Microbiology (2014), 160, 752–765
DOI 10.1099/mic.0.075242-0
Identification of genes essential for anaerobic
growth of Listeria monocytogenes
Stefanie Müller-Herbst,1,2 Stefanie Wüstner,23 Anna Mühlig,2
Daniela Eder,14 Thilo M. Fuchs,1,2 Claudia Held,3 Armin Ehrenreich3
and Siegfried Scherer1,2
Correspondence
Stefanie Müller-Herbst
[email protected]
1
Abteilung Mikrobiologie, Zentralinstitut für Ernährungs- und Lebensmittelforschung,
Technische Universität München, Freising, Germany
2
Lehrstuhl für Mikrobielle Ökologie, Department Biowissenschaften,
Wissenschaftszentrum Weihenstephan, Technische Universität München, Freising, Germany
3
Lehrstuhl für Mikrobiologie, Department Biowissenschaften,
Wissenschaftszentrum Weihenstephan, Technische Universität München, Freising, Germany
Received 20 November 2013
Accepted 22 January 2014
The facultative anaerobic bacterium Listeria monocytogenes encounters microaerophilic or
anaerobic conditions in various environments, e.g. in soil, in decaying plant material, in food
products and in the host gut. To elucidate the adaptation of Listeria monocytogenes to variations
in oxygen tension, global transcription analyses using DNA microarrays were performed. In total,
139 genes were found to be transcribed differently during aerobic and anaerobic growth; 111
genes were downregulated and 28 genes were upregulated anaerobically. The oxygendependent transcription of central metabolic genes is in agreement with results from earlier
physiological studies. Of those genes more strongly expressed under lower oxygen tension, 20
were knocked out individually. Growth analysis of these knock out mutants did not indicate an
essential function for the respective genes during anaerobiosis. However, even if not essential,
transcriptional induction of several genes might optimize the bacterial fitness of Listeria
monocytogenes in anaerobic niches, e.g. during colonization of the gut. For example, expression
of the anaerobically upregulated gene lmo0355, encoding a fumarate reductase a chain,
supported growth on 10 mM fumarate under anaerobic but not under aerobic growth conditions.
Genes essential for anaerobic growth were identified by screening a mutant library. Eleven out of
1360 investigated mutants were sensitive to anaerobiosis. All 11 mutants were interrupted in the
atp locus. These results were further confirmed by phenotypic analysis of respective in-frame
deletion and complementation mutants, suggesting that the generation of a proton motive force
via F1F0-ATPase is essential for anaerobic proliferation of Listeria monocytogenes.
INTRODUCTION
Listeria monocytogenes is a rod-shaped, non-sporulating,
low-GC Gram-positive facultative anaerobic bacterium. It
can be isolated from many environmental niches, such as
3Present address: Institut für Medizinische Mikrobiologie, Immunologie
und Hygiene, Technische Universität München, München, Germany.
4Present address: BLB GmbH Brau-Labor & Beratung, Berching,
Germany.
Abbreviations: BHI, brain heart infusion; DEPC, diethylpyrocarbonate;
LAP, Listeria adhesion protein; q, quantitative; RT, real-time; RTL, relative
transcription level.
The Gene Expression Omnibus accession number for the microarray
experiments reported in this paper is GSE52325.
Three supplementary figures and three supplementary tables are
available with the online version of this paper.
752
soil, water and decaying plant material, where it lives
saprophytically (Fox et al., 2009; Lyautey et al., 2007; Weis
& Seeliger, 1975; Welshimer & Donker-Voet, 1971). One
reason for the ubiquitous distribution of Listeria monocytogenes is its resistance to many adverse environmental
conditions. Listeria monocytogenes grows at temperatures
from 1.7 to 45.0 uC in a pH range from 4.7 to 9.2 (Junttila
et al., 1988; Petran & Zottola, 1989) and it tolerates high salt
concentrations of up to 10 % sodium chloride (McClure
et al., 1991). This resistance spectrum also allows Listeria
monocytogenes to circumvent well-established food conservation barriers like acidification, decrease of water activity or
cooling and is the prerequisite for its relevance as a foodborne pathogen.
Listeria monocytogenes encounters hypoxic or even anaerobic conditions in the environment (soil, sediments), in
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Anaerobic growth of Listeria monocytogenes
food (especially in vacuum packed food) and in the
intestine of its mammalian hosts before the intracellular life
cycle begins. Several studies indicate a link between
anaerobiosis and virulence, suggesting that anaerobiosis
could be an environmental signal that triggers virulence
gene expression. For example, upregulation of the internalins InlA and InlB as well as the Listeria adhesion protein
(LAP) could result in a higher infectivity of anaerobically
grown bacteria in vitro as well as in vivo in the guinea pig
model (Bo Andersen et al., 2007; Burkholder et al., 2009;
Stritzker et al., 2005).
(12 000 g, 10 min, 4 uC). The pellet was washed with 1 ml 70 % (v/v)
EtOH. RNA was dried at room temperature before the pellet was
resuspended in 80 ml diethylpyrocarbonate (DEPC)-treated water
(0.1 % v/v). For DNA digestion, 10 ml DNase I (1 U ml21) and 10 ml
106 DNase I buffer (Promega) were added, and the reaction mix was
incubated at 37 uC for 45 min. After an additional chloroform
extraction (500 ml chloroform), RNA was purified using the RNeasy
Mini kit (Qiagen) according to the manufacturer’s protocol for RNA
clean-up. An additional on-column DNA digest (Qiagen RNase-free
DNase, 2 U ml21) was performed according to the manufacturer’s
instructions before RNA was eluted in a final volume of 50 ml
nuclease-free water.
Although early physiological studies on the anaerobic
metabolism of Listeria monocytogenes (Jydegaard-Axelsen
et al., 2004; Pine et al., 1989; Romick et al., 1996) were
completed using global approaches, such as comparative
genome (Buchrieser et al., 2003; Glaser et al., 2001) and
transcriptome (Toledo-Arana et al., 2009) analyses, the
aerobic growth of Listeria monocytogenes is still much
better understood than the anaerobic growth (reviewed by
Lungu et al., 2009).
Microarray analyses. Transcriptome analyses were performed for six
In the present work, we wanted to further characterize the
adaptation of the food-borne pathogen Listeria monocytogenes to anaerobiosis.
METHODS
Bacterial strains and growth conditions. Strains used in this study
are listed in Table S1 (available in the online Supplementary
Material). Listeria monocytogenes was grown in brain heart infusion
(BHI) broth or on BHI agar plates at 37 uC if not stated otherwise.
When necessary, erythromycin was added at a final concentration of
10 mg ml21. Aerobic growth in broth was conducted in Erlenmeyer
flasks with constant shaking at 150 r.p.m.; anaerobic growth
was performed in sealed Falcon tubes. Growth was monitored
by measuring OD600 with an Ultraspec 2000 spectrophotometer
(Pharmacia Biotech). After reaching OD600 1.0, the culture was
diluted 1 : 10 for further measurements. Pre-tests with cultures to
which the redox indicator resazurin was added [0.0001 % (w/v)]
revealed that oxygen was used up by the growing culture in Falcon
tubes 3 h post-inoculation of 50.5 ml BHI medium with 500 ml of an
overnight culture of Listeria monocytogenes EGD. Generation of an
anaerobic atmosphere for bacteria grown on agar plates was acheived
using the Anaerocult A kit (Merck) in an anaerobic jar (Oxoid).
Anaerobiosis was confirmed via colour change of an Anaerotest strip
(Merck). Escherichia coli strain DH5a was grown in LB broth or on LB
agar plates at 37 uC. When necessary, erythromycin was added at a
final concentration of 300 mg ml21.
RNA isolation. Total bacterial RNA was isolated from Listeria
monocytogenes EGD cell pellets that were shock-frozen in liquid
nitrogen. The pellet of a 50 ml culture was resuspended in 1 ml
TRIzol/TRI Reagent (Invitrogen/Sigma) and transferred into a 2 ml
shredder tube (Sarstedt) containing silica beads (diameter 0.1 mm).
