Download Genome-wide transcription profiling of aerobic and anaerobic

FEMS Microbiology Letters, 364, 2017, fnx006
doi: 10.1093/femsle/fnx006
Advance Access Publication Date: 13 January 2017
Research Letter
R E S E A R C H L E T T E R – Physiology & Biochemistry
Genome-wide transcription profiling of aerobic
and anaerobic Escherichia coli biofilm and
planktonic cultures
Bihter Bayramoglu, David Toubiana and Osnat Gillor∗
Zuckerberg Institute for Water Research, Blaustein Institutes for Desert Research, Ben Gurion University
of the Negev, Midreshet Ben Gurion 84990, Israel
∗
Corresponding author: Zuckerberg Institute for Water Research, Blaustein Institutes for Desert Research, Ben Gurion University of the Negev, Sde Boker
Campus, Midreshet Ben Gurion 84990, Israel. Tel: +972-8-6596986; E-mail: [email protected]
One sentence summary: This study explores global regulation of Escherichia coli biofilm cultured with and without oxygen.
Editor: Jana Jass
ABSTRACT
Many studies have described the response of the facultative anaerobe, Escherichia coli, to anaerobic conditions, yet they all
investigated free-living (planktonic) cells because attempts to cultivate anaerobic E. coli biofilm were mostly unsuccessful.
We challenged these findings and cultivated E. coli strain MG1655 biofilm under both aerobic and anaerobic conditions,
characterizing the mature biofilm architecture and global gene expression profile. We used RNA sequencing technology to
compare stationary phase planktonic cells with mature biofilm, cultured with and without oxygen. Our results suggest that
gene expression patterns significantly differ between biofilm and planktonic cultures cultivated under the same oxygenic
conditions. The anaerobic E. coli biofilms were slow growing and patchy compared to aerobic biofilms, yet some features
were unchanged like the production of extracellular polymeric substances. A closer inspection of the mRNA data revealed
that essential cell processes were attenuated in anaerobic biofilms, including protein synthesis, information transfer, cell
structure, regulation and transport. Our results suggest that lack of oxygen imposes severe stress on mature biofilms thus
limiting the cells’ activity. We further propose that E. coli does not favor growing in anaerobic biofilms and when forced to
do so, the cells prevail by attenuating their activity in order to survive.
Keywords: sessile; flowcell; transcriptomics
INTRODUCTION
The gastrointestinal (GI) tract is dominated by diverse anaerobic bacterial communities, even though it is an open system
with an oxygen gradient running from its proximal to distal
end (Macfarlane and Dillon 2007). The task of sustaining an
oxygen-free environment for the dominating gram-positive obligate anaerobes falls onto facultative anaerobes such as Escherichia coli and its close relatives, which metabolize traces of
oxygen in the GI biofilm (Jones et al. 2011). In the GI tract, E.
coli resides in biofilms—sessile communities of microorganisms
associated with the GI epithelial mucosa (Beloin, Roux and Ghigo
2008). Global expression profiles of E. coli biofilms and freeliving (planktonic) cultures showed distinct patterns, negating
the assumption that biofilms are sessile cells in stationary phase
(Schembri, Kjaergaard and Klemm 2003). Moreover, E. coli cells in
a given biofilm may experience different growth conditions and
depending on the cells’ position within the biofilm, may also experience aerobic, microaerobic or anaerobic conditions (Karatan
and Watnick 2009). The kinetics of E. coli strain MG1655 biofilms
grown under aerobic and microaerobic conditions have been
Received: 7 May 2016; Accepted: 11 January 2017
C FEMS 2016. All rights reserved. For permissions, please e-mail: [email protected]
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FEMS Microbiology Letters, 2017, Vol. 364, No. 3
described (Beloin et al. 2003; Bjergbæk et al. 2006; Hancock and
Klemm 2007), but anaerobic conditions have been suggested to
abate this strain’s biofilm formation (Landini and Zehnder 2002;
Colón-González, Méndez-Ortiz and Membrillo-Hernández 2004).
Under controlled laboratory conditions, planktonic E. coli
cells grow rapidly when respiring oxygen. In the absence of
oxygen and alternative electron acceptors, the cells switch to
anaerobic respiration and fermentation (Partridge et al. 2006).
