended in 20 ml SDS (4% w/v), and boiled for another
20 min. In order to remove the SDS, the pellet was washed six times with hot water (60°C).
The remaining solids were washed with 20 ml acetone, centrifuged, air-dried, and stored at 20°C until they were used.
For the determination of Rpf lytic activity towards the cell walls, 1.47 g of prepared
cell wall were suspended in 40 ml LMM, and diluted 8 times (in LMM) to get a final cell wall
concentration below 5 mg ml-1.
34
4.2.4 Supernatant activity determination by absorbance
Both the supernatant and the boiled aliquot (control) were mixed in the wells of a
microtiter plate (0,25 ml total volume in each well) with the diluted cell wall preparation
(target 1) and the autoclaved E. coli suspension (target 2), in the following ratios (supernatant
or boiled supernatant: target 1 or 2): 1:20, 1:15, 1:10, 1:5, 1:2, 3:1, 4:1, 5:1. These ratios were
prepared in triplicates. For the ratio 3:1 onward, the only target used were the cell walls.
Absorbance was also measured (in triplicates) for the E. coli suspension, the cell wall
suspension (<5 mg ml-1 in LMM), as well as the supernatants (fresh and boiled), and the
lactate medium. To measure the lytic activity towards the bacterial cell wall, a procedure
similar to that reported by Telkov et al (2006) was performed. The change in absorbance (600
nm) after the mixtures was followed for 2 hours in 2 minutes intervals in an EON microplate
spectrophotometer (Biotek®) at 28°C and 180 rpm.
4.2.5 Supernatant activity determination by fluorometry
Supernatant activity was also determined using the fluorogenic substrate 4methylumbelliferyl- -D-N,N’,N’’-triacetylchitotrioside (MUF tri-NAG), as described by
Mukamolova et al (2006). Fluorescence was measured with a Fluoroskan Ascent FL
microplate fluorometer and luminometer with one dispenser (ThermoLabsystems), with an
excitation wavelength of 355 nm. In each well, 250 l of supernatant were placed together
with MUF tri-NAG in a concentration of 80 M. As a negative control for the supernatant
activity, boiled supernatant (10 min) was used. As positive control, lysozyme from chicken
egg white (1x105 units mg-1) was used (2.50 mg of lysozyme were used per well, dissolved in
LMM). The fluorescence of the supernatant, the boiled supernatant and the lactate medium
(background fluorescence) were also measured. Measurements were done in triplicates for 4
hours, at 28°C and 480 rpm.
Supernatant activity was measured at different times during the M. luteus culture
growth to determine the point where supernatant lytic activity was at its maximum: 18, 41, 64,
71, 88, 95, and 256 hours after inoculation in LMM (200 ml).
35
4.2.6 Supernatant purification
After collecting the supernatant from the Micrococcus luteus culture (4.2.2) its high
molecular weight content was concentrated through ultrafiltration using Millpore™
centrifugal filter units (10 kDa) and CENTRIPLUS® disposable centrifugal concentrators (3
kDa). For the filtration in the 10 kDa filters, 12 ml of sterile M. luteus supernatant were
filtered at 4000 × g for 10 minutes.
In the case of the 3 kDa filters, 13 ml of supernatant were filtered for 2,5 hours at 3000
× g to remove the excess lactate. This retentate was then washed with 5 ml LMM without
lithium lactate and further concentrated for 2 more hours. The activity of the retentate and the
filtrate in both filtrations was measured using the fluorogenic substrate MUF tri-NAG as
previously described (4.2.5). Boiled retentate and lysozyme were used as negative and
positive controls, respectively. The washed retentate resulting from the filtration with 3 kDa
filters was used for the enhanced extraction from soil (4.2.8). The background fluorescence of
the retentates and the filtrates was measured as previously described (4.2.5).
4.2.7 Rpf identification
To identify the Rpf protein from the M. luteus culture, the supernatant was purified as
previously described (4.2.6 in the 3 kDa filters), except that the retentate was washed with 5
ml distilled water to reduce its salt content. This purified supernatant (containing the high
molecular weight molecules) was lyophilized for 24 hours. 2 mg of the lyophilized, purified
supernatant were dissolved in 25
l final sample buffer (Laemmli 1970). SDS-PAGE gel
electrophoresis was done according to Laemmli (1970) (modified).
The protein fractions were excised from the gel and cut in small pieces (approximately
1 mm2). A piece of empty gel was also excised and used as a blank. The gel pieces were
placed in Eppendorf tubes. The pieces were then washed for 5 minutes with water and with
0.1 M NH4HCO3/acetonitrile 1:1 solution three times for 5 minutes. The solution was
discarded and pieces were washed with acetonitrile three times for 5 minutes until they
became white. The acetonitrile was discarded and the pieces were incubated for 45 min at 56
ºC in 10 mM dithiotreitol (DTT) solution in 0,1 M NH4HCO3. DTT solution was discarded,
and freshly prepared 55 mM solution iodacetamide (IAA) IN 0.1 M NH4HCO3 was added to
the gel pieces for 30 minutes. After the IAA treatment, gel pieces were washed again with 0.1
36
M NH4HCO3/acetonitrile 1:1 solution three times for 5 minutes and then with acetonitrile also
three times for five minutes, until they became white.
Trypsin solution (12.5 ng l-1 in 50 mM NH4HCO3) was added to the gel pieces and
incubated in ice for 30 minutes. The trypsin solution that did not get into the gel was
discarded, and the gel pieces were covered with 30 l of 50 mM NH4HCO3 solution and
incubated overnight at 37 ºC. The next day, the gel pieces were spinned and sonicated to
extract the peptides. The aliquots extracted from the gels were purified using ZipTip pipette
tips. The peptides were analyzed by LC-ESI-Q-TOF mass spectrometry (4.2.10).
