Download Production and consumption of nitrous oxide in nitrate

Microbiology (2014), 160, 1749–1759
DOI 10.1099/mic.0.079293-0
Production and consumption of nitrous oxide in
nitrate-ammonifying Wolinella succinogenes cells
Monique Luckmann,1 Daniel Mania,2 Melanie Kern,1 Lars R. Bakken,3
Åsa Frostegård2 and Jörg Simon1
Correspondence
Jörg Simon
[email protected]
1
Microbial Energy Conversion and Biotechnology, Department of Biology, Technische Universität
Darmstadt, Schnittspahnstraße 10, 64287 Darmstadt, Germany
2
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life
Sciences, Chr Falsens vei 1, N1432 Ås, Norway
3
Department of Plant and Environmental Sciences, Norwegian University of Life Sciences, PO Box
5003, N1432 Ås, Norway
Received 19 March 2014
Accepted 27 April 2014
Global warming is moving more and more into the public consciousness. Besides the commonly
mentioned carbon dioxide and methane, nitrous oxide (N2O) is a powerful greenhouse gas in
addition to its contribution to depletion of stratospheric ozone. The increasing concern about N2O
emission has focused interest on underlying microbial energy-converting processes and
organisms harbouring N2O reductase (NosZ), such as denitrifiers and ammonifiers of nitrate and
nitrite. Here, the epsilonproteobacterial model organism Wolinella succinogenes is investigated
with regard to its capacity to produce and consume N2O during growth by anaerobic nitrate
ammonification. This organism synthesizes an unconventional cytochrome c nitrous oxide
reductase (cNosZ), which is encoded by the first gene of an atypical nos gene cluster. However,
W. succinogenes lacks a nitric oxide (NO)-producing nitrite reductase of the NirS- or NirK-type
as well as an NO reductase of the Nor-type. Using a robotized incubation system, the wild-type
strain and suitable mutants of W. succinogenes that either produced or lacked cNosZ were
analysed as to their production of NO, N2O and N2 in both nitrate-sufficient and nitrate-limited
growth medium using formate as electron donor. It was found that cells growing in
nitrate-sufficient medium produced small amounts of N2O, which derived from nitrite and, most
likely, from the presence of NO. Furthermore, cells employing cNosZ were able to reduce N2O to
N2. This reaction, which was fully inhibited by acetylene, was also observed after adding N2O to
the culture headspace. The results indicate that W. succinogenes cells are competent in N2O and
N2 production despite being correctly grouped as respiratory nitrate ammonifiers. N2O production
is assumed to result from NO detoxification and nitrosative stress defence, while N2O serves as a
terminal electron acceptor in anaerobic respiration. The ecological implications of these findings
are discussed.
INTRODUCTION
Nitrous oxide (N2O, also known as laughing gas) is a
chemically rather inert trace gas in Earth’s atmosphere
whose level has been consistently increasing over the last
decades (Smith, 2010), largely driven by high input of
reactive nitrogen in agriculture (Reay et al., 2012). N2O is
involved in stratospheric ozone depletion and, moreover, it
is a strong greenhouse gas. Its capacity to absorb infrared
radiation is about 300 times greater than that of CO2 and
therefore anthropogenic N2O emissions, unless balanced by
Abbreviations: cNosZ, cytochrome c nitrous oxide reductase; Nrf,
cytochrome c nitrite reductase; PMF, proton motive force.
Supplementary material is available with the online version of this paper.
079293 G 2014 The Authors
N2O-consuming processes, contribute significantly to climate change. This is even more important in the light of the
fact that an ever increasing amount of synthetic nitrogenbased fertilizers is used to raise agricultural efficiency.
For a long time N2O has been known as a crucial intermediate in the biogeochemical nitrogen cycle, and the
majority of atmospheric N2O is of microbial origin (for
recent reviews see Richardson et al., 2009 and Thomson
et al., 2012). The most important processes in biological
N2O production and turnover are thought to be denitrification [reduction of nitrate or nitrite to N2 via nitric
oxide (NO) and N2O as intermediates] and nitrification
(oxidation of ammonium or nitrite to nitrate with N2O
being a side product under aerobic conditions in a process
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M. Luckmann and others
referred to as nitrifier denitrification) (Stein, 2011). Notably,
some denitrifying organisms lack N2O reductase activity
and release N2O as an end product. Recently, nitrate- (or
nitrite-) ammonifying cells have also been reported to
release N2O (Stremińska et al., 2012; Giles et al., 2012). In
bacterial denitrification and in denitrifying nitrifiers, NO is
reduced by one of various membrane-bound respiratory NO
reductases (predominantly cNor and qNor enzymes) (Simon
& Klotz, 2013). Since NO is a highly toxic compound that
exerts nitrosative stress on cells and organisms, it needs to be
detoxified (Poole, 2005). It is therefore not surprising that
N2O production from NO has been described for numerous
non-respiratory enzymes, including flavodiiron proteins
(Fdp), flavorubredoxin (NorVW), cytochrome c554 (CycA;
in nitrifiers), cytochrome c9-beta (CytS) and cytochrome c9alpha (CytP) (Simon & Klotz, 2013). In these cases, NO
reduction is thought to serve predominantly in NO
detoxification, i.e. in nitrosative stress defence.
In many bacterial and archaeal prokaryotes, N2O is further
reduced to N2 by a homodimeric N2O reductase (NosZ),
which is a copper-containing enzyme located on the outside
of the cytoplasmic membrane (i.e. the periplasm in Gramnegative bacteria) (Zumft, 1997; Zumft & Kroneck, 2007;
Pomowski et al., 2011; Wüst et al., 2012; Pauleta et al., 2013).
Various NosZ enzymes carry a C-terminal extension that
forms a monohaem cytochrome c domain, which was first
described for non-denitrifying Epsilonproteobacteria (Teraguchi
& Hollocher, 1989; Simon et al., 2004). The cytochrome c
domain of such cNosZ enzymes is thought to donate electrons to the CuA site of NosZ (Dell’Acqua et al., 2011; Simon
& Klotz, 2013). Recently, so-called atypical NosZ proteins
(including cNosZ enzymes) and atypical nos gene clusters
from non-denitrifying bacteria and archaea were found to be
widespread in terrestrial environments, where they form an
evolutionarily distinct clade of N2O reductases (Sanford et al.,
2012; Jones et al., 2013). Based on the abundance of these
genes in soils, it was suggested that the corresponding
organisms could be a significant sink for N2O (Jones et al.,
2013). Furthermore, genome sequences suggest that ammonification and N2 production from nitrate/nitrite may coexist
in some bacteria (Sanford et al., 2012; Mania et al., 2014).
The metabolism of N2O in nitrate- or nitrite-ammonifying
organisms is poorly understood. The respective organisms
reduce nitrate to nitrite using a membrane-bound nitrate
reductase (Nar) and/or a periplasmic nitrate reductase
(Nap) (Richardson et al., 2001; Kern & Simon, 2009; Simon
& Klotz, 2013). Prominent examples are Gamma-, Deltaand Epsilonproteobacteria such as Escherichia coli, Salmonella
enterica serovar Typhimurium, Shewanella oneidensis,
Anaeromyxobacter dehalogenans and Wolinella succinogenes.