The solution was incubated at room temperature for 1 min before
cell walls were disrupted mechanically in a FastPrep cell disrupter
(MP Biomedicals; 6.5 m s21 for 45 s, three times). After cell lysis,
silica beads were removed by centrifugation (16 100 g, 3 min, room
temperature). The supernatant was transferred into a 2 ml Eppendorf
tube. After chloroform extraction (200 ml chloroform), RNA was
precipitated with 2-propanol (500 ml). The reaction mixture was
inverted, incubated at room temperature for 10 min and centrifuged
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biologically independent samples by using whole-genome DNA
microarrays. The arrays consisted of 70mer oligodeoxyribonucleotides
(Operon Biotechnologies) covering all 2857 ORFs of the Listeria
monocytogenes EGDe genome (Glaser et al., 2001), which were spotted
in duplicate on epoxy-coated glass slides (BioCat). RNA for microarray
experiments was isolated from Listeria monocytogenes EGD 50 ml
cultures grown to OD600 0.70–0.75 either aerobically or anaerobically.
In total, six biologically independent samples were analysed. In the
cDNA synthesis reaction for four RNA sets, cDNA derived from
anaerobically grown cultures was labelled with Cy3–dCTP and cDNA
derived from aerobically grown cultures was labelled with Cy5–dCTP
(GE Healthcare). Dye swap was performed for the two other RNA sets.
For cDNA synthesis, 30 mg bacterial RNA was mixed with reverse
transcription reaction mix [1 ml Superscript III Reverse Transcriptase
(200 U ml21), 8 ml 56 reaction buffer, 4 ml DTT (0.1 M; Invitrogen),
3 ml random nonamer oligonucleotides (GE Healthcare) and 1 ml
dNTP mix (20 mM dATP, 20 mM dGTP, 20 mM dTTP and 16 mM
dCTP (Fermentas)]. The volume was adjusted to 38 ml with DEPCtreated water and 2 ml Cy3- or Cy5-labelled dCTP analogue was added.
Samples were incubated at 42 uC for 2 h. Then, 2 ml DNase-free RNase
(.30 U mg21; Roche Diagnostics) was added and the sample was
incubated for 45 min at 37 uC. cDNA was purified using the QIAquick
PCR Purification kit (Qiagen) and eluted in 40 ml distilled H2O.
Differently labelled samples derived from one RNA set were combined.
The volume was reduced to 30 ml in a SpeedVac Concentrator 5301
(Eppendorf). Then, 6 ml 206 SSC and 4 ml 1 % (w/v) SDS were added.
The reaction mix was incubated for 1 min at 94 uC and subsequently
put on ice. The cDNA sample was then pipetted on a slide, the slide was
covered with a coverslip and hybridization was carried out at 64 uC for
16–18 h. After hybridization, slides were washed in washing solution 1
[26 SSC and 0.2 % (w/v) SDS, 60 uC, 10 min], washing solution 2 (26
SSC, room temperature, 10 min) and twice with washing solution 3
(0.26 SSC, room temperature, 10 min). Slides were then dipped in
ice-cold 2-propanol and dried by centrifugation (1600 g, 3 min, room
temperature). Slides were scanned with a GenePix 4000B scanner
(MDS Analytical Technologies) and data were analysed using GenePix
6.0 software (MDS Analytical Technologies). For normalization, the
arithmetic mean of the ratios was set to 1 (GenePix Pro 6.0 software).
In general, only spots with a fluorescence signal larger than the local
background (+1 SD) were included in the final analysis. Spots with
irregular morphology were in general excluded from the interpretation when the ratio of medians, the ratio of means and the regression
ratio differed by .30 %. To minimize the possibility of incorrect
exclusion of spots due to automated interpretation, raw data of spots
with a log2 ratio of medians ¢1 or ¡–1 were further analysed. If
either the Cy5 or Cy3 fluorescence intensity reached at least 50 % of
the intensity mean of all spots of the respective array, the spot was
included in the final analysis. As each experiment included technical
duplicates, 12 data points were obtained for the calculation of the
expression level of each gene. Genes were treated as upregulated or
downregulated if (i) the log2RTL (relative transcription level) was
calculated based on at least eight valid spots, each with a log2 ratio of
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S. Müller-Herbst and others
medians (anaerobic/aerobic) ¡–1.0 or ¢1.0, and (ii) the arithmetic
mean of the log2 ratio of medians of valid spots was ¡–1.0 or ¢1.0.
log2RTL indicates the arithmetic mean of the log2 ratios of medians of
all valid spots. log2RTL¡–1.0 indicates a stronger transcription under
aerobic growth conditions, i.e. indicates anaerobically downregulated
genes; log2RTL¢1.0 indicates a stronger transcription of the
respective gene under anaerobic growth conditions, i.e. anaerobically
upregulated genes. The data discussed in this paper have been
deposited in NCBI9s Gene Expression Omnibus (Edgar et al., 2002)
and are accessible through GEO Series accession number GSE52325
(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE52325).
Quantitative real-time (qRT)-PCR. cDNA was prepared from 1 mg
listerial RNA with an all-in-one reaction system (qScript cDNA
SuperMix; Quanta BioSciences). For RT-PCR, a ready-to-use reaction
mix (PerfeCTa SYBR Green FastMix; Quanta BioSciences) was used.
The reaction was carried out in a Smart Cycler (Cepheid). Each analysis
was performed in duplicate for three biologically independent
experiments. Data were analysed using the relative expression software
tool REST-MCS version 2 (Pfaffl et al., 2002). log2RTL indicates the
arithmetic mean of the log2 expression ratios (anaerobic/aerobic)
derived from the three independent experiments. Transcription of the
housekeeping gene rpoB was used for normalization. Oligonucleotides
used for qRT-PCR are summarized in Table S2.
Construction of mutants. Oligonucleotides and plasmids used for
the construction and characterization of mutants are listed in Tables
S2 and S3.
Construction of in-frame deletion mutants. Deletion mutants
were constructed in the genetic background of the sequenced strain
Listeria monocytogenes EGDe (Glaser et al., 2001) using a two-step
integration/excision procedure, which is based on transformation with
the mutagenesis plasmid pLSV101 (Joseph et al., 2006), derived from
pLSV1 (Wuenscher et al., 1991), mainly as described previously
(Williams et al., 2005). Two flanking fragments located upstream and
downstream of the gene to be deleted (x) of ~1000 bp were amplified
from chromosomal DNA from Listeria monocytogenes EGDe with the
primer pairs x_A/x_B and x_C/x_D. After ligation of the two fragments
via an introduced restriction site, a nested PCR with the oligonucleotides x_nestAB and x_nestCD was performed, resulting in a fragment of
~1200 bp. The respective fragment was cloned in the pLSV101 vector.
The resulting deletion plasmid pLSV101-Dx was then electroporated in
Listeria monocytogenes EGDe. Growth at 30 uC on BHI/erythromycin
allowed selection for successful electroporation. The pLSV101 vector
has a temperature-sensitive origin of replication for Gram-positive
bacteria. Therefore, growth at the non-permissive temperature of 42 uC
on BHI/erythromycin allowed for selection of clones that had the
deletion plasmid integrated in the bacterial chromosome via homologous recombination. During reiterated growth at 30 uC without
antibiotic pressure, the plasmid can re-excise from the bacterial
chromosome and be lost, resulting in either the WT or the respective
deletion mutant Dx. Screening for such strains was performed at 30 uC,
searching for erythromycin-sensitive mutants. Successful deletion was
confirmed via PCR and subsequent sequencing.
growth on BHI/erythromycin at the non-permissive temperature of
42 uC. Insertion in the correct target locus was verified by PCR using
plasmid-specific oligonucleotides LSV3 and LSV-4380rev in combination with oligonucleotides x_KI_5 and x_KI_3, located upstream
and downstream of the cloned fragment. Growth analysis of the
insertion mutants was performed in the presence of erythromycin at
42 uC to avoid re-excision of the plasmid out of the chromosome.