4.2.8 Soil extractions and cultivation
For the soil extraction experiments, extraction buffer was mixed with soil in a 1:10
ratio (1.0 g of soil, 9.0 ml of extraction buffer) in falcon tubes. Two independent experiments
consisting of 3 different extractions were performed. Each extraction was done in triplicate
(biological replicates).
In the first experiment, the 3 extractions were (1) extraction with PBS amended with
purified supernatant, (2) extraction with PBS amended with M. luteus cell walls (obtained
from 4.2.3), and (3) extraction with PBS alone (control).
1. Extraction with purified M. luteus supernatant: 1.67 ml of the supernatant after
purification (4.2.6) was added to 3 falcon tubes (three biological replicates, 1 g
of soil each) together with 7.33 ml of extraction buffer (PBS). The
concentration of the high molecular weight fraction of this extraction buffer
was slightly lower than the concentration in the original M. luteus culture
supernatant due to the filtration process.
2. Extraction with M. luteus cell wall: prepared M. luteus cell wall (4.2.3) was
dissolved in PBS (cell wall concentration: 100
g ml-1). 1.67 ml of LMM
without lithium lactate and 7.33 ml of PBS with dissolved cell wall were added
to the falcon tube (1 g of soil).
3. Control extraction: 1.67 ml LMM without lithium lactate, and 7.33 ml PBS.
LMM without lithium lactate was included in the cell wall-amended extraction (2) and
in the control (3) to discard any effect of the salts in the bacteria resuscitation.
37
In a second extraction experiment, the effect of the boiled supernatant was tested.
After purification of the supernatant (4.2.6), half of the supernatant volume was boiled for 10
min. The same ratio of soil to extraction buffer was used:
1. Extraction with purified M. luteus supernatant: extraction with 2.91 ml of
purified supernatant, 6.09 ml of PBS.
2. Extraction with boiled, purified M. luteus supernatant: 2.91 ml boiled, purified
supernatant, and 6.09 ml of PBS.
3. Control extraction: 9 ml PBS.
In all the cases, the extraction proceeded at 120 rpm and 28°C. Three extraction times
were evaluated: 4, 24 and 48 hours in the first experiment, 2, 24 and 48 hours in the second.
The dilutions used for the total viable counts were 103, 104, and 105 in physiological solution.
There were no technical replicates for each dilution. Total viable counts of the 3 biological
replicates of each extraction (for both experiments three times) were done between 24-48
hours of cultivation on modified R2 agar medium.
4.2.9 Diversity screening
The diversity screening was performed only for the first extraction experiment
described in the previous section (extraction purified supernatant, cell wall and control). All
the colonies which grew at the highest dilution plates (that is the 105 dilution, 3 plates after 4
hours, 3 plates after 24 hours, and 3 plates after 48 hours, 9 plates in total) for each treatment
were recollected and inoculated in 96-well microtiter plates in modified R2 broth. For the
purified supernatant treatment, only colonies in 8 out of the 9 plates were collected, since
recollection of single colonies in the last plate was not possible. In total, 763 colonies from
the 105 dilution plates were collected in microtiter plates, which were cultivated at 28 °C and
120 rpm.
All the bacteria were further re-inoculated on modified R2 agar. The analysis of the
763 isolates was performed by Autoflex speed MALDI-TOF mass spectrometer (Bruker
Daltonik GmbH, Germany). Bacterial samples were prepared from the fresh R2 agar cultures
using direct transfer procedure and mixed with HCCA matrix, both according to the
manufacturer's instructions (Bruker Daltonik GmbH, Germany). Spectra were measured
automatically by the Real Time Classification software (Bruker Daltonik GmbH, Germany).
To identify the microorganisms the spectra were processed and analyzed in Biotyper software
38
version 2.0 (Bruker Daltonik GmbH, Germany) by the MALDI Biotyper standard
preprocessing method and MALDI Biotyper standard identification method.
Furthermore, to determine richness of the isolated microorganisms in different
treatments, cluster analysis of the raw spectra was carried out. Spectral processing and
clustering were done in R project following an in-house R script with the use of MALDIquant
and MALDIforeign packages (Gibb and Strimmer 2012). In short, the procedure was: (1) m/z
values were trimmed to the range of 3000 – 15000 Da; (2) intensities were square root
transformed and (3) smoothed by Savitzky-Golay method; (4) baseline correction was done
by SNIP algorithm; and (4) finally data were normalized by the Total Ion Current method.
Peaks in all spectra were detected and transformed into a sample-feature matrix. The Pearson
product-moment correlation coefficient was then computed for all pairs of samples. The
distance matrix was constructed by subtracting absolute values of correlation coefficients
from 1. Hierarchal clustering with average linkage was conducted and clusters or Operation
Taxonomic Units (OTU) were created by applying cut-off distance of 0.2. If two replicates of
the same sample resulted in different clusters, the OTUs were merged together.
4.2.10 LC-ESI-Q-TOF mass spectrometry analyses
Mass spectrometry measurements were carried out using UHPLC Dionex
Ultimate3000 RSLC nano (Dionex, Germany) connected with mass spectrometer ESI-Q-TOF
Maxis Impact (Bruker, Germany). Samples were dissolved in a 10 μl mixture of water :
acetonitrile : formic acid (97:3:0.1%), and 3 μl were loaded on a trap column Acclaim
PepMap 100 C18 (100 μm x 2 cm, particle size 5 μm, Dionex, Germany) with mobile phase A
(0.1% formic acid in water) and flow rate of 5 μL/min for 5 min. The peptides were eluted
from the trap column to the analytical column Acclaim PepMap RSLC C18 (75 μm x 150
mm, particle size 2 μm) by mobile phase B (0.1% formic acid in acetonitrile) using the
following gradient: 0-5 min 3 % B, 5-35 min 5-35 % B, 37 min 90 % B, 37-50 90 %B, 51 min
3 % B, 51-60 min 3 % B. The flow rate during gradient separation was set to 0.3 μL/min.