Subsequently, nitrite is reduced to ammonium by cytochrome c nitrite reductase (NrfA), which obtains electrons
from the quinone/quinol pool through several different
electron transport redox enzyme systems, depending on the
organism (Simon, 2002; Kern & Simon, 2009; Simon &
Klotz, 2013). Many NrfA-containing ammonifiers that also
reduce N2O encode cNosZ, for example W. succinogenes,
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Campylobacter fetus, Desulfitobacterium dehalogenans and A.
dehalogenans (Payne et al., 1982; Kern & Simon, 2009;
Sanford et al., 2012).
The epsilonproteobacterium W. succinogenes is a versatile
respiratory rumen bacterium that typically uses hydrogen
gas or formate as electron donor combined with a
multitude of electron donor substrates such as nitrate,
nitrite, sulfite, polysulfide and fumarate (Kröger et al.,
2002; Kern & Simon, 2009; Kern et al., 2011a; Simon &
Kroneck, 2013). The cells stoichiometrically produce
ammonium from both nitrate and nitrite and the
corresponding cell yields (normalized on the basis of
formate consumption) were found to be similar using
either of these terminal electron acceptors (Bokranz et al.,
1983). W. succinogenes employs the Nap and Nrf systems
for respiratory nitrate ammonification but lacks NOproducing nitrite reductases of the NirK- (copper nitrite
reductase) or NirS-type (cytochrome cd1 nitrite reductase)
known from denitrifiers as well as a respiratory NO
reductase of the cNor and qNor families (Kern & Simon,
2009). In addition to its function as terminal reductase in
respiratory nitrite ammonification, NrfA was recently
shown to function in nitrosative stress defence, implying
that NrfA efficiently reduces NO to ammonium (Kern &
Simon, 2009; Kern et al., 2011b; Simon & Klotz, 2013).
W. succinogenes cells were previously reported to grow with
N2O as sole electron acceptor using formate as electron
donor although growth parameters have not been reported
(Yoshinari, 1980; Schumacher et al., 1992). The W. succinogenes cNosZ enzyme has been purified from N2O-grown cells
and shown to contain N2O reductase activity using reduced
benzyl viologen as electron donor (Teraguchi & Hollocher,
1989). When the genome of the W. succinogenes type strain
(DSM 1740T) was sequenced, an atypical nos gene cluster was
found whose first gene encodes the cNosZ enzyme (Baar
et al., 2003; Simon et al., 2004). Interestingly, this gene was
found to be disrupted in the genome sequence by a copy of
insertion element IS1302 (Simon & Kröger, 1998), thus
raising the question whether this strain was comparable to
the strains used in previous studies. Here, we report the
construction of mutants that either lack nosZ or contain a
genetically stable nos gene cluster. The capacity of these
mutants and of the wild-type strain to produce and to
consume N2O during growth by nitrate ammonification was
examined using nitrate-sufficient or nitrate-limited medium
containing formate as electron donor. Using a robotized cell
incubation system, the concentrations of nitrate, nitrite, NO,
N2O and N2 were quantified with high sensitivity. The
applied growth conditions for nitrate respiration are
regarded to be more physiologically relevant than previously
conducted growth experiments in the presence of externally
added N2O serving as sole electron acceptor.
METHODS
Organisms and liquid media. W. succinogenes cells used in this
study are described in Fig. 1. Cultures were routinely grown in liquid
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Microbiology 160
Nitrous oxide turnover in W. succinogenes
W. succinogenes wild-type (DSM 1740T )
Pnos
nosZ ′
nssC
EcoRI
nosZ ′
IS1302
BamHI
nosB
BamHI
nosD
nosG C1
C2
nosH
F
Y
L
HindIII
1 kb
W. succinogenes DnosZ
Pnos
nssC
EcoRI
cat
nosB
BamHI
BamHI
nosD
nosG
C1
C2
nosH
F
Y
L
HindIII
W. succinogenes kompPnosnosZ
Pnos
Pnos
nssC
nosZ
kan
nosB
nosD
nosG
C1 C2 nosH
F
Y
L
Fig. 1. Physical maps of nos gene loci in the genome of W. succinogenes cells employed in this study. Black bars indicate DNA
regions used for homologous recombination during mutant construction (see Methods for details). Pnos indicates the position of
the nos promoter. The (putative) function of individual nos gene products was discussed previously by Simon et al. (2004) and
van Spanning (2011). The open reading frame located between nosF and nosY encodes a hypothetical protein of 151 amino
acid residues (WS0925) that has not yet been designated.
medium at 37 uC containing formate as sole electron donor and
either fumarate (Kröger et al., 1994) or nitrate as terminal electron
acceptor. Nitrate-sufficient (80 mM sodium formate/50 mM potassium nitrate) or nitrate-limited (80 mM sodium formate/10 mM
potassium nitrate) medium was used. In addition to formate and
nitrate, each medium (pH 7.5) contained 1 mM K2HPO4, 5 mM
fumaric acid, 5 mM ammonium sulfate, 1 mM MgCl2, 0.2 mM
CaCl2, 0.68 mM glutamic acid, 0.57 mM cysteine and 0.2 ml trace
element solution l21 as described by Pfennig & Trüper (1981). Media
were degassed and flushed with either N2 gas or helium several times
to reduce the oxygen content. Brain heart infusion broth (BHIB;
0.5 %, w/v), kanamycin (25 mg l21) and/or chloramphenicol
(12.5 mg l21) were added to the medium as appropriate.
Growth conditions, determination of cytochrome c and
nitrogen compounds. Standard growth curves were generated by
measuring the OD578 of cultures (50 ml) inoculated with overnight
pre-cultures at an initial OD578 of about 0.05.
Nitrate and nitrite concentrations in cell supernatants were
determined using colorimetric assays based on the formation of
spectrophotometrically detectable compounds. Nitrate was converted
to 4-nitro-2,6-dimethylphenol under acidic conditions (Hartley &
Asai, 1963). For nitrite determination, diazotization with sulfanilic
acid and subsequent coupling with N-(1-naphthyl)ethylenediamine
was performed (Rider & Mellon, 1946).
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Detection of cytochromes c in SDS-polyacrylamide gels was
performed by a haem staining procedure using 3,39-dimethoxybenzidine as described previously (Kern et al., 2011a).
For monitoring gas kinetics 120 ml serum vials (50 ml medium)
equipped with a triangular magnetic stirring bar were used. The vials
were tightly sealed with rubber septa and aluminium caps. Prior to
inoculation, the air was replaced by helium gas (six cycles of
evacuation and helium-filling) using a semi-automated system
(Molstad et al., 2007). During this process, the medium was stirred
at 950 r.p.m. to ensure sufficient gas exchange between the liquid and
the gas phase. Excess pressure was relieved using a water-filled
syringe. The vials were incubated at a constant temperature of 37 uC
in a robotized incubation system designed for measuring gas kinetics
(Molstad et al., 2007; Bergaust et al., 2008). In short, this system
monitors NO, N2O and N2 by frequent sampling with a syringe
through the septum using a peristaltic pump coupled to the inlet of a
gas chromatograph and a chemiluminescence NO analyser. After each
sampling, the pump is reversed and pumps back helium to ensure
near-constant pressure (1 atm) during the entire incubation period.