However, it was shown that E. coli anaerobic biofilms could
not be formed even when cultures were supplemented with
various electron acceptors (Colón-González, Méndez-Ortiz and
Membrillo-Hernández 2004), or when longer incubation periods
were imposed (Colón-González, Méndez-Ortiz and MembrilloHernández 2004). Furthermore, a survey of over 45 000 random
mutants failed to identify an E. coli strain that was able to form
a biofilm under anaerobic conditions (Cabellos-Avelar, Souza
and Membrillo-Hernández 2006). But, when 77 E. coli strains isolated from various environments were tested, 7 formed a biofilm
under anaerobic conditions (Colón-González, Méndez-Ortiz and
Membrillo-Hernández 2004). It was further suggested that laboratory E. coli strains could not form a biofilm under anaerobic
conditions because lipopolysaccharide and flagellum biosynthesis are attenuated and, therefore, E. coli’s initial attachment efficiency decreases (Landini and Zehnder 2002).
As the growth of anaerobic biofilms of laboratory E. coli
strains was unsuccessful, studies focused on planktonic cultures, elucidating the transition patterns from aerobic to microaerobic and anaerobic environments (Partridge et al. 2006;
Trotter et al. 2011). But, E. coli genome-wide analyses have
demonstrated marked differences between the transcriptome of
planktonic and biofilm cultures (Beloin, Roux and Ghigo 2008;
Wood 2009). Therefore, we hypothesized that E. coli biofilms
would respond differently to oxygen limitation than planktonic
cells. To address this hypothesis, we developed a method that
enables the growth of E. coli strain MG1655 biofilm under anaerobic conditions. We then compared biofilm structure and global
gene expression profiles of mature biofilms to stationary-phase
planktonic E. coli cultivated with and without oxygen. We hypothesize that the structure and molecular characteristics of the
anaerobic biofilm significantly differ from those of the aerobic
biofilm, yet the differences between planktonic and biofilm cultures would be more prominent.
MATERIAL AND METHODS
For detailed descriptions of the material and methods used in
this study, see supplementary information file S1.
Bacterial strain and culture conditions
All experiments were performed with Escherichia coli strain
MG1655 (F- lambda- ilvG- rfb-50 rph-1) cultured in M9 minimal
medium (Sigma, Rehovot, Israel) supplemented with 4 g L−1 Casein Digest (BD, Franklin Lakes, NJ, USA) at 25◦ C. For each treatment, two biological replicates were prepared and each was
replicated at least three times.
Overnight planktonic E. coli cultures were refreshed and
grown to early exponential phase and then divided for aerobic
and anaerobic incubation. The duration of cultivation differs because E. coli cultures grow faster in aerobic compared to anaerobic conditions. The specified growth rates were verified in preliminary experiments (data not shown).
Attempts at cultivating E. coli anaerobic biofilms were
proven to be unsuccessful when using microtiter plates
(Colón-González, Méndez-Ortiz and Membrillo-Hernández 2004)
or sand columns (Landini and Zehnder 2002). We applied a flow
cell system to culture the biofilms, yet commercial systems are
limited by their surface area (as they mostly include a single
slide) and by the frequent formation of air bubbles that can lead
to the destruction of biofilm architecture (Crusz et al. 2012). To
overcome these limitations, we used a custom designed flow cell
system (Majeed et al. 2014) in an incubator or anaerobic chamber (Coy Laboratory Products, Grass Lake, MI, USA) for 3 and 6
days, respectively. The duration of cultivation was determined
by preliminary experiments (Supplementary files).
Extracellular polymeric substances extraction
and analysis
Extracellular polymeric substances (EPS) was extracted from aerobic and anaerobic biofilms as previously described (Sweity et al.
2011).
Confocal laser-scanning microscopy
To visualize the biofilm cultures, slides were stained with 300 nM
4-6-diamidino-2-phenylindole (DAPI; Sigma), imaged by confocal laser-scanning microscopy (Zeiss M510, Oberkochen, Germany) and the 3D images were reconstituted using IMARIS software (Bitplane, Zurich, Switzerland).
RNA extraction and sequencing
Total RNA was extracted (Epicentre, Madison, WI) from two biological replicates of about 1010 planktonic cells and from the
slides supporting 35 ± 5 μm3 μm−2 aerobic biofilm and 2.7 ±
0.3 μm3 μm−2 anaerobic biofilm. High-throughput sequencing
of the mRNA was carried out by the Genome Center at the Israel
Institute of Technology as previously described (Brownstein et al.
2014).
Data acquisition and analysis
About 90%–95% of all reads were aligned to non-rRNA regions
of the E. coli MG1655 genome (accession number U00096.2) using Rockhopper analysis tool (McClure et al. 2013). The sequence
reads of all samples were deposited in NCBI’s Gene Expression
Omnibus (Edgar 2002) and are accessible through GEO Series accession number GSE72113.