Peptides were eluted directly to the ESI source – Captive spray (Bruker Daltonics, Germany).
Measurements were carried out in positive ion mode with precursor ion selection in the range
of 400–1400 m/z; up to ten precursor ions were selected for fragmentation from each MS
spectrum. Peak lists were extracted from raw data by Data Analysis version 4.1 (Bruker
Daltonics, Germany). Proteins were identified using Mascot version 2.4.1 (Matrix Science,
UK).
39
40
Chapter 5.0. Results
5.1 Micrococcus luteus growth curve
The growth of M. luteus in LMM medium was monitored for 250 hours. Growth was
measured by the increase in absorbance at 600 nm.
Figure 1. M. luteus growth curve.
The beginning of the stationary phase corresponds to an absorbance value of 11.40
(95.5 hours). The longer the M. luteus colonies stayed in PCA plates (before cultivation in
LMM), the longer was the lag phase observed in the liquid culture.
5.2 Micrococcus luteus supernatant activity to lyse peptidoglycan
The ability of the M. luteus culture supernatant to lyse peptidoglycan was determined
by the absorbance (at 600 nm) change of a cell wall suspension in LMM in the presence of the
bacterial supernatant (4.2.4). The supernatant was collected at the beginning of the stationary
phase (figure 1). A decrease in the measured absorbance is caused by the lysis of cell wall
fragments in the suspension (Telkov et al 2006). As a negative control, boiled supernatant was
used.
41
A)
B)
Figure 2. Absorbance change for the ratios A) 1:20 and B) 1:5 (supernatant : cell wall suspension). Absorbances
were measured in triplicates. Means (points) and standard deviations (bars) are shown.
All the different ratios (4.2.4), exhibited an increase in absorbance over time (figure
2). This can be due to the sedimentation of the cell walls (section 6.1). Nevertheless, the
higher the proportion of supernatant in the mixtures was, the lower were the absorbance
values for the unboiled supernatant compared to the boiled supernatant (plot B, figure 2). The
absorbance of the sole boiled and unboiled supernatants did not change in time. For both
supernatants the mean absorbance value for the 61 measurements in time and for each
triplicate was 0.094 (SD=0.001) for the unboiled supernatant and 0.095 (SD= 0.010) for the
boiled one.
Significant differences in the mean between the absorbances of the boiled and the
unboiled supernatant/target mixtures were tested for each ratio and both targets, either M.
luteus cell wall suspension or autoclaved E. coli suspension (for absorbance values after 120
minutes). Results are displayed in table 1.
Significant differences between the mixtures with boiled and unboiled supernatant
started to be observable for increased supernatant contents. Causes for non-significance and
drawbacks from this experiment will be further analyzed in the Discussion chapter 6.1.
Table 1. Results of the t-tests for independent samples, testing the difference in absorbance (600 nm) for the
targets mixed either with boiled or unboiled supernatant after 120 minutes of reaction (figure 2).
Target
Ratio
(Supernatant:Target)
1:20
1:15
Autoclaved
E. coli
suspension
1:10
1:5
1:2
1:20
1:15
1:10
Cell wall
suspension
1:5
1:2
3:1
4:1
5:1
t-test for
independent
samples
t-value = 0.9705
p-value=0.386756
t-value = -0.7372
p-value=0.501899
t-value = -1.9385
p-value=0.12459
t-value = -2.8235
p-value=0.04765
t-value = -3.0632
p-value=0.037542
t-value = -0.6256
p-value=0.565507
t-value = -0.1928
p-value=0.85647
t-value = -1.0067
p-value=0.37103
t-value = -3.4213
p-value=0.026748
t-value = -1.7801
p-value=0.149658
t-value = -13.795
p-value=0.00016
t-value = -1.7693
p-value=0.15156
t-value = -4.6663
p-value=0.009544
43
Mean differences for the targets
with boiled/unboiled supernatant
at p<0.05
No significant difference observed
No significant difference observed
No significant difference observed
Unboiled supernatant has a
significantly lower absorbance
Unboiled supernatant has a
significantly lower absorbance
No significant difference observed
No significant difference observed
No significant difference observed
Unboiled supernatant has a
significantly lower absorbance
No significant difference observed
Unboiled supernatant has a
significantly lower absorbance
No significant difference observed
Unboiled supernatant has a
significantly lower absorbance
5.3 Micrococcus luteus supernatant activity against fluorogenic substrate
The lytic activity of M. luteus supernatant was also determined by its ability to lyse
fluorogenic glycanase substrate MUF tri-NAG (figure 3). In peptidoglycan, Rpf cleaves the glycosidic bond between N-acetylglucosamine and N-acetyl-muramic acid (Mukamolova et al
2006). In MUF tri-NAG, Rpf cleaves the -glycosidic bond between triacetylchitotriose and
4-methylumbelliferone (figure 3), which emits fluorescence when excited (Chen 1968). The
excitation wavelength used in this experiment was 355 nm. Supernatant lytic activity was
determined by an increase in fluorescence in time due to the increase of coumarin
concentration in the medium (4-methylumbelliferone emission wavelength = 460nm).
Figure 3. Molecule of 4-methylumbelliferyl- -D-N,N’,N’’-triacetylchitotrioside (MUF tri-NAG). In this
state, the fluorescence of the molecule is quenched. Rpf cleaves the molecule, releasing the coumarin portion (4methylumbelliferone, top-right), which emits fluorescence when excited (maximum absorbance wavelength in
0.01 M NaOH is 360 nm (Chen 1968)).
For the first activity determination by fluorescence, the supernatant was retrieved
when M. luteus culture absorbance was 11.38 (early stationary phase, figure 1). The result of
this experiment is depicted in figure 4. During the first 50 minutes of the experiment,
background fluorescence (i.e., fluorescence of the supernatant without MUF tri-NAG
substrate) dropped exponentially (data not shown). Since the background fluorescence was
substracted from the recorded fluorescence (where the MUF tri-NAG lysis reaction took
place), the effect of the exponential drop in the relative fluorescence can be seen in the first 50
minutes of the experiment (figure 4). The background fluorescence was in some cases higher
than the recorded fluorescence, explaining the existance of negative relative fluorescence
values. The background fluorescence stabilized after 50 minutes, following a linear trend.