The sampling thus dilutes the headspace with helium. This dilution
was 3.4 % per sampling in the original system (Molstad et al., 2007),
but only 0.9 % in the new system used for most of the experiments in
the present study. There is also a marginal but constant leakage of
traces of N2 and O2 with each sampling (14 nmol O2 and 42 nmol N2
per sampling). Dilution by sampling and the minor leakage of N2 into
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M. Luckmann and others
the vials are taken into account when estimating gas transformation
rates for each time increment between two samplings (see Molstad
et al., 2007 for details), and when calculating cumulative N2
production (reported N2 is invariably cumulative N2 production,
corrected for leakage and sampling dilution). Several batch cultivation
experiments were run with a variety of treatments designed to
characterize the gas kinetics in the various cultures. Replication was
secured by repeating entire experiments, rather than including
replicate vials in each run.
Construction of W. succinogenes mutants. Standard genetic
procedures were used (Sambrook et al., 1989). Genomic DNA was
isolated from W. succinogenes using the DNeasy Tissue kit (Qiagen).
PCR was carried out using Phusion High Fidelity DNA polymerase
(Finnzymes) for cloning procedures or Biotaq Red DNA polymerase
(Bioline) for mutant and plasmid screening with standard amplification protocols.
W. succinogenes DnosZ was constructed through double homologous
recombination of the wild-type (strain DSM 1740T) genome with a
deletion plasmid (pDnosZ cat) designed to replace the entire nosZ
gene in the nos operon by a chloramphenicol resistance gene cartridge
(cat). For homologous recombination, the respective deletion plasmid
contained cat flanked by two DNA segments obtained by PCR that
were identical to appropriate regions in the W. succinogenes genome
(Fig. 1). The two PCR fragments were synthesized using the following
primer pairs: 59-GCGAATTCCGCAGACGGATTTGAGG-39 and 59CCGGATCCTTACCCACTCCTTTTTGTTAATTTGG-39 for amplifying the upstream fragment, and 59-CCGGATCCAACCCTCTCCTCTCC-39 and 59-CCAAGCTTGGGATACATCCCGATGTAGTGATTG39 for the downstream fragment (black bars in Fig. 1). Primers carried
EcoRI, BamHI or HindIII restriction sites (underlined) for cloning.
Both fragments and the cat fragment were consecutively inserted
into plasmid pPR-IBA1 (IBA Lifesciences) using E. coli XL-1 Blue as
cloning host, grown in LB medium at 37 uC. PCR analysis was used to
confirm that the plasmid contained cat in the same orientation as
its surrounding regions. Transformation of W. succinogenes DSM
1740T cells was performed by electroporation as described previously
(Simon et al., 1998, 2000). Transformants of W. succinogenes were
selected in the presence of chloramphenicol (12.5 mg l21) and the
desired gene deletion was confirmed by PCR. The integrity of DNA
stretches involved in recombination events was confirmed by
sequencing suitable PCR products.
To construct W. succinogenes kompPnosnosZ, the nosZ gene lacking
IS1302 was restored on the genome of W. succinogenes DnosZ upon
integration of plasmid pkompPnosnosZ kan (Fig. 1) This plasmid
contained an intact copy of nosZ surrounded by the upstream and
downstream cat-flanking fragments of pDnosZ cat. In addition, a
kanamycin resistance gene cassette (kan), obtained by BamHI excision from pUC4K, was inserted between nosZ and the downstream
flanking fragment. Finally, a nos promoter element was inserted
downstream of kan to ensure transcription of the residual nos operon
(Fig. 1). In a first step to create pkompPnosnosZ kan, the cat gene was
replaced by kan in pDnosZ cat, resulting in pDnosZ kan. PCR analysis
was used to confirm that the plasmid contained kan in the reverse
orientation relative to the nos sequence. The intact nosZ gene was
amplified using the primer pair 5-ATGCAAAGATTACTAAAGCAATCTTTGGTTG-39 and 59-TTAGAAGAGACCCGCGTTCTCATC-39
using genomic DNA of wild-type cells as template (note that a small
percentage of the cells transiently lack the copy of IS1302 within
nosZ). To avoid any adverse effects of adding additional restriction
sites between the native nos promoter and the nosZ gene, the
amplified gene was blunt-end ligated with a linear plasmid fragment
obtained by PCR from pDnosZ kan with the primer pair 59-GGGGATCCCGCTGAGGTCTGCCTCGTGAAGAAGGTG-39 and 59-TTACCCACTCCTTTTTGTTAATTTGGCATTAAC-39, resulting in pkompnosZ
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kan. In the last step, the additional nos promoter fragment (Pnos) was
amplified using the primer pair 59-GGGCGTGGGGAAGAAAATCTCC-39 and 59-TTACCCACTCCTTTTTGTTAATTTGGCATTAAC39 and blunt-end ligated with a linear plasmid fragment obtained by
PCR from pkompnosZ kan with the primer pair 5-GGGGATCCAAAGCCACGTTGTGTCTCAAAATCTCTG-39 and 59-ATGGAAAACAGAGGAATCGCCAAAT-39, to give pkompPnosnosZ kan. The corresponding mutant, W. succinogenes kompPnosnosZ kan, was constructed
by transforming W. succinogenes DnosZ cat with pkompPnosnosZ kan.
Transformants were selected in the presence of kanamycin (25 mg l21).
The desired double homologous recombination was verified by PCR
and the integrity of DNA stretches involved in recombination events as
well as the entire nosZ gene was confirmed by sequencing suitable PCR
products.
RESULTS
Construction of W. succinogenes mutants and
characterization of cultures
As mentioned in the Introduction, the cNosZ-encoding
gene from W. succinogenes wild-type cells (strain DSM
1740T) was found to be disrupted by a copy of insertion
element IS1302. Therefore, it was assumed that the vast
majority of these cells were unable to produce functional
cNosZ. To overcome the problem of genetic variability,
mutants W. succinogenes DnosZ and W. succinogenes
kompPnosnosZ were constructed through double homologous recombination using suitable plasmids as described in
Methods (Fig. 1). The DnosZ deletion mutant lacks the
entire nosZ gene, which is replaced by a chloramphenicol
resistance gene cartridge (cat), while mutant kompPnosnosZ
possesses an intact nosZ gene, a kanamycin resistance gene
cartridge (kan) and an additional nos promoter to ensure
transcription of accessory nos genes (Fig. 1). The absence of
IS1302 in the nosZ gene of mutant kompPnosnosZ was
routinely examined by PCR and confirmed that the mutant
was genetically stable. The presence of the cNosZ protein
was shown in cells of mutant kompPnosnosZ by haem
staining, whereas it proved undetectable in cells of the
wild-type and of mutant W. succinogenes DnosZ (Fig. 2).