Construction of complementation mutants. The in cis complementation mutant Dlmo0355-comp was constructed by transforming
the deletion mutant Dlmo0355 (this study) with the complementation
plasmid pLSV101-lmo0355 and subsequently following the same twostep integration/excision procedure as described for the construction
of the deletion mutants. The complementation plasmid pLSV101lmo0355 carried a 2677 bp SalI/Cfr9I fragment, containing the
lmo0355 gene and upstream and downstream flanking regions of
~600 bp. The respective fragment was amplified via PCR with the
primer pair lmo0355_nestAB and lmo0355_nestCD with chromosomal DNA from Listeria monocytogenes EGDe as template.
The atp deletion mutants were complemented in trans. A gene fragment
containing the respective gene with 20 bp of upstream and downstream
flanking regions was amplified via PCR with the oligonucleotides
atpA_comp_5/atpA_comp_3, atpB_comp_5/atpB_comp_3 or atpD_
comp_5/atpD_comp_3. The fragments were ligated in the Gramnegative/Gram-positive shuttle vector pHPS9 via PstI/Cfr9I restriction
sites. This vector has already been used successfully for the construction
of complementation mutants in Listeria monocytogenes (Chico-Calero
et al., 2002). Transcription of the investigated genes is under the control of
the strong promoter P59 from Lactococcus lactis subsp. cremoris (Haima
et al., 1990). The deletion mutants DatpA, DatpB and DatpD (this study)
were electroporated with the complementation plasmids pHPS9-atpA,
pHPS9-atpB and pHPS9-atpD, resulting in the erythromycin-resistant
complementation mutants DatpA-comp, DatpB-comp and DatpD-comp.
High-throughput screening of a Listeria monocytogenes
insertion mutant bank. An insertion mutant library of Listeria
monocytogenes EGD PkpI, which was produced via insertion
duplication mutagenesis with a random fragment library in the
backbone of pLSV101 (Joseph et al., 2006; Schauer et al., 2010), was
screened for mutants showing a growth-defective phenotype under
anaerobic conditions (Fig. S1a). The 1360 mutants tested here
represented a knock out fraction of ~23 % of the Listeria
monocytogenes genome (Joseph et al., 2006; Lee et al., 1999; Schauer
et al., 2010). The mutants were incubated on BHI agar plates
containing erythromycin (10 mg ml21) at the non-permissive
temperature of 42 uC under aerobic and anaerobic conditions in
parallel. Mutants inserted in genes essential for anaerobic growth were
identified. For the identification of the interrupted gene locus, these
mutants were grown aerobically at the permissive temperature of
30 uC. Under these conditions the plasmid pLSV101-x9 (where x9 is a
DNA fragment corresponding to insertion locus) could be re-excised
from the bacterial chromosome and replicated independently. PCR
was performed using the pLSV101-specific oligonucleotides LSV3 and
LSV-4380rev. The received PCR products were sequenced by GATC
Biotech using oligonucleotides LSV3 or LSV-4380rev (Fig. S1b).
Construction of insertion mutants. Insertion mutants were
constructed in the genetic background of Listeria monocytogenes
EGDe via insertion duplication mutagenesis. Fragments of 250–
300 bp located in the middle of the target gene x were amplified via
PCR with the oligonucleotides x_KI_5_int/x_KI_3_int from chromosomal DNA from Listeria monocytogenes EGDe. The respective
fragments were cloned in the vector pLSV101 via BamHI and EcoRI
restriction sites. The resulting insertion plasmid pLSV101-x was
electroporated in Listeria monocytogenes EGDe. Then, insertion
mutants x : : pLSV101 that had the plasmid integrated in the bacterial
chromosome after homologous recombination were selected by
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RESULTS AND DISCUSSION
Adaptation of Listeria monocytogenes to oxygen
availability
Aerobic and anaerobic growth of Listeria monocytogenes
EGD was analysed in BHI medium (Fig. 1). Although Listeria
monocytogenes EGD is able to grow under oxygen-deprived
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Microbiology 160
Anaerobic growth of Listeria monocytogenes
3.5
3.0
OD600
2.5
2.0
1.5
1.0
0.5
0
0
100
200
300
400
Time (min)
500
600
Fig. 1. Growth of Listeria monocytogenes EGD in BHI under
aerobic and anaerobic culture conditions. Listeria monocytogenes
EGD was incubated aerobically (solid curve) or anaerobically
(dashed curve) at 37 6C. Data are the mean±SD of four
biologically independent replicates. Arrows indicate the growth
stage where samples for transcriptome analyses were taken.
conditions, the anaerobic growth rate was decreased and the
maximum OD600 reached under anaerobiosis was only half
of the aerobic value.
To better understand the adaptation of Listeria monocytogenes EGD to oxygen availability, global transcriptional
analysis was performed using whole-genome DNA microarrays. Comparing aerobic and anaerobic growth, a total of
139 genes showed a differential transcriptional pattern
(Table 1). Anaerobically, transcription of 111 genes was
downregulated (Table 1a, log2RTL¡–1), whereas transcription of 28 genes was upregulated compared with
aerobic growth (Table 1b, log2RTL¢1). The transcriptional pattern determined via microarray analysis was
verified via qRT-PCR (Fig. 2) for 11 anaerobically downregulated and four anaerobically upregulated genes. The
coefficient of determination (R250.91) indicates a high
reliability of the microarray data.
Oxygen-dependent expression of the respiratory
chain
Based on predictions from the genome sequence (Glaser
et al., 2001), the facultative anaerobic bacterium Listeria
monocytogenes is able to generate ATP either via oxidative
phosphorylation or via substrate-level phosphorylation. As
no anaerobic respiration has been described for Listeria
monocytogenes, oxidative phosphorylation that results in a
higher ATP yield than substrate-level phosphorylation and
therefore leads to improved growth occurs only in the
presence of oxygen. A prerequisite for ATP synthesis by
oxidative phosphorylation via F1F0-ATP synthetase is the
establishment of a proton motive force via the respiratory
chain. The respiratory chain in Listeria monocytogenes is
considered to be quite simple (Glaser et al., 2001; Patchett
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et al., 1991). Electrons from NADH dehydrogenases
(lmo2389, lmo2638), glycerol 3-phosphate dehydrogenase
(glpD, lmo1293) and, perhaps, a putative formate dehydrogenase (lmo2586) are transferred via menaquinone to
either the cytochrome aa3 (lmo0013–lmo0016, qoxABCD)
or the cytochrome bd (lmo2718–lmo2715, cydABCD)
terminal (mena)quinol-oxidases. Transcription of several
genes of this respiratory chain, i.e. the dehydrogenase genes
lmo2389 (log2RTL 21.7), glpD (log2RTL 24.4) and lmo2586
(log2RTL 22.3), and the qox genes (log2RTL 22.6, 23.4,
23.6 and 23.5), is regulated in an oxygen-dependent
manner. In the absence of oxygen as the terminal electron
acceptor, the expression of the respiratory chain is downregulated in Listeria monocytogenes, indicating a lower ATP
yield and therefore decreased growth.