Figure 4. Fluorescence increases for MUF tri-NAG solution in the prescence of supernatant (●); boiled
supernatant (●); chicken white egg lysozyme in LMM (positive control) (●). Each measurement was done in
triplicates at 28 ºC. Points are the mean values and bars are standard deviations. Background fluorescence (from
the supernatants and the lysozyme in LMM without fluorogenic substrate) was substracted from the recorded
fluorescence.
M. luteus supernatant is likely a very complex mixture, so it is difficult to ascertain the cause
of this exponential drop in fluorescence, which masks the lytic activity in the first 50 minutes
of the experiment. It must be noted that only few minutes after the supernatant retrieval
(4.2.2), a color change in the sterile supernatant could be observed.
The relative fluorescence trend after 50 minutes was analyzed with a simple linear
regression to determine if there was a functional dependancy of the increase of relative
fluorescence in time (H0: =0, no linear dependence of relative fluorescence on time; HA:
1≠1,
linear dependence of relative fluorescence on time) for each curve (figure 4). The linear
regression (dependence of relative fluorescence on the elapsed time) is highly significant at
= 5% for the supernatant (Y = -0.3457 (±0.2468) + 0.01735 (±0.0014) X; r2 = 0.8836; F1,19 =
144.2; p < 2.56 × 10-10) and the chicken egg white lysozyme (Y = 4.1511 (±0.451) + 0.1353
(±0.0026) X; r2 = 0.993; F1,19 = 2627; p < 2.2 × 10-16); for the boiled supernatant, it is not
significant (Y = 3.6830 (±1.0467) - 0.00244 (±0.00613) X; r2 = 0.0083; F1,19 = 0.1591; p=
0.6944).
In order to determine the bacterial growth stage at which the supernatant had
maximum lytic activity, M. luteus supernatant was retrieved at different times during its
45
growth: 18, 41, 64, 71, 88, 95, and 256 hours after inoculation in LMM (200 ml, figure 5).
Since a linear model could be approximated to the supernatant fluorescence change over time
after 50-minute reaction (r2 = 0.8836), the growth stage with maximum lytic activity was that
with the highest increase of relative fluorescence per hour (highest slope). In this experiment,
the fluorescence changes for the supernatant at different growth stages were measured from
50 minutes (initial time) to 240 minutes.
Figure 5. Relative fluorescence per hour (●) at different growth stages (●) for the supernatant. Means
and standard deviations are shown.
Supernatant activity against MUF tri-NAG was higher during the exponential growth,
and diminished starting the stationary phase. At late stages of the stationary phase (256 hours,
absorbance = 11.375), the supernatant had no remarkable activity. The supernatant activity
after purification (4.2.6) was also determined by fluorimetry. After ultrafiltration using both
the 10 and 3 kDa filters, the lytic activity of the retentates (high molecular weight fractions)
and of the filtrates was determined. The results of both filtrations are shown in figure 6.
It can be observed in figure 6 A) that the boiling impaired the increase in fluorescence
(supernatant lytic activity). Both the filtrate (<10 kDa) and the retentate (>10 kDa) fractions
exhibited an increase in relative fluorescence in time. In the case of the 3 kDa filtration (figure
6 B), the retentate had activity, but the filtrate’s activity was reduced as compared with the
activity of the filtrate obtained by the filtration with 10 kDa (figures 6 and 7). It must be noted
that a fluorescence decay is reported for the coumarin 4-methylumbeliferone (figure 3) (Chen
1968). This fluorescence decay can be observed in figure 7 B). In this figure, production of 4methylumbelliferone is stopped in the 3 kDa filtrate (figure 7 B), while in the 10 kDa filtrate
(figure 7 A) the fluorescence keeps increasing for the measured reaction time.
46
A)
B)
Figure 6. Fluorescence increase for retentate (●);boiled retentate (●);and filtrate (●). A) Filtration with 10 kDa
filter; B) filtration with 3 kDa filter. Measurements done in triplicates (means and standard deviations shown).
The determination of the lytic capacities of the retentate and filtrate is a necessary step
for the extraction experiments (4.2.8). The presence of lithium lactate or the salts contained in
the LMM (still likely present in the supernatant) can contribute to an increase in the number
of CFUs observed on solid media, masking the effect of the growth factors secreted by M.
luteus (like Rpf). Since the filtration with the 10 kDa filter still exhibited activity in its filtrate
fraction, the 3 kDa filter was chosen for the supernatant purification to: (1) get rid of the
excess lithium lactate and other low-molecular-weight compounds that could favor bacterial
growth (and maybe resuscitation); and (2) to concentrate the growth factors in the retentate.
B)
5
Relative fluorescnce
unit
Relative
fluorescence unit
A)
4
3
2
1
0
0
100
200
Time (minutes)
300
6
4
2
0
-2
-4
0
100
200
Time (minutes)
300
Figure 7. Fluorescence increases for filtrates (♦) after 50 minutes of reaction. A) Filtration with 10 kDa filter; B)
filtration with 3 kDa filter.
47
5.4 Rpf detection
The Rpf identification procedure (4.2.7) was carried out when M. luteus was in
exponential phase (absorbance
8). Telkov et al (2006) reported that the size of secreted Rpf
is 19 kDa. In the SDS-PAGE gel, two bands were observed around this size (figure 8, white
arrow). The bands were excised from the gel (4.2.7) and its protein content was analyzed by
LC-ESI-Q-TOF-MS (4.2.10). By the time of the publication of this work, the determination of
the identity of the bands was still ongoing.