All three W. succinogenes organisms were grown in anoxic
batch cultures with formate and nitrate as energy sources in
either formate-limited/nitrate-sufficient (80 mM formate/
50 mM nitrate) or formate-sufficient/nitrate-limited (80 mM
formate/10 mM nitrate) medium, each supplemented with
0.5 % (w/v) BHIB. In both media, the cells showed comparable growth behaviour (Fig. 3). The stationary growth
phase was reached after 7–8 h in nitrate-sufficient and 5–6 h
in nitrate-limited medium and similar doubling times of
about 1.6 h were determined. However, in nitrate-sufficient
cultures large amounts of nitrite (30 to 40 mM) accumulated (Fig. 3a), whereas in nitrate-limited medium nitrite
was only transiently produced at a maximal concentration of
about 5 mM (Fig. 3b). Under the latter conditions,
accumulating nitrite is assumed to be rapidly converted to
ammonium by cytochrome c nitrite reductase, NrfA, as
described previously (Bokranz et al., 1983, Lorenzen et al.,
1993; Simon 2002). Production of the gaseous nitrogen
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Nitrous oxide turnover in W. succinogenes
DnosZ
Wild-type
NS
NL
M
M
kompPnosnosZ
NL
NS
M
kDa
kDa
100
100
70
70
NL
NS
cNosZ
(95 kDa)
NrfA
(58 kDa)
55
55
40
40
35
35
25
25
Fig. 2. Detection of cNosZ and NrfA in different W. succinogenes cells grown in either formate-limited/nitrate-sufficient
(80 mM formate/50 mM nitrate, designated NS) or formate-sufficient/nitrate-limited (80 mM formate/10 mM nitrate,
designated NL) medium (see text for details). Cells were harvested in the late exponential growth phase and whole-cell
extracts were subjected to denaturing SDS-PAGE (100 mg protein was applied to each lane). Subsequently, cytochrome c was
detected by haem staining. Arrows indicate cNosZ and NrfA proteins (Kern et al., 2010). M, size marker proteins.
OD578
50
0.1
0
4
6
8
1
1
50
0
40
30
30
30
20
20
20
10 0.1
10 0.1
10
0
10
0
2
4
6
8
1
4
6
Time (h)
8
0
10
10
0
2
4
6
8
1
0
10
10
8
8
8
6
6
6
2
2
50
40
4
0.1
1
40
10
OD578
(b)
2
kompPnosnosZ
ΔnosZ
Wild-type
1
0
10
4
0.1
0
2
2
4
6
Time (h)
8
4
0.1
0
10
0
Nitrate, nitrite (mM)
(a)
after 15 h of incubation), whereas N2 was very close to the
detection limit. The final cumulative N2 production by these
cultures was about 0.3 mmol. This was most likely due to
chemical reactions between nitrite and organic compounds
in the medium, as discussed below. In contrast, the
kompPnosnosZ mutant accumulated N2 but lacked N2O,
indicating the presence of active nitrous oxide reductase.
2
2
4
6
Time (h)
8
Nitrate, nitrite (mM)
compounds NO, N2O and N2 was determined in a series of
experiments with the three strains grown in either nitratesufficient or nitrate-limited medium. Fig. 4 shows a representative result for the three strains (single vial data) grown
in nitrate-sufficient medium. The wild-type and mutant W.
succinogenes DnosZ accumulated low amounts of N2O
during the exponential growth phase (up to 1.7 mmol vial21
0
10
Fig. 3. Growth of W. succinogenes cells and concomitant nitrate and nitrite concentrations in the medium. Cells were grown at
37 6C in sealed 120 ml vials (50 ml medium). (a) Formate-limited, nitrate-sufficient medium (80 mM formate and 50 mM
nitrate). (b) Formate-sufficient, nitrate-limited medium (80 mM formate and 10 mM nitrate). Representative experiments are
shown (performed in triplicate; deviation less than 5 %). m, Optical density; $, nitrate concentration; &, nitrite concentration.
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M. Luckmann and others
5
250 5
250
5
250
4
200 4
200
4
200
3
150 3
150
3
150
2
100 2
100
2
100
1
50
50
1
50
0
0
5
10
Time (h)
0
15
1
0
0
5
10
Time (h)
0
15
0
0
5
10
Time (h)
NO (nmol vial–1)
N2O, N2 (mmol vial–1)
kompPnosnosZ
DnosZ
Wild-type
0
15
Fig. 4. Measured NO, N2O and N2 during growth of three W. succinogenes organisms in nitrate-sufficient medium. N2 is the
cumulative N2 production (corrected for leaks and sampling dilution). Representative experiments are shown (performed in
triplicate; deviation less than 5 %). #, NO (nmol vial”1); g, N2O (mmol vial”1); h, N2 (mmol vial”1).
These differences between the cultures regarding N2O and
N2 accumulation were also observed in two further replicate
experiments (Figs S2 and S3, available in the online
Supplementary Material). Supplementary experiments were
run with 10 % acetylene (an inhibitor of nitrous oxide
reductase) in the headspace, demonstrating that the
reduction of N2O to N2 by mutant kompPnosnosZ was
effectively inhibited by this treatment (Figs S9 and S10). For
all three organisms significant accumulation of NO was
detected, but only during the late exponential and stationary
growth phases (Fig. 4). NO accumulation initiating during
the late exponential/stationary phase was also observed in
several experiments, shown in Figs S9 and S10.
In nitrate-limited medium, the concentrations of NO
invariably remained below 1 nmol vial21and N2O below
0.1 mmol vial21 and the production of N2 was below the
detection limit (~0.5 mmol for cumulative N2 production
over a 15 h incubation period) (not shown). Nonetheless,
the cNosZ and NrfA proteins were detectable in cells of W.
succinogenes kompPnosnosZ (Fig. 2). The very low amounts
of gaseous nitrogen compounds in nitrate-limited medium
suggest that the appearance of both NO and N2O depended
on nitrite accumulation in nitrate-sufficient medium.
Therefore, control experiments were conducted in which
varying amounts of nitrite (10, 20 or 30 mM) were added
to sterile nitrate-sufficient medium (Fig. 5). In addition,
vials with medium lacking BHIB were also analysed. N2O
production could not be detected in any of the treatments.
On the other hand, NO was found to accumulate in the
medium with BHIB at a rate largely proportional to the
nitrite concentration but the rate declined with time [initial
rate of NO formation ~1024 mol NO (mol nitrite)21 h21;
see Fig. S1 for a closer inspection of the kinetics]. In the
medium without BHIB, however, NO production was
marginal (~5 % of that in the presence of BHIB). The
production of N2 was also significant and independent of
BHIB. The rate of N2 production was a linear function of
nitrite concentration [~1024 mol N2 (mol nitrite)21 h21;
see Fig. S1]. These results imply that NO is formed by
chemical reactions between nitrite and BHIB compounds,
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whereas the chemical N2 formation appears to be due to
reactions between nitrite and compounds within the basal
medium. In theory, one could use the observed kinetics of
chemical N2 formation from nitrite to correct the observed
N2 formation in the cultures, thus estimating the enzymic
production of N2. However, this turned out to be problematic since the measured N2 accumulation in the wildtype and in mutant W. succinogenes DnosZ was lower than
that predicted (Fig. 4). In fact, these two organisms accumulated only about 0.3 mmol N2 vial21, which was much
lower than predicted by the observed reaction rate between
nitrite and fresh sterile medium. Assuming that the mean
concentration of nitrite was 35 mM for a time span of 7 h,
the predicted chemical N2 formation would amount to
1.2 mmol N2 vial21. Thus, N2 formation by nitrite reacting
with ‘spent medium’ (i.e. after 7 h of growth) appears to be
much lower than with fresh medium.