Significant differences in gene expression were considered
only when expression changed at least 2.5-fold (Fitzgerald,
Bonocora and Wade 2014; Shao et al. 2015). Genes were classified with MultiFun (Serres and Riley 2000) and annotated using
EcoCyc database (Keseler et al. 2013). An overview of the differentially regulated genes was constructed in heatmaps using
R software (Ihaka and Gentleman 1996). Differences of expression levels between the different environments were attempted
with statistically stringent approach, integrated in DESeq package (Anders and Huber 2010), and a less stringent approach,
integrated in limma package (Ritchie et al. 2015) and Biobase (Gentleman et al. 2004) in R environment. Gene expression distribution among treatments was visualized by boxplot showing
that normalized gene counts were reproducible between biological replicates (Fig. S1, Supporting Information). Partitioning
around medoids (PAM) clustering was performed to determine
the optimal number of clusters of the dataset (Fig. S2, Supporting Information) and principal component analyses (PCA)
were performed on the multivariate datasets using pcaMethods
Bayramoglu et al.
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Figure 1. Biofilms of E. coli strain MG1655. Biofilms cultured (A) for 3 days under aerobic conditions and (B) for 6 days under anaerobic conditions. The reconstructed
confocal images were obtained after culturing the biofilms in continuous-flow chambers. Each square on the grid of image A is 50 μm and of image B, 80 μm.
Table 1. Properties of EPS in E. coli biofilms cultured under aerobic
and anaerobic conditions.
EPS contents (ppm)
Total organic carbon
Polysaccharides
Total nitrogen
Protein
Aerobic
44.08
41.2
10.96
12.1
±
±
±
±
5.27
3.43
1.28
1.51
indicate that E. coli cells maintain their overall biofilm structure
despite the oxygen-related stress.
Anaerobic
54.24
40.5
13.25
15.6
±
±
±
±
7.02
3.68
1.79
1.76
package from Bioconductor (http://bioconductor.org) (Stacklies
et al. 2007) in the R environment.
RESULTS AND DISCUSSION
Biofilm formation and structure under
anaerobic conditions
We cultured the Escherichia coli biofilm with and without
oxygen and noted that under anaerobic conditions biofilm development was significantly slower. The mature, 6-days-old,
anaerobic biofilm was patchier and thinner compared to the
aerobic biofilm (Fig. 1). While the biovolume of aerobic biofilm
was 60 ± 10 μm3 μm−2 , anaerobic biofilm had a biovolume of
4 ± 1 μm3 μm−2 . Aerobic biofilms maximum thickness went
up to 69.1 ± 15.5 μm with 89.8 ± 5.4% substratum coverage.
On the other hand, anaerobic biofilms had a maximum thickness of 50.8 ± 11.5 μm with 70.2 ± 9.5% substratum coverage. This suggests that lack of oxygen limits E. coli biofilm formation but, in contrast to conclusions drawn from previous
studies (Landini and Zehnder 2002; Colón-González, MéndezOrtiz and Membrillo-Hernández 2004; Cabellos-Avelar, Souza
and Membrillo- Hernández 2006), it does not prevent it. Surprisingly, the EPS properties of the aerobic and anaerobic biofilms
including the total organic carbon, polysaccharide, total nitrogen and protein contents (Table 1) were similar in E. coli biofilms
grown under both the aerobic and anaerobic conditions (P >
0.05). Moreover, RNA sequencing revealed that the genes promoting EPS production (Ionescu and Belkin 2009), such as the
wca, pga and yjb gene operons as well as the dfc pseudo-operon,
were similarly expressed in aerobic and anaerobic biofilms (2>
q value > 0.5; Table S1, Supporting Information). These results
Quantitative analysis of planktonic and biofilm
Escherichia coli transcriptomes
We first investigated whether the oxygen treatments or growth
conditions (planktonic vs biofilm) play a more significant role
in differential gene expression profiles of E. coli cells. This was
achieved by portraying hierarchal cluster heatmaps of the normalized gene expression (Fig. 2) and by using PCA clustering (Fig. 3). The comparative analyses of the genes differential expression are provided by the heatmaps (Fig. 2) revealing major differences between growth conditions, and to a
lesser extent oxygen treatment (Fig. S3, Supporting Information). The optimal number of clusters estimated by the PAM
clustering PCA plot suggested a separation into two groups
corresponding to the discrepancies between planktonic and
biofilm gene expression regardless of the oxygen treatment
(Fig. 3).