Figure 8. SDS-PAGE gel of the lyophilized M. luteus supernatant.
5.5 Soil extractions and cultivation: quantitative analyses
The first extraction experiment consisted of the extraction of soil bacteria from soil
using three different buffers: 1) PBS and purified M. luteus supernatant (4.2.6), the “SRpfenhanced extraction”; 2) PBS and M. luteus cell wall suspension (4.2.3, 100 g ml-1), the “cell
wall-enhanced extraction”; and 3) PBS alone, as a control. LMM without lithium lactate was
added to both the cell wall-enhanced extraction and the control extraction in the same volume
as the volume of purified supernatant added to the supernatant-enhanced extraction; in this
way, any effect of the LMM salts in the experiment was excluded. The extractions proceeded
for 4, 24 and 48 hours at 120 rpm shaking and 28 °C. Plating on modified R2 agar at different
dilutions (103, 104, and 105) was performed. CFUs were counted in the 103 and 104 dilutions.
Figure 10 shows the CFUs observed on R2 modified agar for the different extractions,
extraction times, and dilutions. In the 105 dilution plates it can be seen that the increase in
48
CFUs due to the presence of SRpf was ca. one order of magnitude (figure 10 B). This increase
can also be seen after 48 hours of extraction (figure 10 C). After 4 hours of extraction (figure
10 A), no relevant difference was observed between the control and the two enhanced
extractions. In the case of the cell wall-enhanced extraction after 24 and 48 hours (figure 10 B
and C, respectively), the CFU number increased in the dilution 104, but sharply decreased in
the 105 dilution (even slightly lower than the PBS extraction). In figure 10 B) in the 104
dilution, only one of the 3 plates was counted (single point of the cell wall-enhanced
extraction), because the other two were already overgrown.
The second extraction experiment, where the effect of the boiled supernatant was
tested, yielded a similar increase in the number of observed CFUs for the SRpf-enhanced
compared to the control extraction. In this experiment, the extraction times were 2, 24 and 28
hours. The extraction attempt with the boiled supernatant suggests that boiling reduces the
efficiency of the extraction. For the 105 dilution and after 24 hours of extraction, the SRpfenhanced extraction plates were already overgrown (+200 CFU), whereas for the boiled
supernatant extraction the CFU number was 131 (±64.58). After 48 hours of extraction and
for the same dilution, the supernatant-enhanced extraction yielded on average 163.67 CFU
(±24.94), while the boiled supernatant extraction yielded on average 138 CFU (±37.27). In
order to prove the differences to be statistically significant, the experiment will have to be
repeated with more dilutions.
Figure 9. Modified R2 agar plates for the SRpf-enhanced extraction (top three) and the PBS (control) extraction.
The plates correspond to the 105 dilution and 24 hours extraction.
49
A)
B)
-
C)
Figure 10. CFUs observed on modified
R2 agar after 4 (A), 24 (B) and 48 (C)
hours
of
extraction
for
the
PBS
extraction (Ctrl), cell wall-enhanced
extraction (Pg) and SRpf-enhanced
extraction (Sup). Horizontal line at CFU
= 200 expresses overgrowth.
5.6 Soil extractions and cultivation: qualitative analyses (microbial diversity)
The CFUs from the 105 dilution from all the extraction times were inoculated for
storage at 28 °C in microtiter plates containing R2 modified broth. For the control extraction
and the cell wall-enhanced extraction, colonies were selected from all the 9 plates for the 105
dilution (three for each extraction time, three for each extraction, control or cell wallenhanced). For the supernatant-enhanced extraction only colonies from 8 plates were isolated
50
due to overgrowth in one plate. In total, 763 isolates (161 colonies from the cell wallenhanced extraction, 127 from the control, and 475 from the SRpf-enhanced extraction) were
retrieved from the 26 plates. The isolates were then reinoculated on R2 modified agar to
screen for their diversity using Autoflex speed MALDI-TOF mass spectrometry (4.2.9, each
isolate analyzed in duplicate).
From the 763 isolates, after the spectral processing and clustering (4.2.9), 241
operational taxonomical units (OTUs) were determined. The supernatant-enhanced extraction
not only did increase the total number of CFUs observed, but also the richness in terms of
different OTUs. The number of OTUs isolated with the different extraction approaches is
shown in figure 11 (for more comprehensive results refer to the Appendix, OTU table). A
total of 119 OTUs from the 241 identified OTUs across the three extractions were observed
only in the SRpf-enhanced extraction; that is almost half of all the observed OTUs (49.38%).
Diversity indices (Simpson’s index, Shannon’s entropy, total number of OTUs, and Chao1
estimator) (Chao 1984, Hill 1973) were obtained for each of the extraction methods (table 2).
Figure 11. OTUs observed for the 3 different extractions: cell wall- and supernatant-enhanced extraction, and
PBS extraction (control).
Table 2. Diversity indexes for the different extraction approaches
SRpf-enhanced
Cell wall-enhanced
Control
0.9655
0.9710
0.9511
Shannon’s entropy
4.2048
4.0208
3.6158
Total OTU number
161
88
64
Chao1 Estimator
255
161
103
Simpson’s index of
diversity
51
All the treatments shared some common OTUs which clustered to taxa with the
highest number of CFUs observed. In the SRpf-enhanced extraction, the CFU number of these
common OTUs increased. The bacterial community observed in the SRpf-enhanced extraction
was highly uneven, with some of the SRpf-exclusive OTUs having only one CFU, thus being
“rare”. The richness increase observed for the SRpf-enhanced extraction can also be observed
OTU Richness
in figure 12.
Figure 12. Rarefaction curves for the cell wall-enhanced (Pg), SRpf-enhanced (Sup) and PBS (ctrl)
extractions.
52
Chapter 6.0. Discussion
6.1 Supernatant activity
As reported by Telkov et al (2006) and Mukamolova et al (2006), M. luteus
supernatant has lytic activity towards its cell wall suspension and the substrate MUF tri-NAG.