Link between transcriptomic data and early
physiological studies
A characteristic of carbon metabolism in Listeria monocytogenes is the split of the citric acid cycle into an oxidative
branch and a reductive branch due to a lack of aketoglutarate dehydrogenase (Eisenreich et al., 2006; Trivett
& Meyer, 1971). The implication is that glucose as a carbon
source cannot be completely oxidized to carbon dioxide
aerobically. Alternative end products during growth on
glucose have been described in early physiological studies
(Pine et al., 1989; Romick et al., 1996). Under aerobic growth
conditions these end products (in the strain Listeria
monocytogenes F5069 in terms of per cent carbon recovery)
are lactate (28 %), acetate (23 %), acetoin (26 %) and carbon
dioxide (23 %). Anaerobically, lactate (79 %) is the major end
product, but small amounts of acetate (2 %), formate (5.4 %),
ethanol (7.8 %) and carbon dioxide (2.3 %) are also formed
(Romick et al., 1996). Our transcriptional data fit very well
with these early physiological studies and the theoretical
metabolic capability of Listeria monocytogenes according to
the genome sequence (Glaser et al., 2001) and the KEGG
database (http://www.genome.jp/kegg/). A model of the
glucose catabolism of Listeria monocytogenes EGD grown in
BHI medium aerobically or anaerobically is depicted in Fig. 3.
Glucose can be catabolized initially by Listeria monocytogenes
either via glycolysis or via the pentose phosphate cycle to
pyruvate (Eisenreich et al., 2006; Joseph et al., 2006). Pyruvate
then serves as a substrate for various reactions.
One major end product of glucose catabolism during aerobic
growth is acetoin, which is not produced during anaerobic
growth (Romick et al., 1996). Transcription of alsS
(lmo2006), encoding the acetolactate synthase AlsS, which
catalyses the synthesis of the acetoin precursor 2-acetolactate,
is downregulated anaerobically (log2RTL 21.5). This downregulation probably contributes to the observed difference in
acetoin production depending on oxygen availability.
Another major aerobic end product from glucose catabolism is acetate. Acetate can be generated from pyruvate via
acetyl-P. The first step in this reaction is the oxidative decarboxylation of pyruvate by a pyruvate oxidase
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S. Müller-Herbst and others
Table 1. Oxygen-dependent transcription in Listeria monocytogenes EGD
Genes showing a differential expression (log2RTL: ¡–1.0; ¢1.0) in Listeria monocytogenes EGD grown anaerobically and aerobically. Descriptions
and functional annotations of the respective genes are according to ListiList database (http://genolist.pasteur.fr/ListiList/). Previously described
operon organizations (Toledo-Arana et al., 2009) are indicated. Toledo-Arana et al. (2009) performed transcriptional analysis under hypoxic
conditions for the strain Listeria monocytogenes EGDe; genes that were regulated equivalently dependent on oxygen availability in their study and in
the present study in strain Listeria monocytogenes EGD are indicated in bold. CS, coding sequence; PTS, phosphotransferase system.
Name
Gene
Description
(a) Anaerobically downregulated genes
lmo0013
qoxA
aa 3–600 quinol oxidase subunit II
lmo0014
qoxB
aa 3–600 quinol oxidase subunit I
lmo0015
qoxC
aa 3–600 quinol oxidase subunit III
lmo0016
qoxD
Highly similar to quinol oxidase aa 3–600 chain IV
lmo0027
Similar to PTS system, b-glucoside-specific enzyme IIABC
lmo0109*
Similar to transcriptional regulatory proteins, AraC family
lmo0115
lmaD
Similar to Antigen D
lmo0116
lmaC
Similar to Antigen C
lmo0121
Similar to bacteriophage minor tail proteins
lmo0126
CS
lmo0130
Similar to 59-nucleotidase, putative peptidoglycan-bound protein
(LPXTG motif)
lmo0278
Similar to sugar ABC transporter, ATP-binding protein
lmo0299
Similar to PTS b-glucoside-specific enzyme IIB component
lmo0300
Similar to phospho-b-glucosidase and phospho-b-galactosidase
lmo0319
Similar to phospho-b-glucosidase
lmo0391*
CS
lmo0392*
Highly similar to Bacillus subtilis YqfA protein
lmo0393*
CS
lmo0425*
Similar to transcription antiterminator BglG family
lmo0426*
Similar to PTS fructose-specific enzyme IIA component
lmo0427*
Similar to PTS fructose-specific enzyme IIB component
lmo0428*
Similar to PTS fructose-specific enzyme IIC component
lmo0429*
Similar to sugar hydrolase
lmo0536
Similar to 6-phospho-b-glucosidase
lmo0590
Similar to a fusion of two types of conserved hypothetical proteins
lmo0781
Similar to mannose-specific PTS component IID
lmo0782
Similar to mannose-specific PTS component IIC
lmo0783
Similar to mannose-specific PTS component IIB
lmo0784
Similar to mannose-specific PTS component IIA
lmo0791
CS
lmo0914
Similar to PTS system, IIB component
lmo0915
Similar to PTS enzyme IIC
lmo0916
Similar to PTS enzyme IIA
lmo0917
Similar to b-glucosidase
lmo0918
Similar to transcription antiterminator BglG family
lmo1042*
moeA
Similar to molybdopterin biosynthesis protein MoeA
lmo1043
mobB
Similar to molybdopterin-guanine dinucleotide biosynthesis MobB
lmo1044*
Similar to molybdopterin converting factor subunit 2
lmo1045*
Similar to molybdopterin converting factor subunit 1
lmo1046*
Similar to molybdenum cofactor biosynthesis protein C
lmo1052
pdhA
Highly similar to pyruvate dehydrogenase (E1 a subunit)
lmo1053
pdhB
Highly similar to pyruvate dehydrogenase (E1 b subunit)
lmo1054
pdhC
Highly similar to pyruvate dehydrogenase (dihydrolipoamide
acetyltransferase E2 subunit)
lmo1055
pdhD
Highly similar to dihydrolipoamide dehydrogenase (E3 subunit of
pyruvate dehydrogenase complex)
lmo1254
Similar to a,a-phosphotrehalase
lmo1255
Similar to PTS system trehalose-specific enzyme IIBC
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Operon organization
qoxABCD
qoxABCD
qoxABCD
qoxABCD
lmo0109–lmo0110
lmo0115–lmo0129
lmo0115–lmo0129
lmo0115–lmo0129
lmo0115–lmo0129
log2RTL
22.6
23.4
23.6
23.5
23.1
21.3
21.2
21.3
21.5
21.2
22.2
lmo0914–lmo0918
lmo0914–lmo0918
lmo0914–lmo0918
lmo0914–lmo0918
lmo0914–lmo0918
lmo1041–lmo1047
lmo1041–lmo1047
lmo1041–lmo1047
lmo1041–lmo1047
lmo1041–lmo1047
pdhABCD
pdhABCD
pdhABCD
22.1
22.4
23.2
22.0
21.3
21.4
21.3
22.5
22.4
22.1
22.4
21.9
21.7
21.3
21.6
22.1
22.1
21.7
21.6
23.8
23.7
24.0
24.0
22.3
21.7
21.7
21.8
22.0
21.7
24.6
24.7
24.6
pdhABCD
24.6
lmo1255–lmo1254
lmo1255–lmo1254
22.4
22.5
lmo0391–lmo0393
lmo0391–lmo0393
lmo0391–lmo0393
lmo0425–lmo0429
lmo0425–lmo0429
lmo0425–lmo0429
lmo0425–lmo0429
lmo0425–lmo0429
lmo0589–lmo0591
lmo0782–lmo0781
lmo0782–lmo0781
lmo0784–lmo0783
lmo0784–lmo0783
Microbiology 160
Anaerobic growth of Listeria monocytogenes
Table 1. cont.