Both clustering analyses (Figs 2 and 3) validated our hypotheses that there are major differences between the E. coli
expression profiles under the two cultivation modes (biofilm
and planktonic). These differences were considerable amounting to over 40% of the E. coli genome under aerobic conditions and over 60% of the genome under anaerobic conditions
(Fig. S4; Tables S2 and S3, Supporting Information). Similarly,
global gene expression comparison portrayed by microarray
analysis between aerobic biofilm and planktonic Pseudomonas
aeruginosa cells revealed dramatic changes, with 26% of the
genes showing altered expression rates (Waite et al. 2006). In
contrast, a study testing the differences between E. coli cells in
the stationary phase and in mature biofilms under aerobic conditions using the microarray technology revealed only a 4% difference in gene expression (Beloin et al. 2003). We could not attribute these dramatic differences among studies to the difference in applied analyses, as an agreement between microarray and RNA-seq has been demonstrated (Malone and Oliver
2011); rather, the different outcomes could be attributed to differences in the biofilm-culturing protocols. Dötsch et al. (2012)
reported that there is little consistency in gene expression regulation patterns across various studies comparing planktonic
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Figure 2. Whole-gene expression profile of planktonic and biofilm cultures growing under aerobic and anaerobic conditions. A heatmap of a hierarchal cluster of
normalized gene expression for genes that were differentially regulated in planktonic and biofilm E. coli cultured with and without oxygen. The color key represents
the overall normalized gene expression pattern indicated by Z-score analysis. A comprehensive list of absolute and differential gene expression for all genes included
in this figure is provided in Table S1.
Quantitative analysis of aerobic and anaerobic
Escherichia coli transcriptomes
Figure 3. Cluster analysis of normalized gene expression in E. coli biofilm and
planktonic cultures for genes expressed under aerobic and anaerobic conditions.
PCA of gene expression levels of biofilms cultured under aerobic and anaerobic
conditions (red circles); planktonic cultures under aerobic and anaerobic conditions (blue triangles). PC1 accounts for 77% and PC2 for 16.1% of the variance
observed between expression levels. All experiments exert similar contributions
to PC1 as indicated by the direction and length of the arrows. Bio = biofilm; Pl =
planktonic; aero = aerobic; anaero = anaerobic.
and biofilm growth conditions in P. aeruginosa. The authors compared five studies testing the global gene expression under the
two growth conditions and pointed to substantial differences
among studies. They suggested that culturing conditions of the
tested biofilms have a major impact on the emerging transcriptome profiles (Dötsch et al. 2012). This may provide an explanation for the differences between our results and Beloin et al.
(2003) as we used a custom-made flow-cell system (Majeed et al.
2014), while they used a continuous-culture system in which the
biofilm is formed in a microfermenter. We thus suggest that further research is required to standardize biofilm-culturing conditions.
Planktonic cultures
Our results show that 80 genes out of 4321 significantly differ in expression and were either up- or downregulated in the
stationary-phase planktonic E. coli cultures grown under aerobic
vs anaerobic conditions (Fig. 4a; Table S4, Supporting Information). Among the 52 E. coli genes downregulated under anaerobic conditions, 23 were of unknown or putative function, including 4 that were identified as Salmonella orthologs regulated
by rpoS (Keseler et al. 2013). In addition, at least seven of the
known downregulated genes were identified as rpoS-regulated
(Table S4).
A total of 28 genes were upregulated in anaerobic stationaryphase cultures (Fig. 4a; Table S4) including stress-related genes
involving cold shock (cspAG) and osmoprotectants (proVW) (Keseler et al. 2013). In addition, genes encoding a multisubunit
complex responsible for anaerobic oxidation of formic acid to
carbon dioxide and molecular hydrogen (hycABCDEFGHI and
fdhF) (Sanchez-Torres et al. 2009) were upregulated. Our results
correspond with other research done on E. coli (Myers et al. 2013)
where the expression of the hyc gene family was also increased
when the strains were cultured to exponential phase under
anaerobic conditions.
Biofilm cultures
The RNA-seq analyses of aerobic and anaerobic mature biofilms
showed very different gene expression profiles compared to
planktonic stationary-phase cultures (Figs 2 and 3) and exponential cultures (Myers et al. 2013). In fact, the majority of the
differentially expressed genes were downregulated in the anaerobic biofilms, with only three upregulated genes (Fig. 4b; Tables 2
and S5, Supporting Information). Only one of the three upregulated genes was characterized safA, while the other two yobI and
yqgC are of an unknown function (Keseler et al. 2013).
The analysis of the downregulated genes in anaerobic
compared to aerobic E. coli biofilm showed that most are
Bayramoglu et al.