The lytic activity against the cell wall suspension and the autoclaved E. coli suspension is
more apparent when the supernatant fraction increases (table 1). Absorbance (600 nm) is
significantly lower for the supernatant/cell wall or E. coli suspension mixtures with higher
supernatant content, as compared against the mixtures with boiled supernatant (table 1).
Boiling does not change supernatant’s absorbance, but it seems to impair its lytic properties
(figure 2, table 1). The enzyme in the supernatant responsible for its lytic activity, most likely
Rpf (Mukamolova et al 2006, Telkov et al 2006), is denaturated by the supernatant boiling.
The increase in absorbance observed in figure 2 is a trend that is present in all the
ratios; it is relatively faster in the beginning, and then it tends to stabilize at the end of the
measurements. This increase can be the result of the cell wall sedimentation. The cell wall
suspension sedimentedin the wells of the microtiter plate several hours after the beginning of
the experiments (4.2.4). The fact that the cell wall preparation (4.2.3) resulted in a suspension
rather than a solution decreases the reliability of the experiment and increases variability in
the absorbance (figure 2). This increased variability can be the cause of non-significance
between supernatant and boiled supernatant mixtures observed even at some highly diluted
cell wall concentrations (table 1). Telkov et al (2006) determined the ability of Rpf to lyse M.
luteus cell walls. They observed a decrease in absorbance at 600 nm, but the absorbance of
their control also decreased. They also saw lysis zones in polyacrylamide gels containing cell
walls from M. luteus (Telkov et al 2006). In the case of E. coli, when it expresses the rpf gene,
Rpf accumulates in its periplasm and causes cell lysis (Mukamolova et al 2006). In this work,
a decreased absorbance for the E. coli suspension mixed with supernatant as compared with
the boiled supernatant mixture was also observed (table 1).
To better determine the lytic activity of the supernatant, its ability to lyse MUF triNAG was measured. Fluorescence increased in MUF tri-NAG solutions in the presence of
chicken egg white lysozyme (positive control) and M. luteus supernatant, whereas in the
presence of boiled supernatant it did not (figure 4). The fluorescence increase in the presence
of supernatant was smaller compared to that of the positive control (figure 4), but reported
specific activity of M. luteus Rpf is 50-fold less than that for chicken egg white lysozyme
53
(Mukamolova et al 2006). Boiling seems again to impair the lytic activity of the supernatant
(figures 4 and 6).
Telkov et al (2006) determined the dynamics of Rpf accumulation in M. luteus culture
medium. They observed that Rpf accumulation increases after the onset of the exponential
phase, reaches a maximum at the late exponential phase, and decreases dramatically before
the start of the stationary phase (Telkov et al 2006). Results of this work were similar to those
observed for the supernatant activity at different growth stages of M. luteus (figure 5).
Activity increased markedly at the onset of the exponential phase, and decreased once the
culture entered the stationary phase. Since the observed activity trend along the M. luteus
growth stages (figure 5) matched the reported Rpf accumulation in liquid medium (Telkov et
al 2006), it is very likely that Rpf was responsible for the lytic activity of the supernatant
against MUF tri-NAG.
6.2 Enhanced extraction experiments
The medium chosen for culturing bacteria from the enhanced extraction experiments,
the modified R2 agar, has three important features: (1) it contains a wide variety of
compounds, to allow the growth of a broad range of soil microorganisms; (2) it has a low
nutrient concentration to resemble rather oligotrophic conditions in the environment, thus
enabling the growth of oligotrophs; and (3) it contains sodium pyruvate, whose presence can
reduce the formation of oxidizing agents such as H2O2 (Bogosian et al 2000) and thus enable
also the growth of injured bacteria present in the soil.
Compared to the CFU numbers observed in the extraction with PBS (control), the
SRpf-enhanced extraction increased the number of colonies observed on solid media about
one order of magnitude (figures 9 and 10). This increase must have been promoted by the
presence of a M. luteus growth factor, most likely Rpf (Mukamolova et al 1998), not by any
low molecular metabolites present in the LMM medium after cultivation as the supernatant
was deprived of its low molecular weight fraction, and the LMM salts were included in the
control and the cell-wall-enhanced extraction.
In the case of the cell wall-enhanced extraction, a slight increase of CFU numbers on
media was observed compared with the control extraction. Nevertheless, the cell wallenhanced extraction approach needs to be improved by testing the effect of different cell wall
concentrations on the extraction efficiency. In a Mycobacterium tuberculosis and
54
Mycobacterium smegmatis resuscitation experiments with peptidoglycan fragments from their
cell walls, Nikitushkin et al (2013) determined a peptidoglycan concentration at which the
resuscitation of these bacteria was maximum (0.1
g ml-1). Reducing the concentration 5
times or increasing it 2 times decreased the number of resuscitated cells.
A reduction in the CFU number observed for the boiled supernatant extraction
compared to the SRpf-enhanced extraction indicated that the effectiveness of M. luteus
growth factors to promote cell division, at least for some bacteria, was linked to its enzymatic
activity. This favors the resuscitation model (section 2.3.2) in which M. luteus Rpf hydrolyzes
bacterial cell wall and, in consequence, triggers resuscitation (Pinto et al 2015). Boiling the
supernatant reduced its lytic activity against the cell wall suspensions and the MUF tri-NAG
substrate (sections 5.2 and 5.3), so it can also reduce its ability to hydrolyze the bacterial cell
wall of dormant cells, reducing the number of resuscitated CFUs observed on solid medium
(Telkov et al 2006). Nevertheless, not all bacteria must have benefited from the presence of
supernatant in the extraction approach in the same way. Although the presence of M. luteus
supernatant enhanced the growth of CFU on medium (figures 9 and 10), more research is
needed to correctly ascertain how M. luteus growth factors promote resuscitation and bacterial
growth.