Name
lmo1257
lmo1293*
lmo1348*
lmo1349*
lmo1350*
lmo1390
lmo1391
lmo1538*
lmo1539*
lmo1587
lmo1591
lmo1729
lmo1879*
lmo1954
lmo1955*
lmo1998
lmo1999
lmo2006
lmo2057
lmo2090
lmo2124
lmo2158
lmo2159
lmo2160
lmo2161
lmo2162
lmo2163
lmo2169
lmo2335
lmo2336
lmo2337
lmo2340
lmo2344
lmo2389
lmo2436
lmo2569
lmo2584*
lmo2585*
lmo2586*
lmo2590*
lmo2645
lmo2646*
lmo2647
lmo2648
lmo2649
lmo2650
lmo2651*
lmo2665
lmo2666*
lmo2667
lmo2695
lmo2708
lmo2743
lmo2755
lmo2761*
lmo2763*
Gene
glpD
argF
argC
cspD
drm
alsS
ctaB
argG
fruA
fruB
fdhD
http://mic.sgmjournals.org
Description
CS
Similar to glycerol-3-phosphate dehydrogenase
Similar to aminomethyltransferase
Similar to glycine dehydrogenase (decarboxylating) subunit 1
Similar to glycine dehydrogenase (decarboxylating) subunit 2
Similar to ABC transporter (permease proteins)
Similar to sugar ABC transporter, permease protein
Similar to glycerol kinase
Similar to glycerol uptake facilitator
Highly similar to ornithine carbamoyltransferase
Similar to N-acetylglutamate c-semialdehyde dehydrogenases
Similar to b-glucosidases
Similar to cold shock protein
Similar to phosphopentomutase
Similar to integrase/recombinase
Similar to opine catabolism protein
Weakly similar to glucosamine-fructose-6-phosphate aminotransferase
Similar to a-acetolactate synthase protein AlsS
Highly similar to haem A farnesyltransferase
Similar to argininosuccinate synthase
Similar to maltodextrin ABC transport system (permease)
Similar to Bacillus subtilis YwmG protein
Similar to oxidoreductase
Similar to unknown proteins
CS
Similar to unknown proteins
Similar to oxidoreductase
CS
Highly similar to PTS fructose-specific enzyme IIABC component
Fructose-1-phosphate kinase
Similar to regulatory protein DeoR family
Similar to Erwinia chrysanthemi IndA protein
Similar to Bacillus subtilis YtnI protein
Similar to NADH dehydrogenase
Similar to transcription antiterminator
Similar to dipeptide ABC transporter (dipeptide-binding protein)
Similar to formate dehydrogenase associated protein
Similar to Bacillus subtilis YrhD protein
Similar to formate dehydrogenase a chain
Similar to ATP-binding proteins
CS
CS
Similar to creatinine amidohydrolase
Similar to phosphotriesterase
Similar to hypothetical PTS enzyme IIC component
Similar to hypothetical PTS enzyme IIB component
Similar to mannitol-specific PTS enzyme IIA component
Similar to PTS system galactitol-specific enzyme IIC component
Similar to PTS system galactitol-specific enzyme IIB component
Similar to PTS system galactitol-specific enzyme IIA component
Similar to dihydroxyacetone kinase
Similar to PTS cellobiose-specific enzyme IIC
Similar to transaldolase
Similar to acylase and diesterase
Similar to b-glucosidase
Similar to PTS cellobiose-specific enzyme IIC
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Operon organization
lmo1348–lmo1350
lmo1348–lmo1350
lmo1348–lmo1350
lmo1389–lmo1391
lmo1389–lmo1391
lmo1539–lmo1538
lmo1539–lmo1538
argCJBDF
argCJBDF
lmo1729–lmo1726
lmo1955–lmo1953
lmo1955–lmo1953
lmo2004–lmo1997
lmo2004–lmo1997
argGH
lmo2126–lmo2121
lmo2163–lmo2159
lmo2163–lmo2159
lmo2163–lmo2159
lmo2163–lmo2159
lmo2163–lmo2159
lmo2168–lmo2169
lmo2337–lmo2335
lmo2337–lmo2335
lmo2337–lmo2335
lmo2341–lmo2340
lmo2351–lmo2343
lmo2586–lmo2585
lmo2586–lmo2585
lmo2651–lmo2645
lmo2651–lmo2645
lmo2651–lmo2645
lmo2651–lmo2645
lmo2651–lmo2645
lmo2651–lmo2645
lmo2651–lmo2645
lmo2668–lmo2659
lmo2668–lmo2659
lmo2668–lmo2659
lmo2695–lmo2697
lmo2741–lmo2743
lmo2755–lmo2754
lmo2761–lmo2765
lmo2761–lmo2765
log2RTL
22.0
24.4
22.9
23.0
23.0
21.5
21.6
23.9
25.1
22.3
22.7
21.5
22.9
21.2
21.3
22.9
21.7
21.5
21.4
22.6
21.9
21.6
22.5
22.8
23.0
22.6
23.1
21.2
22.1
22.2
22.3
21.1
21.6
21.7
22.2
22.8
21.7
22.4
22.3
21.2
23.8
25.1
24.0
24.7
24.9
24.3
23.7
21.2
21.5
21.4
21.6
22.5
21.7
21.5
21.2
21.6
757
S. Müller-Herbst and others
Table 1. cont.
Name
Gene
Description
lmo2764*
Similar to xylose operon regulatory protein and to glucose kinase
lmo2785
kat
Catalase
lmo2788*
bvrA
Transcription antiterminator
lmo2797
Similar to PTS mannitol-specific enzyme IIA
lmo2798
Similar to phosphatase
lmo2799*
Similar to PTS mannitol-specific enzyme IIBC
lmo2800
Similar to dehydrogenase
lmo2831
Similar to phosphoglucomutase
lmo2832
Similar to unknown proteins
(b) Anaerobically upregulated genes
lmo0279
Highly similar to anaerobic ribonucleoside-triphosphate reductase
lmo0280
Highly similar to anaerobic ribonucleotide reductase activator protein
lmo0355
Similar to flavocytochrome c fumarate reductase chain A
lmo0434
inlB
Internalin B
lmo0471
CS
lmo0641
Similar to heavy-metal-transporting ATPase
lmo0814
Similar to oxidoreductases
lmo0897
Similar to transport proteins
lmo0903
Conserved hypothetical protein
lmo0912
Similar to transporters (formate)
lmo0971
dltD
DltD protein for D-alanine esterification of lipoteichoic acid and wall
teichoic acid
D-Alanyl carrier protein
lmo0972
dltC
lmo0973
dltB
DltB protein for D-alanine esterification of lipoteichoic acid and wall
teichoic acid
lmo1251
Similar to regulator of the Fnr CRP family (including PrfA)
lmo1310
Similar to Escherichia coli YbdN protein
lmo1475
hrcA
Transcription repressor of class I heat shock gene HrcA
lmo1634
adh
Similar to alcohol acetaldehyde dehydrogenase
lmo1901
panC
Similar to pantothenate synthetases
lmo1902
panB
Similar to ketopantoate hydroxymethyltransferases
lmo2114
Similar to ABC transporter (ATP-binding protein)
lmo2115
Similar to ABC transporter (permease)
lmo2361
Conserved hypothetical protein
lmo2467
Similar to chitinase and chitin-binding protein
lmo2485
Similar to Bacillus subtilis YvlC protein
lmo2486
CS
lmo2487
Similar to Bacillus subtilis YvlB protein
lmo2669
CS
lmo2805
Hypothetical secreted protein
Operon organization
log2RTL
lmo2761–lmo2765
21.6
21.6
21.5
22.6
22.5
23.4
22.5
21.5
21.3
lmo2801–lmo2795
lmo2801–lmo2795
lmo2801–lmo2795
lmo2801–lmo2795
lmo2832–lmo2830
lmo2832–lmo2830
lmo0279–lmo0280
lmo0279–lmo0280
dltABCD
2.5
2.3
2.3
1.3
1.0
2.1
1.1
2.3
1.6
1.3
1.2
dltABCD
dltABCD
1.3
1.5
inlAB
lmo1312–lmo1307
lmo1475–lmo1470
panBCD
panBCD
lmo2114–lmo2115
lmo2114–lmo2115
lmo2361–lmo2360
lmo2487–lmo2484
lmo2487–lmo2484
lmo2487–lmo2484
lmo2805–lmo2803
1.6
1.4
1.1
1.4
1.1
1.5
1.2
1.2
1.4
1.4
1.2
1.6
1.5
1.5
1.8
*Transcription has been shown to be upregulated in Listeria monocytogenes in both hprK and ccpA insertion mutants (Mertins et al., 2007).