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Figure 4. Overview of differential gene expression in (A) planktonic and (B) biofilm E. coli under oxic compared to anoxic conditions. Genes displaying >2.5-fold or
<2.5-fold change ratios were significantly altered (q < 0.01). Each data points correspond to b-numbers and plotted in chronological order. Genes displaying >10-fold
or <10-fold change are at the outer limits that enable the data to be viewed at a reasonable scale.
essential to basic cell functions thereby the downregulation of
these genes may attenuate key cell processes (Table 2) cutting down on the cells’ energy costs. This included the expression of genes encoding major fundamental components of
rRNA function such as 30S (rpsBCDEFGHIJKLMNOPQRSTU) and
50S (rplABCDEFGIJKLMNOPQRSTUVY and rpmABCDEFGHIJ) ribosomal subunit proteins (Keseler et al. 2013). Similarly, some
tRNA-related genes were downregulated including those for
methionine (metTWZ), isoleucine (ileTUV), lysine (lysT), leucine
(leuQ), threonine (thrU), valine (valTUXYZ), argenine (argX), glutamate (gltUV), serine (serS) and alanine (alaW) as well as essential
tRNA-encoding factors (trmD, def and frr) (Table 2). Additionally,
genes encoding key enzymes in the biosynthesis of fatty acids
(accCD and fabABFGI) were downregulated together with genes
involved in ATP synthase (atpABCDEFGH) and lipid biosynthesis
(lpxACD, skp and fabZ). The expressions of genes that regulate a
myriad of other functions were downregulated, such as motility (flxA, flgBCKLM, fliAC, motA), cell division (ftsJ, sulA, zapA and
zipA), DNA recombination and repair (dinI, ihfA, ihfB, recA, yebG,
ssb and lexA) and cold-shock-related genes (cspABCDG) (Keseler
et al. 2013).
Among the downregulated genes we detected major transcription factors (Table 2) regulating genes involved in numerous processes and responses (Table S6, Supporting Information).
In fact, over 10% of regulators in the E. coli genome (Fis, H-NS
and Lrp) were repressed. Part of this group included transcription factor genes that play a major role in biofilm formation (Fis,
Crp and IscR) along with factor regulating genes involved in the
transition from aerobic to anaerobic growth (Fnr and ArcA) (Keseler et al. 2013). In addition, bacterial cell surface components
playing and important role in biofilm development and maturation including type 1 fimbriae and flagellin [fimA (Rodrigues and
Elimelech 2009) and fliC (Zhou et al. 2014), respectively] were significantly downregulated (Table S2). The inhibition of these essential factors may have imposed the attenuated growth and the
patchy morphology of the anaerobic biofilms (Fig. 1).
A large number (∼5%) of small RNA (sRNA) and related genes
were repressed (Table S6) in the anaerobic E. coli biofilms. Prominent among them was hfq, an RNA chaperone that facilitates
sRNA base pairing with their target, has been found necessary for the translation of RpoS (Gottesman and Storz 2015)
and is also a master regulator of the general stress responses
in E. coli (Chambers and Sauer 2013). Moreover, we detected
downregulated sRNAs (including srsD, ryeA and psrD) that are associated to hfq and by proxy to rpoS (Gottesman and Storz 2015).
The downregulation of the hfq proteins could be linked with the
overall repression of key regulatory mechanisms and key cell
functions (Fig. 4b; Table S6) as well as the slower growth rate
(Vytvytska et al. 1998) of the anaerobic biofilms.
CONCLUSION
Facultative anaerobes, like Escherichia coli, consume oxygen and
modify their hosts’ environments yet, efficient oxygen scavenging may impose anaerobic respiration conditions on E. coli cells
(Morris and Schmidt 2013). In this study, we demonstrated that
under anaerobic conditions E. coli biofilm attenuates essential
processes but could still persist as a biofilm (Fig. 1), whereas
planktonic cells were less affected by the lack of oxygen. We report that unlike the sessile cultures only a few pathways were
affected by oxygen depletion in the planktonic cultures (Figs 3
and 4). In an attempt to alleviate the oxygen-depletion stress
in the anaerobic biofilm, E. coli cells minimize activity and mitigate the expression of essential genes (Table 2). The effect of
oxygen availability was likewise tested in Pseudomonas aeruginosa, where cells were grown under aerobic, microaerobic and
anaerobic conditions. It was demonstrated that the P. aeruginosa
cells favored microaerobic conditions, while anaerobic conditions inhibited planktonic growth and biofilm development thus
significantly altering gene expression profiles (Alvarez-Ortega
and Harwood 2007). In contrast to previous reports, the results
presented here may suggest that E. coli cells are constrained
by anaerobic conditions. Perhaps E. coli cells, like P. aeruginosa,
could better tolerate microaerobic over anaerobic conditions
that might better serve the enterics’ main role in the mammalian colon, i.e. removal of oxygen traces and providing an
oxygen-free environment to the dominant anaerobic populations (Jones et al. 2011). Hence, we aim to examine E. coli biofilm
under microaerobic conditions exploring the means by which
oxygen-limited (but probably not deprived) E. coli cells successfully populate the colon.