The fact that NO concentrations during the first 8–10 h of
the batch cultivations were invariably lower than those
predicted by the chemical decomposition of the accumulating nitrite suggests that W. succinogenes cells are able
to efficiently reduce NO in order to maintain non-toxic
levels. The enzymic inventory putatively serving in the
underlying NO detoxification processes is considered in
the Discussion.
Addition of N2O and NO to W. succinogenes
cultures
Additional experiments analogous to that shown in Fig. 4
were conducted, in which 1 % N2O was added to the
headspace of cultures grown in nitrate-sufficient medium
(Fig. 6). Under these conditions, only cells of W. succinogenes
kompPnosnosZ converted N2O stoichiometrically to N2
(replicate experiments are shown in Figs S4 and S5). In
nitrate-limited medium, cells of the kompPnosnosZ mutant
also reduced added N2O to N2, but at a considerably lower
rate (about 30 %) compared with nitrate-sufficient medium
(Figs S4–S6). Conceivably, this might be due to the
apparently lower amount of cNosZ detectable in cells grown
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Microbiology 160
Nitrous oxide turnover in W. succinogenes
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NO (nmol vial–1)
30 mM Nitrite
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N2O, N2 (µmol vial–1)
20 mM Nitrite
2000
0
NO (nmol vial–1)
N2O, N2 (µmol vial–1)
10 mM Nitrite
5
0
Time (h)
Time (h)
Fig. 5. Production of NO, N2O and N2 by chemical reactions (control experiments related to data shown in Fig. 3). Sterile
nitrate-sufficient medium was incubated in the presence (upper panel) or absence (lower panel) of 0.5 % (w/v) BHIB. The
indicated amounts of nitrite were added. Mean values from three independent experiments are presented. The figure shows
measured NO (#), N2O (g) and N2 (h). Error bars indicate the standard error of the mean.
in nitrate-limited medium (Fig. 2). Additional experiments
with 10 % acetylene in the headspace demonstrated efficient
acetylene inhibition of N2 formation by strain kompPnosnosZ
(Fig. S11). Finally, we conducted an experiment where a
bolus of NO (~1 mmol NO vial21) was injected at the onset
of the incubations. All three organisms reduced NO to N2O
within the first 4 h but only cells of mutant kompPnosnosZ
reduced this N2O further to N2 (Figs S7 and S8).
Estimated specific cellular N2O reduction rates
The data for N2O reduction or N2 production rates in the
various batch cultivations of mutant kompPnosnosZ and the
measured OD (converted to cell dry weight vial21
assuming 0.4 mg cell dry weight ml21 at an OD578 of 1)
were used to estimate the specific N2 production rate
throughout the incubations. The result is shown in Fig. 7,
where the rate expressed as mmol N2 (g cell dry weight)21
h21 is plotted against time for the different treatments.
In vials without NO or N2O injected, low and relatively
constant specific rates were recorded throughout the initial
8–15 h. In vials with NO injected, high rates were recorded
initially, coinciding with a rapid reduction of the entire
injected NO, which caused a transient peak in N2O during
the first 4 h (see Fig. S8). In vials with excess N2O in the
headspace, the specific rates increased gradually during the
first 10 h, and declined rapidly after 12 to 15 h. The N2O
concentration in the headspace was only reduced by about
15 %; thus the sharp decline in N2 production after 15 h
was not due to substrate depletion. It is currently unknown
whether the presence of NO enhances the production of
cNosZ in nitrate-grown W. succinogenes cells.
http://mic.sgmjournals.org
DISCUSSION
The results demonstrate that nitrate-ammonifying cells of
the W. succinogenes cultures tested catalyse both N2Oproducing and N2O-consuming reactions that eventually
result in N2 formation. Nonetheless, W. succinogenes
cannot be regarded as a denitrifier since the vast majority
of nitrate has previously been shown to end up in the
respiratory ammonification pathway using the respiratory
Nap and Nrf systems (see Introduction). The production of
N2O seems to be mainly associated with the detoxification
of the NO generated by chemical reactions between nitrite
and components of the medium, whereas N2O consumption is envisaged as an additional mode of anaerobic
respiration, as discussed in the following sections.
Production of NO and N2O
In nitrate-sufficient cultures of W. succinogenes, the
accumulation of nitrite causes the emergence of small
amounts of NO. This reaction is chemically obvious and
apparently facilitated by BHIB ingredients, presumably
involving nitrosation and decomposition of nitroso compounds. It has not been elucidated to date whether W.
succinogenes synthesizes an enzyme that produces NO from
nitrite (assuming that NrfA is unlikely to release NO as a
by-product). The formation of NO from nitrite was
reported for membrane-bound nitrate reductase (NarGHI
complex) from Salmonella enterica serovar Typhimurium,
which, however, has no counterpart in W. succinogenes
(Gilberthorpe & Poole, 2008; Rowley et al., 2012). In
contrast, there is no report that a periplasmic nitrate
reductase of the Nap-type is able to generate NO.
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1755
M. Luckmann and others
Specific rate of N2 production
[mmol (g dw)–1 h–1]
N2O (mmol vial–1)
45
40
35
30
0
5
10
15
20
25
120
100
80
60
40
20
0
0
5
10
15
20
Time (h)
Time (h)
10
Fig. 7. Estimated specific cellular rates of N2 production throughout
the batch cultivation of W. succinogenes kompPnosnosZ in nitratesufficient medium with or without either NO or N2O in the
headspace at the onset of cultivation. ($, &, red) NO added (two
individual vials), (m, ., ¤, blue) N2O added (three individual vials),
($, green), no NO or N2O added (for clarity only one representative
vial, out of three, is shown). Details of the experiments with added
NO are shown in Figs S7 and S8. dw, Dry weight.
N2 (mmol vial–1)
8
6
4
2
0
0
5
10
15
20
25
Time (h)
Fig. 6. Concentrations of N2O and N2 in W. succinogenes
cultures with N2O added to the headspace. The experiment
corresponds to that shown in Fig. 4 but 1 % N2O was injected into
the headspace prior to inoculation. Representative experiments are
shown (performed in triplicate). (#, red) Wild-type in nitratesufficient medium, ($, red) wild-type in nitrate-limited medium, (e,
green) mutant DnosZ in nitrate-sufficient medium, (X, green)
mutant DnosZ in nitrate-limited medium, (g, blue) mutant
kompPnosnosZ in nitrate-sufficient medium, (m, blue) mutant
kompPnosnosZ in nitrate-limited medium.