(lmo0722). We have seen no oxygen-dependent transcriptional regulation of lmo0722, but the enzymic reaction
depends strictly on the presence of oxygen, indicating
that this pathway is only active during aerobic growth.
Furthermore, pyruvate can be metabolized to acetate via
acetyl-CoA. The conversion from pyruvate to acetyl-CoA is
catalysed by the pyruvate dehydrogenase complex, encoded
by pdhABCD. Interestingly, this pathway seems to be
favoured aerobically, as transcription of the pdhABCD
operon is clearly downregulated anaerobically (log2RTL
24.6, 24.7, 24.6 and 24.6). During this reaction, NAD+ is
reduced to NADH. NADH can be reoxidized aerobically to
758
NAD+ via the activity of NADH dehydrogenases, thereby
contributing to the establishment of a proton motive force
via the respiratory chain. It is worth mentioning that during
all of the reactions described so far, carbon dioxide, a major
aerobic end product, is released. Another possibility for
acetyl-CoA formation is the cleavage of pyruvate via
pyruvate formate lyase PflA or PflB. We have no indications
for an oxygen-dependent regulation of pflA/pflB (lmo1917/
lmo1406) transcription, suggesting that this reaction might
take place both aerobically and anaerobically. Acetyl-CoA
can be converted to acetate via acetyl-P via the activity of
phosphotransacetylase and acetate kinase.
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Microbiology 160
Anaerobic growth of Listeria monocytogenes
log2RTL
qRT-PCR
4
lmo0279
Fig. 2. Validation of microarray analysis
by qRT-PCR. We investigated the oxygendependent transcription of the anaerobically
downregulated genes lmo0014, lmo0914,
lmo1044, lmo1293, lmo1538, lmo1879,
lmo2006, lmo2161, lmo2389, lmo2586 and
lmo2650 as well as the transcription of the
anaerobically upregulated genes lmo0279,
lmo0355, lmo0434 and lmo0971 in Listeria
monocytogenes EGD. The log2RTLs of the
respective genes in anaerobically versus aerobically grown cultures of the microarray analysis are plotted against qRT-PCR data.
The coefficient of determination R250.91
was calculated using Microsoft Excel.
3
lmo0434
2
lmo0355
lmo0971
1
0
–6
–5
–4
–3
–2
–1
0
1
2
–1
lmo2586 lmo2389
lmo1044–2
lmo1879 lmo2006
lmo0914
–3
lmo2161
lmo1538
lmo1293
–4
lmo0014
lmo2650
–5
3
log2RTL
4 microarray
R 2=0.91
–6
Although lactate is the major end product during anaerobic
growth, we did not observe enhanced transcription of
lmo0210 (ldh), encoding lactate dehydrogenase, under
these growth conditions. However, as several branches of
pyruvate metabolism (pyruvate dehydrogenase, acetolactate
synthase, pyruvate oxidase) are repressed anaerobically,
we suggest that this channels pyruvate to the lactate
formation pathway. NADH can be reoxidized to NAD+
via this fermentative reaction in the absence of a respiratory
chain.
acetoin
CO2 + H2O2
CO2
lmo0722
lmo1992
CO2
2-acetolactate
NADH
NAD+
pdhABCD
CO2
ADP
acetyl-P
pta
alsS
2x
glycolysis
pyruvate
carbohydrate
pentose phosphate cycle
NADH
NADH
ldh*
ackA1/ackA2
acs
acetyl-CoA
pflA/pflB
NAD+ NADH
fdh
NAD+
adh
CO2
formate
ATP
acetate
NAD+
acetaldehyde
NADH
adh
NAD+
ethanol
lactate
Fig. 3. Model of aerobic and anaerobic glucose catabolism in Listeria monocytogenes. The model of Listeria monocytogenes
carbohydrate catabolism is based on the physiological study of Romick et al. (1996) and the KEGG database. Main aerobic
pathways are highlighted by black dotted lines, main anaerobic pathways by grey dashed lines. The major end products of
aerobic carbohydrate catabolism are acetate, acetoin, lactate and carbon dioxide (highlighted in bold). Anaerobically, the major
end product of carbon metabolism is lactate. Acetate, ethanol and formate are minor end products. Anaerobically generated end
products are underlined. Genes whose products are involved in the respective metabolic pathways are marked; those showing
oxygen-dependent regulation are highlighted in bold. Furthermore, lmo0722 is highlighted in bold. It encodes pyruvate oxidase
– an enzyme that requires oxygen for its activity. *Pyruvate is reduced to lactate by lactate dehydrogenase both aerobically and
anaerobically. However, this pathway is more active anaerobically. ackA1/ackA2, acetate kinase; acs, acetyl-CoA synthetase;
adh, bifunctional acetaldehyde-CoA/alcohol dehydrogenase; alsS, acetolactate synthase; fdh, formate dehydrogenase
(a subunit); ldh, lactate dehydrogenase; pdhABCD, pyruvate dehydrogenase; pflA/pflB, pyruvate formate lyase; pta,
phosphotransacetylase; lmo0722, pyruvate oxidase; lmo1992, acetolactate decarboxylase.
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759
S. Müller-Herbst and others
An alternative pathway, in which NADH is reoxidized to
NAD+, is the reduction of acetyl-CoA to acetaldehyde and
further to ethanol via the bifunctional acetaldehyde-CoA/
alcohol dehydrogenase (adh, lmo1634). Transcription of
adh was found to be upregulated anaerobically in the
present study (log2RTL 1.4). A transcriptional induction of
adh under conditions of oxygen deficiency has also been
described previously (Burkholder et al., 2009).
Another end product specific for anaerobic metabolism is
formate. We speculate that during aerobic growth formate
is completely oxidized to carbon dioxide by formate
dehydrogenase (fdh, lmo2586). Transcription of fdh is
downregulated anaerobically (log2RTL 22.3), indicating
that oxidation of formate to carbon dioxide might not
occur anaerobically, leading to potentially toxic formate
accumulation. This hypothesis is further underlined by the
observation that during anaerobic growth transcription of
a gene encoding a protein of the formate/nitrite transporter
family (lmo0912) is upregulated (log2RTL 1.3), suggesting
that formate is exported.
Taken together, our transcriptional data fit very well with
previous physiological data on the metabolism of Listeria
monocytogenes and give us a general view about which of
these metabolic adaptations are regulated, at least in part,
at the transcriptional level.
It is worth mentioning at this point that amongst the
111 genes found to be downregulated anaerobically, 33
genes (marked by an asterisk in Table 1) were described
previously as being upregulated in both a hprK and ccpA
insertion mutant in Listeria monocytogenes (Mertins et al.,
2007). The Hpr kinase (encoded by hprK) phosphorylates
the cofactor of the catabolite control protein A (CcpA,
encoded by ccpA), Hpr (encoded by ptsH), at position
Ser46. CcpA in complex with its phosphorylated cofactor
Hpr-Ser46~P, is the key regulator during catabolite
repression in low-GC Gram-positive bacteria (reviewed
by Deutscher et al., 2006). A stronger transcription of
catabolite-controlled genes during aerobic growth suggests
a more stringent catabolite repression during anaerobic
growth. This makes sense in the way that we performed
our experiments in the complex BHI medium, in
which, besides glucose (2 %), other carbon sources are
also present. Anaerobically, the use of the preferred
carbon source glucose allows maximal ATP generation.
Aerobically, when cells perform respiration, ATP yield is
probably not limiting and other carbon sources can also be
exploited for growth.
Anaerobically upregulated genes are not
essential for anaerobic growth
In total, 28 genes were found in the microarray analysis to
be anaerobically upregulated (Table 1b). To investigate
whether some of these are essential for anaerobic growth,
knock out mutants for 20 of the respective genes were
constructed and their anaerobic growth was analysed
760
(Table 2). Mutants were streaked out in parallel on two
BHI agar plates, the plates were incubated for 48 h at 42 uC
either in an aerobic or an anaerobic atmosphere and then
plates were checked for a growth/no-growth phenotype of
the respective mutants.