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FEMS Microbiology Letters, 2017, Vol. 364, No. 3
Table 2. Key differentially expressed genes in anaerobic E. coli biofilm (for the complete genes list, please see Table S4).
Function
Amino acids
Asparagine
Aspartate
Glutamine
Histidine
Isoleucine
and valine
Lysine
Phenylalanine
Serine
Threonine
Tyrosine
Carbon
metabolism
Asparagine
Carbohydrate
transport
Carbon
catabolite
repression
Fructose
Glucose
Glycine
Pentose
Pyruvate
Succinate
Elongation
factor
Fatty
acids/lipids
Biotin
biosynthesis
Key protein
Synthesis of
unsaturated
fatty acids
Lipid
biosynthesis
Membrane
structure
Name
asnA & B
pyrB
glnB
glnA
gltD
hisL
ilvC
ilvL
dapA
dapD
pheL
aroG
serA
serC
glyA
thrL
aroF
Product
Asparagine synthetase A & B
Aspartate carbamoyltransferase, catalytic subunit
Regulatory protein P-II for glutamine synthetase
Glutamine synthetase
Glutamate synthase, 4Fe-4S protein, small subunit
His operon leader peptide
Ketol-acid reductoisomerase, NAD(P) binding
ilvG operon leader peptide
Dihydrodipicolinate synthase
2,3,4,5-Tetrahydropyridine-2-carboxylate N-succinyltransferase
pheA gene leader peptide
3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase, phenylalanine
repressible
D-3-Phosphoglycerate dehydrogenase
3-Phosphoserine/phosphohydroxythreonine aminotransferase
Serine hydroxymethyltransferase
thr operon leader peptide
3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase,
tyrosine-repressible
Fold
difference
∗
− 2.69
− 2.79
− 5.17
− 2.54
− 3.55
− 3.04
− 6.57
− 2.66
− 4.1
− 4.4
− 2.69
− 4.21
− 2.62
− 4.47
− 4.39
− 4.1
− 2.66
− 4.02
gpmA
Isoaspartyl peptidase
Phosphohistidinoprotein-hexose phosphotransferase component of PTS
system (Hpr)
Phosphoglyceromutase 1
fbaA
crr
eno
tpiA
gapA
gcvH
talB
aceE
aceF
cptB
Fructose-bisphosphate aldolase, class II
Glucose-specific enzyme IIA component of PTS
Enolase
Triosephosphate isomerase
Glyceraldehyde-3-phosphate dehydrogenase A
Glycine cleavage complex lipoylprotein
Transaldolase B
Pyruvate dehydrogenase, decarboxylase component E1, thiamin-binding
Pyruvate dehydrogenase, dihydrolipoyltransacetylase component E2
Antitoxin of CptAB toxin-antitoxin pair
− 4.13
− 3.31
− 4.08
− 2.72
− 7.04
− 3.31
− 4.35
− 5.4
− 3.92
− 2.66
efp
ihfA & B
tsf
tufa & B
tufB
Polyproline-specific translation elongation factor EF-P
Integration host factor (IHF), DNA-binding protein, subunits α & β
Potein chain elongation factor EF-Ts
Protein chain elongation factor EF-Tu
Protein chain elongation factor EF-Tu (duplicate of)
− 2.57
accC
accD
acpP
fabA
fabB & F
fabG
fabI
fabZ
lpxA
lpxC
lpxD
cvpA
gnsB
lpp
pal
slyB
spr
mipA
safA
Acetyl-CoA carboxylase, biotin carboxylase subunit
Acetyl-CoA carboxylase, beta (carboxyltransferase) subunit
Acyl carrier protein (ACP)
Beta-hydroxydecanoyl thioester dehydrase
3-Oxoacyl-[acyl-carrier-protein] synthase I, II
3-Oxoacyl-[acyl-carrier-protein] reductase
Enoyl-[acyl-carrier-protein] reductase, NADH-dependent
(3R)-Hydroxymyristol acyl carrier protein dehydratase
UDP-N-acetylglucosamine acetyltransferase
UDP-3-O-acyl N-acetylglucosamine deacetylase
UDP-3-O-(3-hydroxymyristoyl)-glucosamine N-acyltransferase
Membrane protein required for colicin V production
Qin prophage; multicopy suppressor of secG(Cs) and fabA6(Ts)
Murein lipoprotein
Peptidoglycan-associated outer membrane lipoprotein
Outer membrane lipoprotein
Mutational suppressor of prc thermosensitivity, outer membrane lipoprotein
Scaffolding protein for murein synthesizing machinery
Two-component system connector membrane protein, EvgSA to PhoQP
− 2.55
− 2.68
− 9.61
− 4.34
iaaA
ptsH
− 4.71
∗
− 5.73
∗
∗
− 4.5
− 2.69
− 2.86
− 2.61
− 4.01
− 2.53
− 2.86
− 2.67
− 8.8
− 5.47
− 3.09
− 3.12
− 3.38
4.38
7
Bayramoglu et al.