It is widely accepted that bacteria thriving in different
habitats need to combat environmental NO derived from
nitrogen oxyanions (Poole, 2005). N2O and ammonium
are typical NO detoxification products when oxygen is
absent. In the case of W. succinogenes it has been reported
that at least two proteins are involved in nitrosative stress
defence (Kern et al., 2011b). These are periplasmic
cytochrome c nitrite reductase (NrfA) and a cytoplasmic
flavodiiron protein (Fdp). NrfA is induced in the presence
of nitrate and efficiently catalyses the reduction of nitrite,
NO and hydroxylamine to ammonium in the periplasm
(Simon, 2002; Einsle, 2011). This reaction is thought to
make W. succinogenes cells tolerant to high nitrite concentrations, in line with the fact that nitrite does not need
to be transported into the cytoplasm during nitrate
ammonification. Reduction of NO to N2O by NrfA
proteins from Desulfovibrio desulfuricans, W. succinogenes
and E. coli was also reported, but the physiological
significance of this reaction is questionable (Costa et al.,
1990; Simon et al., 2011). Fdp proteins, on the other hand,
1756
are known to reduce NO to N2O (Saraiva et al., 2004).
However, this reaction has not been demonstrated for W.
succinogenes Fdp since the protein has not been purified.
Further possible NO reductases in W. succinogenes are the
hybrid cluster protein (Hcp) and a homologue (57 %
identity) of the HP0013 protein from Helicobacter pylori
26695 (WS1903, NP_908012) (Justino et al., 2012). Fdp,
Hcp and Ws1903 are all predicted to be cytoplasmic
proteins. Gene deletion strains lacking either nrfA, fdp or
hcp are available and will be examined as regards their N2O
production capacity in future experiments.
The ability of nitrate-ammonifying cells to produce N2O in
pure culture has been reported before (Bleakley & Tiedje,
1982; Schumacher & Kroneck, 1992; Rowley et al., 2012;
Stremińska et al., 2012). In Salmonella enterica serovar
Typhimurium, 20 % of nitrate-N was released as N2O
under nitrate-sufficient conditions when nitrite had accumulated to millimolar levels (Rowley et al., 2012). This
phenotype is envisaged to result from NO production from
nitrite by the Nar-type nitrate reductase (see above) and
subsequent anoxic NO detoxification to N2O. Stremińska
et al. (2012) demonstrated that soil isolates belonging to
the genera Bacillus or Citrobacter formed N2O under
nitrate-sufficient conditions (low C-to-NO2
3 ratio) and
found that a strain of Citrobacter sp. reduced up to 5 % of
nitrate-N to N2O. Kaspar & Tiedje (1981) reported that the
nitrate-ammonifying rumen microbiota accumulated up to
0.3 % of the added nitrate-N as N2O and consequently
stated that denitrification is absent from the rumen. In the
present study, exponentially growing W. succinogenes cells
converted about 0.15 % of nitrate-N to N2O under the
nitrate-sufficient conditions tested (Fig. 3). These percentages, however, should be compared with caution as W.
succinogenes is not a dominant bacterium in cattle rumen.
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Microbiology 160
Nitrous oxide turnover in W. succinogenes
In nitrate-sufficient medium, W. succinogenes cells continued
to produce N2O after the transition from the exponential to
the stationary growth phase, indicating that nitrite-dependent NO production as well as NO detoxification continued
even after cell growth had ceased. This phenomenon was also
reported for other nitrate ammonifiers (Bleakley & Tiedje,
1982; Schumacher & Kroneck, 1992).
Consumption of N2O
W. succinogenes cells were previously reported to grow by
‘N2O respiration’ using N2O as sole electron acceptor of
anaerobic respiration (Yoshinari, 1980). In these experiments the cells were gassed with pure N2O and grew to a
modest optical density with formate as electron donor. In
contrast, our experiments describe N2O turnover during
respiratory nitrate ammonification. The results demonstrate
that the W. succinogenes cytochrome c nitrous oxide
reductase (cNosZ) is able to convert N2O to N2 under the
applied conditions. This reaction is thought to play a minor
role in menaquinone replenishment since menaquinone is
required for formate oxidation, which is catalysed by an
electrogenic, i.e. proton-motive force (PMF)-generating,
membrane-bound formate dehydrogenase complex (Kern &
Simon, 2009; Simon & Kern, 2008; Simon & Klotz, 2013;
Simon et al., 2008). In this way, N2O reduction is equivalent
to formate-dependent nitrate and nitrite reduction performed by the respiratory Nap and Nrf systems. In each case,
menaquinol oxidation seemingly does not contribute to the
generation of a PMF in W. succinogenes (Kern & Simon,
2009; Simon & Klotz, 2013). The precise architecture of the
electron transport chain that catalyses N2O reduction by
menaquinol is not known, but a menaquinol dehydrogenase
of the NapGH-type (named NosGH) might be involved in
order to direct electrons from the membrane to the periplasmic space (Simon et al., 2004; Simon & Klotz, 2013).
Subsequently, electrons are likely to be transferred either
directly or indirectly via the cytochrome c domain of cNosZ
to the copper-containing catalytic site of N2O reduction.
The specific rates of N2O reduction (Fig. 7) are very
low compared to those of regular denitrifying bacteria.
In Paracoccus denitrificans, for instance, the maximum
specific rate of N2O reduction was found to be 2.5 fmol
N2O cell21 h21 (Bergaust et al., 2012), which is equivalent to
12.5 mmol N2O (g cell dry weight)21 h21 (dry weight per
cell52610213 g). This is two orders of magnitude higher
than the maximum specific rates estimated for W. succinogenes kompPnosnosZ. This could be taken to suggest that W.
succinogenes is an insignificant sink for N2O in the environment. It remains to be explored, however, to what extent the
N2O reductase activity of the organism could be enhanced by
conditions other than those tested in the present study.
ammonium and N2 from nitrate, albeit to different extents
(Sanford et al., 2012; Heylen & Keltjens, 2012; Mania et al.,
2014). It remains to be established, however, whether such
cells indeed perform both types of anaerobic respiration
or if one reaction mainly results from detoxification. In
this context, the discrimination between detoxifying and
respiratory processes is not trivial, especially given the fact
that quinol oxidation by nitrate, nitrite and N2O by the
respective Nap, Nrf and cNos systems is thought to be
electroneutral and therefore not directly involved in the
generation of a PMF and hence ATP production (Simon &
Klotz, 2013). The results presented here suggest that N2O
formation in W. succinogenes resulted from NO detoxification and that N2O serves as an additional electron sink
during growth by respiratory nitrate ammonification.
ACKNOWLEDGEMENTS
Work in our laboratories is funded by the Deutsche Forschungsgemeinschaft (grant SI 848/4-1 to J. S.) and the Norwegian Research
Council.
REFERENCES
Baar, C., Eppinger, M., Raddatz, G., Simon, J., Lanz, C., Klimmek, O.,
Nandakumar, R., Gross, R., Rosinus, A. & other authors (2003).