All mutants were able to grow aerobically and anaerobically
(summarized in Table 2, Figs S2 and S3), suggesting that
none of the investigated genes is essential for anaerobic
growth of Listeria monocytogenes EGDe on BHI medium.
Anaerobiosis triggers virulence gene expression
in Listeria monocytogenes
Although none of the anaerobically more strongly transcribed genes is essential for anaerobic survival in vitro, it is
tempting to speculate that the observed adaptation to
anaerobiosis could improve bacterial fitness in low-oxygen
niches in the natural environment. Anaerobiosis could be an
environmental signal to trigger the initial colonization of
Listeria monocytogenes in the intestine and mediate an
advantage during in vivo growth. It has already been shown
that anaerobic pre-culture of Listeria monocytogenes
enhances adhesion in in vitro cell culture assays and
virulence in vivo in the guinea pig model (Bo Andersen
et al., 2007). Anaerobic induction of InlB (inlB, lmo0433,
log2RTL 1.3), involved in adherence to (Lindén et al., 2008)
and invasion in (Pentecost et al., 2010) intestinal tissue, and
LAP (a bifunctional enzyme, also with metabolic capability,
lmo1634, described previously in the text as adh, log2RTL
1.4), involved in adherence (Burkholder et al., 2009) and
paracellular translocation (Burkholder & Bhunia, 2010;
Kim & Bhunia, 2013), was already described previously
(Burkholder et al., 2009; Stritzker et al., 2004, 2005). In
addition, we observed anaerobically an upregulation of
lmo0971–lmo0973, encoding the Dlt proteins involved in Dalanine esterification of lipoteichoic acid and wall teichoic
acid (dltD, dltC and dltB, log2 1.2, 1.3 and 1.5). The D-alanine
esterification has been shown to contribute to adhesion to
host cells and to virulence in Listeria monocytogenes (Abachin
et al., 2002). Furthermore, lmo2467, encoding a protein
similar to chitinase and chitin-binding protein, was more
strongly transcribed anaerobically than aerobically (log2RTL
1.4). It has already been shown that this gene also contributes
to virulence in Listeria monocytogenes (Chaudhuri et al.,
2010). The enhanced transcription of several virulence genes
during anaerobic growth could enhance the initial colonization of the intestine in vivo.
Fumarate supports anaerobic growth of Listeria
monocytogenes
Not only an enhanced expression of virulence genes, but
also metabolic adaptations could improve colonization.
One of the anaerobically upregulated genes is the
monocistronically transcribed gene lmo0355 (log2RTL
2.3), encoding a protein similar to the fumarate reductase
a chain. To analyse the physiological function of the
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Microbiology 160
Anaerobic growth of Listeria monocytogenes
Table 2. Phenotypic growth analysis of knock out mutants
Gene
Mutant
Name
Aerobic growth
Anaerobic growth
Operon organization*
lmo0279
lmo0280
lmo0355
lmo0434
lmo0471
lmo0641
lmo0814
lmo0897
lmo0903
lmo0912
lmo0971
lmo0972
lmo0973
lmo1251
lmo1310
lmo1475
lmo1634
lmo1901
lmo1902
lmo2114
lmo2115
lmo2361
lmo2467
lmo2485
lmo2486
lmo2487
lmo2669
lmo2805
Insertion
Insertion
Deletion
Insertion
No
Deletion
Insertion
Insertion
NoD
Insertion
No
NoD
Deletion
Deletion
Insertion
Nod
Insertion
Insertion
Insertion
Insertion
Insertion
NoD
Insertion
NoD
Insertion
Insertion
Insertion
NoD
lmo0279 : : pLSV101
lmo0280 : : pLSV101
Dlmo0355
lmo0434 : : pLSV101
–
Dlmo0641
lmo0814 : : pLSV101
lmo0897 : : pLSV101
–
lmo0912 : : pLSV101
–
–
Dlmo0973
Dlmo1251
lmo1310 : : pLSV101
–
lmo1634 : : pLSV101
lmo1901 : : pLSV101
lmo1902 : : pLSV101
lmo2114 : : pLSV101
lmo2115 : : pLSV101
–
lmo2467 : : pLSV101
–
lmo2486 : : pLSV101
lmo2487 : : pLSV101
lmo2669 : : pLSV101
–
+
+
+
+
+
+
+
+
lmo0279–lmo0280
lmo0279–lmo0280
ND
ND
+
+
+
+
+
+
ND
ND
+
+
ND
ND
ND
ND
+
+
+
+
+
+
ND
ND
+
+
+
+
+
+
+
+
+
+
ND
ND
+
+
ND
ND
+
+
+
+
+
+
ND
ND
inlAB
dltABCD
dltABCD
dltABCD
lmo1312–lmo1307
lmo1475–lmo1470
panBCD
panBCD
lmo2114–lmo2115
lmo2114–lmo2115
lmo2361–lmo2360
lmo2487–lmo2484
lmo2487–lmo2484
lmo2487–lmo2484
lmo2805–lmo2803
+, Growth; ND, not determined.
*Already described operon organizations (Toledo-Arana et al., 2009) are indicated.
DNo mutant was constructed due to the short gene sequence (,500 bp).
dConstruction of insertion mutant was unsuccessful.
anaerobic upregulation of lmo0355, we investigated
whether the addition of fumarate enhances anaerobic
growth. Growth of Listeria monocytogenes EGDe WT was
analysed in BHI medium under aerobic and anaerobic
conditions in the presence or absence of fumarate
(10 mM). Fumarate slightly supports anaerobic but not
aerobic growth of Listeria monocytogenes EGDe (Fig. 4a).
Aerobically, the deletion mutant Dlmo0355 showed the
same growth characteristics as the WT, i.e. fumarate had
no stimulatory effect. However, the growth-supportive
effect of fumarate during anaerobic growth was completely
lost in the deletion mutant (Fig. 4b). This growth
disadvantage was restored by complementation of the
deletion with the WT allele lmo0355 in cis in the respective
gene locus in the complementation mutant Dlmo0355comp (Fig. 4c). The stimulatory effect of fumarate on
growth of Listeria monocytogenes EGDe under anaerobic
conditions depends, therefore, on the activity of the
fumarate reductase a chain. This growth support during
anaerobiosis was even more pronounced in a chemically
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defined minimal medium (data not shown). Whilst
fumarate respiration has recently been shown to play an
important role during colonization of the mouse intestine
by E. coli (Jones et al., 2007, 2011), Listeria monocytogenes is
not known to perform fumarate respiration. Nevertheless,
reduction of fumarate may be involved in the regeneration
of reduction equivalents during fermentative growth.
Therefore, it is tempting to speculate that such a metabolic
adaptation could improve bacterial fitness during anaerobic
in vivo growth in the presence of fumarate – a hypothesis
that is currently under investigation in our laboratory.