The diversity screening was necessary to prove that the enhanced extractions
improved the efficiency of cultivation in general. Based on the OTUs which were seen only in
the supernatant-enhanced extraction (figure 11), the total OTU numbers for each extraction
and the diversity indices (table 2, figure 12), it is clear that a higher diversity was observed in
the enhanced extractions compared to the control. Importantly, the enhanced extraction
increased the number of different bacteria that appeared in culture.
The Simpson’s diversity index differences among extractions are very small. The
Simpson’s diversity index was higher for the cell wall-enhanced extraction than for the SRpfenhanced extraction. The Simpson index (which is subtracted from 1 to get the Simpson index
of diversity) gives more weight to the more plentiful species (or OTUs) in the samples (Hill
1973). The success of the extraction to bring difficult-to-culture bacteria to culture must be
evaluated in terms of the increased number of “rarities”, rather than the enrichment of
evidently predominant bacteria. In this sense, it is better to consider the Chao1 estimator to
address the differences in the diversity among different extractions (Chao 1984). The SRpfenhanced extraction was thus the extraction with the highest observed diversity (table 2,
figure 12).
55
The increased diversity for the SRpf-enhanced extraction as compared to the cell wallenhanced extraction also evidences an important opportunity area of the former enhanced
extraction: the supernatant can be promoting the growth and detection of “rarities”, but also of
otherwise predominant representatives. Under this scenario, predominant bacterial
representatives could still outcompete slow-growers, and thus thwart an increase in observed
diversity. Higher dilutions could be beneficial to increase the number of “rarities” and to
avoid that copiotrophs overgrow oligotrophs. It is possible that the “rare cells”, most likely
oligotrophs, exist in higher numbers in soil than the fast growers observed, but because of
their slow-growing nature they are easily overgrown by the fast growers (Button et al 1993).
Some OTUs appeared only in the PBS (control) extraction. It is possible that due to
the lytic activity of Rpf, some actively growing bacteria can be lysed in the presence of
supernatant (like E. coli when Rpf accumulates in its periplasm). Though unlikely, this can
include also yet-uncultured representatives. It is also worth to question why bacteria which
only appeared in the PBS extraction did not respond to the M. luteus growth factors or were
inhibited by them. An example is OTU012 (appendix), which appeared several times in the
control extraction, but did not appear either in the supernatant-enhanced or the cell wallenhanced extractions. The identity of all the OTUs found in this work will be subjected to
identity based on the 16S rRNA gene. It is expected that some of these OTUs can result in
new bacterial species, cultured in the laboratory for the first time.
Although this work does not adjudge the lytic effect of supernatant to be a result of its
Rpf content, and by the date of publication of this work its identification was pending
(4.2.10), several hints suggest that this is the case. These hints include the reduced enzymatic
activity of the supernatant after boiling (figures 2, 4 and 6) and the dynamics of supernatant
lytic activity at different growth stages of M. luteus (figure 5). The enhanced extraction was
also caused by a product of the bacterium M. luteus, and not by some medium component
(section 5.5). In the SDS-PAGE gel, two bands were found which corresponded to the
reported size of Rpf (section 5.4, figure 8).
56
Chapter 7.0. Conclusion
Several strategies have been developed in recent years to bring yet-uncultured bacteria
to culture. Notwithstanding the important efforts already made in the field, due to the amount
of uncultured microorganisms, there is still a very long path for researchers to travel. Efforts
are also directed to understand the nature of the VBNC state. The molecular mechanisms
which trigger the entrance into this state are still blurry, and hopefully they will become
clearer in forthcoming years.
Two enhanced approaches for the isolation of difficult-to-culture bacteria were tested
in this work. One of them, the SRpf-enhanced extraction, proved to increase the yield of
cultivation both in number of CFU (figures 9 and 10) and diversity (figures 11 and 12, table
2). A lot of unique OTUs were cultured by enriching the extraction buffer with SRpf (figure
11), and some of these OTUs can be previously-uncultured soil microorganisms. As described
by Telkov et al (2006), and as suggested by the reduced CFU numbers observed for the
extraction with boiled supernatant, Rpf resuscitation potential, at least for some cells, is likely
linked to its enzymatic activity. The diversity increase for the SRpf-enhanced extraction also
suggests that SRpf is useful to increase the culturability of a wide range of different bacteria,
both gram positives and gram negatives.
Dormancy and unculturability may be more intrinsically related than what research
has recognized in recent years. There is a need to address both issues more closely to each
other. Novel media which truly resemble natural environments, as well as high throughput
cultivation techniques have already been developed. In this work, bacteria extracted with the
SRpf-enhanced buffer grew on R2 modified agar. These bacteria which grew on the
laboratory medium may also have been present in the control extraction; although the medium
was suitable for them, they did not grow. The combination of the resuscitation potential of
SRpf, together with novel cultivation media which resemble natural environments and other
cultivation approaches (2.2) can result powerful to culture bacteria which have resisted
cultivation in the laboratory. The VBNC state must be explained as fast as novel laboratory
media have been developed.
If VBNC cells exhibit generalized physiological features, like their thickened cell wall
(Signoretto et al 2000, Zengler et al 2002), then they can also share universal resuscitation
approaches. Knowledge on the molecular background of the VBNC state can also uncover
new strategies to culture bacteria more efficiently. Difficult-to-culture bacteria are like the
57
Briar-Rose, they might already be sleeping in majestic castles. Now the question is, how to
give them the wake-up kiss?
58
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Appendix. OTU Table
69
70
Appendix 1. First 100 OTU (total number 241). NA = not reliable identification. Cell Wall-, Supernatant-enhanced extractions. Control (PBS).
The last 141 OTUs are mainly non-identified (NA) singletons within the Supernatant treatment (not shown).