Complete genome sequence and analysis of Wolinella succinogenes.
Proc Natl Acad Sci U S A 100, 11690–11695.
Bergaust, L., Shapleigh, J., Frostegård, Å. & Bakken, L. (2008).
Transcription and activities of NOx reductases in Agrobacterium
tumefaciens: the influence of nitrate, nitrite and oxygen availability.
Environ Microbiol 10, 3070–3081.
Bergaust, L., van Spanning, R. J. M., Frostegård, Å. & Bakken, L. R.
(2012). Expression of nitrous oxide reductase in Paracoccus
denitrificans is regulated by oxygen and nitric oxide through FnrP
and NNR. Microbiology 158, 826–834.
Bleakley, B. H. & Tiedje, J. M. (1982). Nitrous oxide production by
organisms other than nitrifiers or denitrifiers. Appl Environ Microbiol
44, 1342–1348.
Bokranz, M., Katz, J., Schröder, I., Roberton, A. M. & Kröger, A.
(1983). Energy metabolism and biosynthesis of Vibrio succinogenes
growing with nitrate or nitrite as terminal electron acceptor. Arch
Microbiol 135, 36–41.
Costa, C., Macedo, A., Moura, I., Moura, J. J. G., Le Gall, J., Berlier, Y.,
Liu, M.-Y. & Payne, W. J. (1990). Regulation of the hexaheme nitrite/
nitric oxide reductase of Desulfovibrio desulfuricans, Wolinella
succinogenes and Escherichia coli. A mass spectrometric study. FEBS
Lett 276, 67–70.
Dell’Acqua, S., Moura, I., Moura, J. J. G. & Pauleta, S. R. (2011). The
electron transfer complex between nitrous oxide reductase and its
electron donors. J Biol Inorg Chem 16, 1241–1254.
Einsle, O. (2011). Structure and function of formate-dependent
cytochrome c nitrite reductase, NrfA. Methods Enzymol 496, 399–422.
Gilberthorpe, N. J. & Poole, R. K. (2008). Nitric oxide homeostasis in
Salmonella typhimurium: roles of respiratory nitrate reductase and
flavohemoglobin. J Biol Chem 283, 11146–11154.
CONCLUSION
In recent years the view has emerged that individual
bacterial cells are capable of simultaneously producing
http://mic.sgmjournals.org
Giles, M., Morley, N., Baggs, E. M. & Daniell, T. J. (2012). Soil nitrate
reducing processes - drivers, mechanisms for spatial variation, and
significance for nitrous oxide production. Front Microbiol 3, 407.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 20:07:04
1757
M. Luckmann and others
Hartley, A. M. & Asai, R. J. (1963). Spectrophotometric determination
of nitrate with 2,6-xylenol reagent. Anal Chem 35, 1207–1213.
Heylen, K. & Keltjens, J. (2012). Redundancy and modularity in
Pomowski, A., Zumft, W. G., Kroneck, P. M. H. & Einsle, O. (2011).
N2O binding at a [4Cu : 2S] copper–sulphur cluster in nitrous oxide
reductase. Nature 477, 234–237.
membrane-associated dissimilatory nitrate reduction in Bacillus.
Front Microbiol 3, 371.
Poole, R. K. (2005). Nitric oxide and nitrosative stress tolerance in
Jones, C. M., Graf, D. R. H., Bru, D., Philippot, L. & Hallin, S.
(2013). The unaccounted yet abundant nitrous oxide-reducing
Reay, D. S., Davidson, E. A., Smith, K. A., Smith, P., Melillo, J. M.,
Dentener, F. & Crutzen, P. J. (2012). Global agriculture and nitrous
microbial community: a potential nitrous oxide sink. ISME J 7,
417–426.
oxide emissions. Nature Clim. Change 2, 410–416.
bacteria. Biochem Soc Trans 33, 176–180.
Richardson, D. J., Berks, B. C., Russell, D. A., Spiro, S. & Taylor, C. J.
(2001). Functional, biochemical and genetic diversity of prokaryotic
Justino, M. C., Ecobichon, C., Fernandes, A. F., Boneca, I. G. &
Saraiva, L. M. (2012). Helicobacter pylori has an unprecedented
nitrate reductases. Cell Mol Life Sci 58, 165–178.
nitric oxide detoxifying system. Antioxid Redox Signal 17, 1190–
1200.
Richardson, D., Felgate, H., Watmough, N., Thomson, A. & Baggs, E.
(2009). Mitigating release of the potent greenhouse gas N2O from the
Kaspar, H. F. & Tiedje, J. M. (1981). Dissimilatory reduction of nitrate
and nitrite in the bovine rumen: nitrous oxide production and effect
of acetylene. Appl Environ Microbiol 41, 705–709.
nitrogen cycle – could enzymic regulation hold the key? Trends
Biotechnol 27, 388–397.
Rider, B. F. & Mellon, M. G. (1946). Colorimetric determination of
Kern, M. & Simon, J. (2009). Electron transport chains and
nitrite. Ind Eng Chem 18, 96–98.
bioenergetics of respiratory nitrogen metabolism in Wolinella
succinogenes and other Epsilonproteobacteria. Biochim Biophys Acta
1787, 646–656.
Rowley, G., Hensen, D., Felgate, H., Arkenberg, A., Appia-Ayme, C.,
Prior, K., Harrington, C., Field, S. J., Butt, J. N. & other authors
(2012). Resolving the contributions of the membrane-bound and
Kern, M., Eisel, F., Scheithauer, J., Kranz, R. G. & Simon, J. (2010).
periplasmic nitrate reductase systems to nitric oxide and nitrous
oxide production in Salmonella enterica serovar Typhimurium.
Biochem J 441, 755–762.
Substrate specificity of three cytochrome c haem lyase isoenzymes
from Wolinella succinogenes: unconventional haem c binding motifs
are not sufficient for haem c attachment by NrfI and CcsA1. Mol
Microbiol 75, 122–137.
Kern, M., Klotz, M. G. & Simon, J. (2011a). The Wolinella succinogenes
mcc gene cluster encodes an unconventional respiratory sulphite
reduction system. Mol Microbiol 82, 1515–1530.
Kern, M., Volz, J. & Simon, J. (2011b). The oxidative and nitrosative
stress defence network of Wolinella succinogenes: cytochrome c nitrite
reductase mediates the stress response to nitrite, nitric oxide,
hydroxylamine and hydrogen peroxide. Environ Microbiol 13, 2478–
2494.
Kröger, A., Geisler, V. & Duchêne, A. (1994). Isolation of Wolinella
succinogenes hydrogenase. Chromatofocusing. In A Practical Guide to
Membrane Protein Purification, pp. 141–147. Edited by G. von Jagow
& H. Schägger. London: Academic Press.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning:
a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring
Harbor Laboratory.
Sanford, R. A., Wagner, D. D., Wu, Q., Chee-Sanford, J. C., Thomas,
S. H., Cruz-Garcı́a, C., Rodrı́guez, G., Massol-Deyá, A., Krishnani,
K. K. & other authors (2012). Unexpected nondenitrifier nitrous
oxide reductase gene diversity and abundance in soils. Proc Natl Acad
Sci U S A 109, 19709–19714.