F1F0-ATPase is essential for anaerobic growth of
Listeria monocytogenes
The transcriptional upregulation of specific genes during
anaerobiosis might improve the overall fitness of Listeria
monocytogenes in anaerobic niches. However, none of
the transcriptionally upregulated genes was found to be
essential for growth under oxygen-deprived conditions. To
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S. Müller-Herbst and others
(a)
Listeria monocytogenes EGDe WT
F1
(a)
F0
3.5
3.0
ε
β
γ
α
atpC
atpD
atpG
atpA
δ
b
c
a
i
OD600
2.5
2.0
1.5
(b)
1.0
WT
DatpA
atpH atpF atpE atpB atpI
DatpB
DatpD
0.5
0
0
100
200
300
400
500
aerobic
(b) Dlmo0355
3.5
3.0
anaerobic
OD600
2.5
2.0
1.5
1.0
DatpA-comp DatpB-comp DatpD-comp
(c)
0.5
0
0
100
200
300
400
500
anaerobic
(c) Dlmo0355-comp
3.5
WT
pHPS9
3.0
OD600
2.5
DatpA
pHPS9
DatpB
pHPS9
DatpD
pHPS9
2.0
anaerobic
1.5
1.0
0.5
0
0
100
200 300
Time (min)
400
500
Fig. 4. Fumarate stimulates anaerobic growth of Listeria monocytogenes EGDe. Growth of (a) WT Listeria monocytogenes
EGDe, (b) deletion mutant Dlmo0355 and (c) complementation
mutant Dlmo0355-comp was analysed in BHI medium (black
squares) and in BHI medium supplemented with 10 mM fumarate
(grey triangles) aerobically (solid lines) and anaerobically (dotted
lines). Growth curves were calculated based on the mean±SD
OD600 of three biologically independent growth experiments.
further analyse whether Listeria monocytogenes harbours
genes that might be essential for anaerobic growth, an
insertion mutant library of Listeria monocytogenes EGD
(Schauer et al., 2010) was screened for mutants showing an
anaerobic growth-defective phenotype (Fig. S1). Of 1360
clones screened, 11 mutants did not grow anaerobically,
whilst they did grow aerobically. All of them carry the
insertion in the atp operon (atpIBEFHAGDC) (Fig. 5a) that
encodes F1F0-ATP synthetase/F1F0-ATPase. Seven mutants
762
Fig. 5. F1F0-ATPase is essential for anaerobic growth of Listeria
monocytogenes. (a) Organization of the atp operon in Listeria
monocytogenes EGDe. The atp operon encoding F1F0-ATP
synthetase of Listeria monocytogenes EGDe consists of nine
genes: atpI, encoding a non-structural protein; atpB, atpE and
atpF, encoding the subunits a, c and b of the F0 subcomplex
(proton channel); and atpH, atpA, atpG, atpD and atpC, encoding
the subunits d, a, c, b and e of the F1 subcomplex, containing the
catalytic sites for ATP synthesis or hydrolysis. atpB, atpA and atpD,
the genes found to be essential for anaerobic growth of Listeria
monocytogenes EGD via the high-throughput approach, are
highlighted in dark grey. (b) Phenotypic analysis of the deletion
mutants DatpA, DatpB and DatpD. The WT Listeria monocytogenes EGDe and the mutants were grown aerobically or
anaerobically on BHI for 48 h. (c) Anaerobic growth of the
complementation mutants DatpA-comp, DatpB-comp and DatpDcomp. Growth of the complementation mutants was compared
with that of the WT and the deletion mutants transformed with an
empty pHPS9 vector (WT pHPS9, DatpA pHPS9, DatpB pHPS9
and DatpD pHPS9). Strains were incubated anaerobically on BHI
agar with erythromycin for 48 h.
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Anaerobic growth of Listeria monocytogenes
are interrupted in the atpB gene, three in the atpA gene and
one in the atpD gene.
These results indicate that the expression of a functional
F1F0-ATP synthetase/F1F0-ATPase complex is essential for
anaerobic growth of Listeria monocytogenes. To exclude
polar effects due to the insertion duplication process, the
in-frame deletion mutants DatpA, DatpB and DatpD were
constructed in the genetic background of strain EGDe.
These mutants were able to grow aerobically but not
anaerobically (Fig. 5b), confirming the results from the
growth analysis of the insertion mutants. Next, the in trans
complementation mutants DatpA-comp, DatpB-comp and
DatpD-comp were constructed and analysed (Fig. 5c).
Complementation of the atpB and atpD deletions was
successful, and the mutants DatpB-comp and DatpD-comp
were able to grow anaerobically. Surprisingly, although
exploiting the same experimental setup, complementation
of the atpA deletion was not possible. The mutant DatpAcomp was not able to grow anaerobically, the reason for
this finding being unknown. It might be that a strict
regulation of atpA expression, which is stronger in the WT
Listeria monocytogenes EGDe than in the complementation
mutant DatpA-comp (data not shown), is essential for
complementation of the anaerobic phenotype. However, at
this point we also cannot exclude a secondary mutation in
the DatpA mutant.
It is assumed that during aerobic growth Listeria monocytogenes generates ATP via substrate-level phosphorylation
during glycolysis and via H+-gradient-dependent phosphorylation at F1F0-ATP synthetase. As the deletion mutants
DatpA, DatpB and DatpD are still able to grow aerobically,
ATP generation via substrate-level phosphorylation seems
to be sufficient for aerobic growth of Listeria monocytogenes
EGDe. It has already been shown for the facultative anaerobic bacteria Bacillus subtilis (Santana et al., 1994) and
Corynebacterium glutamicum (Koch-Koerfges et al., 2012)
that F1F0-ATP synthetase and ATP generation via oxidative
phosphorylation are not essential for aerobic growth.
However, F1F0-ATP synthetase can also export protons at the
expense of ATP, thus working as an F1F0-ATPase. This
function of the F1F0 complex seems to be the essential function
for anaerobic growth for two reasons. (1) During anaerobic
growth more organic acid is produced (Romick et al., 1996)
than during aerobic growth, leading to a faster acidification of
the growth medium. Export of protons might help to
maintain a near-neutral cytoplasmic pH. It has been shown
previously that F1F0-ATPase is involved in acid tolerance in
Listeria monocytogenes (Cotter et al., 2000) and other bacteria,
e.g. Helicobacter pylori (Bijlsma et al., 2000), Salmonella
enterica (Turner et al., 2003), Lactococcus lactis (Amachi et al.,
1998), and oral Streptococci (Bender et al., 1986).
(2) It seems plausible that Listeria monocytogenes uses the
proton pumping function of F1F0-ATPase to establish an
essential proton gradient during anaerobic growth, e.g.
driving transport processes over the cytoplasmic membrane. This hypothesis is further strengthened by the fact
http://mic.sgmjournals.org
that the F1F0-ATPase complex is also essential for growth
of Lactococcus lactis. This organism naturally lacks a
complete respiratory chain (Brooijmans et al., 2007) and
it was speculated that F1F0-ATPase is involved in creating
a proton motive force that is sufficient for growth
(Koebmann et al., 2000).
Concluding remarks
A deeper insight in the adaptation of Listeria monocytogenes
to anaerobiosis improves our understanding of the
adaptation of the bacterium to anaerobiosis in its
ecological niches. Our global transcriptional analyses of
adaptation to different oxygen levels are in line with early
physiological studies and provide us with a deeper insight
into how Listeria monocytogenes adapts its metabolism
according to oxygen availability. Furthermore, our data
strengthen the hypothesis that anaerobiosis serves as an
environmental signal that triggers virulence gene expression, which might improve initial colonization of the
mammalian hosts. However, not only virulence gene
expression, but also induced expression of metabolism
genes like lmo0355 might help the bacterium to optimally
adapt to anaerobic niches in its natural environments.
Finally, we identified F1F0-ATPase to be essential for
anaerobic growth of Listeria monocytogenes. Essential
metabolic pathways or genes are promising drug targets.
Indeed, the essential function of the F1F0-ATP synthetase/
F1F0-ATPase complex has been shown to be a promising
drug target in Mycobacterium tuberculosis (reviewed by
Palomino & Martin, 2013) even though in this case ATP
synthesis is the essential function of the complex (Haagsma
et al., 2010).
ACKNOWLEDGEMENTS
Regina Fischer, Lisa Schürch, Tom Schwarzer, Constantin Stautner
and Julia Wiesner are thanked for help with cloning, qRT-PCR and
growth analysis during their microbiological internship or bachelor
theses. Katharina Sturm is thanked for technical assistance. This
research project was supported partly by the German Ministry of
Economics and Technology (via AiF) and the Forschungskreis der
Ernährungsindustrie eV (Bonn). Project AiF 15835N.
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