Pg Ctrl Sup Taxonomy
OTU001 18
24
53 Bacillus pumilus;NA
OTU002 14
16
34 NA;Paenibacillus azoreducens
Arthrobacter pascens;NA;Arthrobacter polychromogenes;Arthrobacter aurescens;Arthrobacter oxydans;
OTU003 10
0
63 Arthrobacter globiformis;Arthrobacter ureafaciens;Arthrobacter nicotinovorans;Pseudomonas syringae
OTU004
6
0
16 NA;Bacillus simplex;Bacillus muralis
OTU005
2
0
38 Arthrobacter polychromogenes;Arthrobacter oxydans;NA;Arthrobacter sulfonivorans
OTU006 14
2
8 NA
OTU007
1
3
12 NA
OTU008
8
7
0 NA
OTU009
2
0
12 Pseudomonas koreensis;Pseudomonas chlororaphis;Pseudomonas corrugate;Pseudomonas thivervalensis
Arthrobacter pascens;Arthrobacter polychromogenes;NA;Arthrobacter oxydans; Arthrobacter aurescens;
OTU010
5
6
10 Arthrobacter globiformis
OTU011
5
4
5 NA
Pseudomonas corrugata;Pseudomonas chlororaphis;Pseudomonas jessenii; Pseudomonas thivervalensis;
OTU012
0
12
0 Pseudomonas koreensis
OTU013
1
0
17 Arthrobacter polychromogenes;Arthrobacter oxydans;Arthrobacter pascens;NA
OTU014
3
1
11 NA
OTU015
0
0
12 NA;Arthrobacter aurescens;Arthrobacter nicotinovorans;Arthrobacter ureafaciens
OTU016
5
0
9 NA
OTU018
5
4
0 Bacillus drentensis;NA
OTU019
0
1
8 Bacillus megaterium
OTU020
5
0
8 NA
OTU022
0
0
9 Arthrobacter globiformis;NA;Arthrobacter oxydans
OTU024
3
0
4 Bacillus niacini;NA
OTU026
3
2
1 NA;Bacillus megaterium
OTU027
0
1
5 Arthrobacter aurescens;NA
OTU029
0
0
6 Bacillus firmus
OTU031
2
2
7 NA
OTU032
OTU033
OTU035
OTU037
OTU038
OTU039
OTU040
OTU042
OTU043
OTU045
OTU046
OTU048
OTU049
OTU050
OTU051
OTU052
OTU054
OTU056
OTU057
OTU058
OTU059
OTU060
OTU061
OTU063
OTU064
OTU065
OTU067
OTU068
Pg Ctrl Sup Taxonomy
1
0
6 NA
0
0
6 NA;Paenibacillus macerans
2
3
0 Bacillus simplex;Bacillus muralis
0
6
0 NA;Arthrobacter pascens
Pseudomonas chlororaphis;Pseudomonas corrugata;Pseudomonas umsongensis;Pseudomonas thivervalensis; Pseudomonas
0
0
5 kilonensis;Pseudomonas jessenii
0
0
5 Pseudomonas umsongensis;Pseudomonas kilonensis
0
0
5 Pseudomonas corrugata;Pseudomonas brassicacearum;Pseudomonas koreensis;Pseudomonas chlororaphis
3
1
0 Bacillus marisflavi
0
4
0 NA;Cupriavidus campinensis
1
1
2 NA
1
3
2 NA
6
1
1 NA
0
4
0 NA
2
0
2 NA;Bacillus indicus
1
0
3 Arthrobacter nicotinovorans;Arthrobacter aurescens;Arthrobacter ureafaciens
1
0
3 NA;Bacillus niacini
Pseudomonas vancouverensis;Pseudomonas corrugata;Pseudomonas jessenii;
4
0
0 Pseudomonas thivervalensis
0
0
4 Arthrobacter polychromogenes;Arthrobacter oxydans;NA
0
0
4 Arthrobacter polychromogenes;Arthrobacter oxydans;NA
0
0
4 NA;Arthrobacter sulfonivorans;Arthrobacter oxydans
0
0
4 NA;Arthrobacter polychromogenes;Arthrobacter oxydans
0
0
4 NA;Arthrobacter oxydans
0
2
3 NA;Arthrobacter polychromogenes;Arthrobacter sulfonivorans
3
0
0 Cupriavidus campinensis
1
2
0 Arthrobacter histidinolovorans;Arthrobacter sulfonivorans;NA
1
0
2 NA
1
0
2 Bacillus indicus;Bacillus idriensis;NA
1
1
1 Arthrobacter polychromogenes;NA
72
OTU069
OTU070
OTU071
OTU072
OTU073
OTU074
OTU075
OTU077
OTU078
OTU079
OTU080
OTU081
OTU082
OTU083
OTU084
OTU085
OTU087
OTU088
OTU090
OTU091
OTU092
OTU093
OTU094
OTU095
OTU096
OTU097
OTU098
OTU100
Pg Ctrl Sup Taxonomy
1
2
0 NA
0
1
2 NA
1
0
2 NA
1
0
4 Arthrobacter sulfonivorans;Arthrobacter oxydans;Arthrobacter pascens
0
0
3 NA
0
0
3 NA;Variovorax paradoxus
0
0
3 NA
0
0
3 NA
1
0
3 Bacillus pumilus
3
2
0 NA
2
0
3 NA
2
0
0 NA
2
0
0 NA
1
1
0 Arthrobacter polychromogenes;Arthrobacter oxydans
1
1
0 Arthrobacter polychromogenes
1
1
0 Arthrobacter polychromogenes;Arthrobacter scleromae
1
1
0 Arthrobacter polychromogenes;Arthrobacter oxydans
1
1
2 Bacillus megaterium;NA
1
1
0 NA
2
0
0 NA
1
1
0 Arthrobacter oxydans;Arthrobacter polychromogenes;NA
1
1
0 NA
0
2
0 NA
0
2
0 NA
1
0
1 NA;Arthrobacter globiformis
0
0
2 Pseudomonas kilonensis
0
0
2 NA
0
0
2 NA
73