Saraiva, L. M., Vicente, J. B. & Teixeira, M. (2004). The role of the
flavodiiron proteins in microbial nitric oxide detoxification. Adv
Microb Physiol 49, 77–129.
Schumacher, W. & Kroneck, P. M. H. (1992). Anaerobic energy
metabolism of the sulfur-reducing bacterim ‘‘Spirillum‘‘ 5175 during
dissimilatory nitrate reduction to ammonia. Arch Microbiol 157, 464–
470.
Kröger, A., Biel, S., Simon, J., Gross, R., Unden, G. & Lancaster,
C. R. D. (2002). Fumarate respiration of Wolinella succinogenes:
Schumacher, W., Kroneck, P. M. H. & Pfennig, N. (1992).
enzymology, energetics and coupling mechanism. Biochim Biophys
Acta 1553, 23–38.
Comparative systematic study on ‘‘Spirillum‘‘ 5175, Campylobacter
and Wolinella species. Arch Microbiol 158, 287–293.
Lorenzen, J. P., Kröger, A. & Unden, G. (1993). Regulation of
Simon, J. (2002). Enzymology and bioenergetics of respiratory nitrite
ammonification. FEMS Microbiol Rev 26, 285–309.
anaerobic respiratory pathways in Wolinella succinogenes by the
presence of electron acceptors. Arch Microbiol 159, 477–483.
Mania, D., Heylen, K., van Spanning, R. J. M. & Frostegård, Å. (2014).
Simon, J. & Kern, M. (2008). Quinone-reactive proteins devoid of
haem b form widespread membrane-bound electron transport
modules in bacterial respiration. Biochem Soc Trans 36, 1011–1016.
The nitrate-ammonifying and nosZ carrying bacterium Bacillus vireti
is a potent source and sink for nitric and nitrous oxide under high
nitrate conditions. Environ Microbiol doi:10.1111/1462-2920.12478
[Epub ahead of print].
Simon, J. & Klotz, M. G. (2013). Diversity and evolution of
Molstad, L., Dörsch, P. & Bakken, L. R. (2007). Robotized incubation
Simon, J. & Kröger, A. (1998). Identification and characterization of
system for monitoring gases (O2, NO, N2O N2) in denitrifying
cultures. J Microbiol Methods 71, 202–211.
IS1302, a novel insertion element from Wolinella succinogenes
belonging to the IS3 family. Arch Microbiol 170, 43–49.
Pauleta, S. R., Dell’Acqua, S. & Moura, I. (2013). Nitrous oxide
Simon, J. & Kroneck, P. M. H. (2013). Microbial sulfite respiration.
reductase. Coord Chem Rev 257, 332–349.
Adv Microb Physiol 62, 45–117.
Payne, W. J., Grant, M. A., Shapleigh, J. & Hoffman, P.
(1982). Nitrogen oxide reduction in Wolinella succinogenes and
Simon, J., Gross, R., Ringel, M., Schmidt, E. & Kröger, A. (1998).
Campylobacter species. J Bacteriol 152, 915–918.
Pfennig, N. & Trüper, H. G. (1981). Isolation of members of the
families Chromatiaceae and Chlorobiaceae. In The Prokaryotes, pp.
279–289. Edited by M. P. Starr, H. Stolp, H. G. Trüper, A. Balous &
H. G. Schlegel. New York, Berlin, Heidelberg: Springer.
1758
bioenergetic systems involved in microbial nitrogen compound
transformations. Biochim Biophys Acta 1827, 114–135.
Deletion and site-directed mutagenesis of the Wolinella succinogenes
fumarate reductase operon. Eur J Biochem 251, 418–426.
Simon, J., Gross, R., Einsle, O., Kroneck, P. M. H., Kröger, A. &
Klimmek, O. (2000). A NapC/NirT-type cytochrome c (NrfH) is the
mediator between the quinone pool and the cytochrome c nitrite
reductase of Wolinella succinogenes. Mol Microbiol 35, 686–696.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Fri, 16 Jun 2017 20:07:04
Microbiology 160
Nitrous oxide turnover in W. succinogenes
Simon, J., Einsle, O., Kroneck, P. M. H. & Zumft, W. G. (2004). The
unprecedented nos gene cluster of Wolinella succinogenes encodes a
novel respiratory electron transfer pathway to cytochrome c nitrous
oxide reductase. FEBS Lett 569, 7–12.
Thomson, A. J., Giannopoulos, G., Pretty, J., Baggs, E. M. &
Richardson, D. J. (2012). Biological sources and sinks of nitrous
oxide and strategies to mitigate emissions. Philos Trans R Soc B Biol
Sci 367, 1157–1168.
Simon, J., van Spanning, R. J. M. & Richardson, D. J. (2008). The
van Spanning, R. J. M. (2011). Structure, function, regulation and
organisation of proton motive and non-proton motive redox loops in
prokaryotic respiratory systems. Biochim Biophys Acta 1777, 1480–
1490.
evolution of the nitrite and nitrous oxide reductases: denitrification
enzymes with a beta-propeller fold. In Nitrogen Cycling in Bacteria,
pp. 135–161. Edited by J. W. B. Moir. Wymondham: Caister
Academic Press.
Simon, J., Kern, M., Hermann, B., Einsle, O. & Butt, J. N. (2011).
Physiological function and catalytic versatility of bacterial multihaem
cytochromes c involved in nitrogen and sulfur cycling. Biochem Soc
Trans 39, 1864–1870.
Wüst, A., Schneider, L., Pomowski, A., Zumft, W. G., Kroneck,
P. M. H. & Einsle, O. (2012). Nature’s way of handling a greenhouse
Smith, K. A. (2010). Nitrous Oxide and Climate Change. London:
gas: the copper–sulfur cluster of purple nitrous oxide reductase. Biol
Chem 393, 1067–1077.
Earthscan.
Yoshinari, T. (1980). N2O reduction by Vibrio succinogenes. Appl
Stein, L. Y. (2011). Surveying N2O-producing pathways in bacteria.
Environ Microbiol 39, 81–84.
Methods Enzymol 486, 131–152.
Zumft, W. G. (1997). Cell biology and molecular basis of denitrifica-
Stremińska, M. A., Felgate, H., Rowley, G., Richardson, D. J. &
Baggs, E. M. (2012). Nitrous oxide production in soil isolates of
Zumft, W. G. & Kroneck, P. M. H. (2007). Respiratory transformation
nitrate-ammonifying bacteria. Environ Microbiol Rep 4, 66–71.
tion. Microbiol Mol Biol Rev 61, 533–616.
Teraguchi, S. & Hollocher, T. C. (1989). Purification and some
of nitrous oxide (N2O) to dinitrogen by Bacteria and Archaea. Adv
Microb Physiol 52, 107–227.
characteristics of a cytochrome c-containing nitrous oxide reductase
from Wolinella succinogenes. J Biol Chem 264, 1972–1979.
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