Table 2. (Continued).
Function
Membranebound
ATP
synthase
Motility
Nucleic acids
Purine
biosynthesis
Component
of ribosome
16S subunit
Component
of ribosome
23S subunit
Components
of ribosome
30S subunit
Components
of ribosome
50S subunit
SOS response
atp B, E, F
Fold
difference
F1 sector, subunit α, ε, β, γ , δ, respectively
∗
F0 sector, subunit a, c, b, respectively
∗
Biofilm-dependent modulation protein
Pleiotropic regulatory protein for carbon source metabolism
Major type 1 subunit fimbrin (pilin)
Flagellar component of cell-proximal portion of basal-body rod
Flagellar hook-filament junction protein 1
RNA polymerase, sigma 28 (sigma F) factor
Flagellar filament structural protein (flagellin)
Proton conductor component of flagella motor
Conserved protein with nucleoside triphosphate hydrolase domain
− 2.61
− 4.4
− 22.52
∗
∗
− 3.79
− 10.48
− 3.01
− 4.13
− 3.2
−3
− 3.26
− 2.93
cmk
pyrC
pyrI
rimM
16S rRNA processing protein
− 4.71
rlmE
23S rRNA U2552 2’-O-ribose methyltransferase, SAM-dependent
− 2.57
rpsB, C, D, E, F,
G, H, I, J, K, L,
M, N, O, P, Q,
R, S, T, U
rplA, B, C, D, E,
F, I, J, K, L, M,
N, O, P, Q, R, S,
T, U, V, W, X, Y
rpmA, B, C, D,
E, F, G, H, I, J
rpoA
rpoZ
frr
hpf
ssb
sulA
tisB
yebG
csrB
glmZ
psrD
psrO
ryeA
ryeB
ryfA
sibC
sibE
tff
Global sRNA
chaperone
Product
Adenylate kinase
Guanylate kinase
Phosphoribosylpyrophosphate synthase
Carbamoyl phosphate synthetase small subunit, glutamine
amidotransferase
Cytidylate kinase
Dihydro-orotase
Aspartate carbamoyltransferase, regulatory subunit
adk
gmk
prs
carA
dinI
lexA
recA
sRNA
Transcriptional
regulator
atp A, C, D, G,
H
bdm
csrA
fimA
flgB & C
flgK & L
fliA
fliC
motA
phoH
Pyrimidine
biosynthesis
rRNA
Name
hfq
− 2.72
− 3.56
− 5.89
Proteins S2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21
respectively
∗
Proteins L1, 2, 3, 4, 5, 6, 9, 10, 11, 7/12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25 respectively
∗
Proteins L27, 28, 29, 30, 31, 32, 33, 34, 35, 36 respectively
∗
RNA polymerase, alpha subunit
RNA polymerase, omega subunit
Ribosome recycling factor
Ribosome hibernation promoting factor HPF; stabilizes 70S dimers (100S)
− 5.96
− 4.194
− 3.74
− 3.11
DNA damage-inducible protein I
DNA-binding transcriptional repressor of SOS regulon
DNA strand exchange and recombination protein with protease and
nuclease activity
Single-stranded DNA-binding protein
SOS cell division inhibitor
Toxic membrane persister formation peptide, LexA-regulated
Conserved protein regulated by LexA
Regulating carbon source metabolism
Posttranscriptional regulation of glmS
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
Unknown
− 6.35
− 4.15
− 6.49
HF-I, host factor for RNA phage Q beta replication
− 6.78
− 2.67
− 4.83
− 2.96
− 4.89
− 6.75
− 6.4
− 4.91
− 2.98
− 4.58
− 5.25
− 2.67
− 3.13
− 2.72
− 9.01
8
FEMS Microbiology Letters, 2017, Vol. 364, No. 3
Table 2. (Continued).
Function
Stress response
Cold shock
Heat shock
High pH
Osmotic
stress
Oxidative
stress
Starvation
and low pH
Temperature
Universal
stress
response
Sulfate
assimilation
Transport
tRNA
Alanine
Arginine
Aspargine
and phenylalanine
Glutamic acid
Isoleucine
Leucine
Lysine
Methionine
Threonine
Name
Product
Fold
difference
− 2.88
− 10.84
− 5.9
− 3.31
− 5.33
− 3.06
− 3.49
− 3.25
− 2.55
ycgZ
cspA
cspB
cspD
cspG
cspI
grpE
cpxP
osmB
RcsB connector protein for regulation of biofilm and acid-resistance
RNA chaperone and anti-terminator, cold-inducible
Cold shock protein
Inhibitor of DNA replication, cold shock protein homolog
Cold shock protein homolog, cold-inducible
Cold shock protein
Heat shock protein
Inhibitor of the cpx response; periplasmic adaptor protein
Lipoprotein
bcp
icd
lpd
tpx
trxA
can
sspA
Peroxiredoxin; thiol peroxidase, thioredoxin-dependent
e14 prophage; isocitrate dehydrogenase, specific for NADP+
Lipoamide dehydrogenase, E3 component is part of three enzyme complexes
Lipid hydroperoxide peroxidase
Thioredoxin 1
Carbonic anhydrase 2
Stringent starvation protein A
cspC
sodA
uspA
sodB
groS
cysA
cysD
cysH
cysJ
cysK
cysN
cysP
cysW
Stress protein, member of the CspA-family
Superoxide dismutase, Mn
Universal stress global response regulator
Superoxide dismutase, Fe
Cpn10 chaperonin GroES, small subunit of GroESL
Sulfate/thiosulfate transporter subunit
Sulfate adenylyltransferase, subunit 2
3’-Phosphoadenosine 5’-phosphosulfate reductase
Sulfite reductase, alpha subunit, flavoprotein
Cysteine synthase A, O-acetylserine sulfhydrolase A subunit
Sulfate adenylyltransferase, subunit 1
Thiosulfate-binding protein
Sulfate/thiosulfate ABC transporter subunit
− 6.09
− 3.7
− 5.59
− 3.54
− 2.9
− 4.8
− 6.2
− 2.81
− 3.56
− 15.47
− 4.22
− 5.11
− 3.52
AcrAB-TolC efflux pump accessory protein, membrane-associated
Cytochrome o ubiquinol oxidase subunit II
Membrane spanning protein in TonB-ExbB-ExbD complex
Cystine transporter subunit
Glutamine transporter subunit
Maltose transporter subunit
Outer membrane porin protein C
Outer membrane porin 1a (Ia;b;F)
Outer membrane protein X
Glycine betaine transporter subunit
Sulfate transporter subunit
Protein export chaperone
Preprotein translocase membrane subunit
Periplasmic chaperone
TatABCE protein translocation system subunit
Periplasmic protein
Nucleoside channel, receptor of phage T6 and colicin K
Ala tRNA
Arg tRNA
tRNA m(1)G37 methyltransferase, SAM-dependent
− 3.84
− 3.159
− 2.679
− 3.19
− 4.084
− 2.835
− 4.72
− 12.51
− 7.32
− 3.55
− 2.88
− 4.235
acrZ
cyoA
exbB
fliY
glnH
malE
ompC
ompF
ompX
proV
sbp
secB
secE & G
skp
tatA
tolB
tsx
alaW
argX
trmD
gltU & V
ileT, U, V
leuQ & W
lysT
metT, W, Z
thrU
Glu tRNA
Ile tRNA
Leu tRNA
Lys tRNA
Met tRNA
Thr tRNA
− 2.51
− 5.23
− 4.71
− 9.22
− 4.49
− 3.85
− 3.54
∗
− 6.4
− 2.63
− 2.606
− 2.56
− 2.917
− 4.726
− 5.1
∗
∗
∗
− 5.44
∗
− 4.16
9
Bayramoglu et al.
Table 2. (Continued).
Function
tRNA
modification
tRNA
modification
Valine
Name
Product
Fold
difference
dusB
tRNA-dihydrouridine synthase B
− 3.363
miaA
Delta(2)-isopentenylpyrophosphate tRNA-adenosine transferase
− 3.46
valT, U, X, Y, Z
∗
Val tRNA
Fold difference values indicate downregulation in anaerobic compared to aerobic conditions.
∗
Please refer to Table S4 for the fold difference values.
SUPPLEMENTARY DATA
Supplementary data are available at FEMSLE online.
ACKNOWLEDGEMENTS
We are grateful to Moshe Herzberg for his help with the EPS analyses. We also thank Ahuva Vonshak and Lusine Ghazaryan for
their help in performing the experiments. We greatly appreciate
the insightful comments of Maya Benami and Adam Stovicek to
the manuscript.
Conflict of interest. None declared.
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