plospcbiplcbPLoS Comput BiolploscompPLoS Computational Biology1553-734X1553-7358Public Library of ScienceSan Francisco, USA10.1371/journal.pcbi.0010055plcb-01-05-07Research ArticleComputational BiologyMicrobiologyMolecular BiologyDissimilatory Metabolism of Nitrogen Oxides in Bacteria: Comparative
Reconstruction of Transcriptional NetworksRegulation of Nitrogen Oxides
MetabolismRodionovDmitry A14*DubchakInna L2ArkinAdam P3AlmEric J3GelfandMikhail S145 Institute for Information Transmission Problems, Russian Academy of
Sciences, Moscow, Russia Genomics Division, Lawrence Berkeley National Laboratory, Berkeley,
California, United States of America Physical Biosciences Division, Lawrence Berkeley National Laboratory,
Berkeley, California, United States of America State Scientific Center GosNIIGenetika, Moscow, Russia Department of Bioengineering and Bioinformatics, Moscow State
University, Moscow, Russia MiyanoSatoruEditorUniversity of Tokyo, Japan
DAR and MSG conceived and designed the experiments. DAR performed the
experiments. DAR, ILD, APA, and EJA analyzed the data. DAR, ILD, APA, EJA,
and MSG wrote the paper.
* To whom correspondence should be addressed. E-mail: rodionov@iitp.ru
The authors have declared that no competing interests exist.
10200528102005299200515e5523200529920052005Rodionov et alThis is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited.
Bacterial response to nitric oxide (NO) is of major importance since NO is an
obligatory intermediate of the nitrogen cycle. Transcriptional regulation of the
dissimilatory nitric oxides metabolism in bacteria is diverse and involves
FNR-like transcription factors HcpR, DNR, and NnrR; two-component systems NarXL
and NarQP; NO-responsive activator NorR; and nitrite-sensitive repressor NsrR.
Using comparative genomics approaches, we predict DNA-binding motifs for these
transcriptional factors and describe corresponding regulons in available
bacterial genomes. Within the FNR family of regulators, we observed a
correlation of two specificity-determining amino acids and contacting bases in
corresponding DNA recognition motif. Highly conserved regulon HcpR for the
hybrid cluster protein and some other redox enzymes is present in diverse
anaerobic bacteria, including Clostridia, Thermotogales, and
delta-proteobacteria. NnrR and DNR control denitrification in alpha- and
beta-proteobacteria, respectively. Sigma-54-dependent NorR regulon found in some
gamma- and beta-proteobacteria contains various enzymes involved in the NO
detoxification. Repressor NsrR, which was previously known to control only
nitrite reductase operon in Nitrosomonas spp., appears to be
the master regulator of the nitric oxides' metabolism, not only in most gamma-
and beta-proteobacteria (including well-studied species such as
Escherichia coli), but also in Gram-positive
Bacillus and Streptomyces species.
Positional analysis and comparison of regulatory regions of NO detoxification
genes allows us to propose the candidate NsrR-binding motif. The most conserved
member of the predicted NsrR regulon is the NO-detoxifying flavohemoglobin Hmp.
In enterobacteria, the regulon also includes two nitrite-responsive loci,
nipAB (hcp-hcr) and nipC (dnrN), thus
confirming the identity of the effector, i.e. nitrite. The proposed NsrR
regulons in Neisseria and some other species are extended to
include denitrification genes. As the result, we demonstrate considerable
interconnection between various nitrogen-oxides-responsive regulatory systems
for the denitrification and NO detoxification genes and evolutionary plasticity
of this transcriptional network.
Synopsis
Comparative genomics is the analysis and comparison of genomes from different
species. More then 100 complete genomes of bacteria are now available.
Comparative analysis of binding sites for transcriptional regulators is a
powerful approach for functional gene annotation. Knowledge of
transcriptional regulatory networks is essential for understanding cellular
processes in bacteria. The global nitrogen cycle includes interconversion of
nitrogen oxides between a number of redox states. Despite the importance of
bacterial nitrogen oxides' metabolism for ecology and medicine, our
understanding of their regulation is limited. In this study, the researchers
have applied comparative genomic approaches to describe a regulatory network
of genes involved in the nitrogen oxides' metabolism in bacteria. The
described regulatory network involves five nitric oxide−responsive
transcription factors with different DNA recognition motifs. Different
combinations of these regulators appear to regulate expression of dozens of
genes involved in nitric oxide detoxification and denitrification. The
reconstructed network demonstrates considerable interconnection and
evolutionary plasticity. Not only are genes shuffled between regulons in
different genomes, but there is also considerable interaction between
regulators. Overall, the system seems to be quite conserved; however, many
regulatory interactions in the identified core regulatory network are
taxon-specific. This study demonstrates the power of comparative genomics in
the analysis of complex regulatory networks and their evolution.
Citation:Rodionov DA, Dubchak IL, Arkin AP, Alm EJ, Gelfand MS (2005)
Dissimilatory metabolism of nitrogen oxides in bacteria: Comparative
reconstruction of transcriptional networks. PLoS Comput Biol 1(5):
e55.Introduction
Interconversion of nitrogen species between a number of redox states forms the
biogeochemical nitrogen cycle, which has multiple environmental impacts and
industrial applications. Bacteria can utilize soluble nitrogen oxides, nitrate and
nitrite, as terminal electron acceptors in oxygen-limiting conditions. Two
dissimilar pathways of nitrate respiration, ammonification and denitrification,
involve formation of a common intermediate, nitrite, but end in different products,
ammonia and gaseous nitrogen oxides or dinitrogen, respectively (Figure 1). At the first step,
nitrite is formed by one of three different types of nitrate reductases: soluble
assimilatory Nas, membrane-associated respiratory Nar, and periplasmic dissimilatory
Nap. The next step of ammonification is conversion of nitrite into ammonia by either
respiratory cytochrome c nitrite reductase NrfA or detoxifying
siroheme-containing enzyme NirBD [1]. In contrast, during denitrification,
nitrite is reduced to nitric oxide (NO), nitrous oxide, and, finally, dinitrogen,
using nitrogen oxide reductases NirK (or NirS), NorB, and NosZ, respectively
[2].
10.1371/journal.pcbi.0010055.g001
The Bacterial Inorganic Nitrogen Cycle
The ammonification, denitrification, detoxification, nitrogen fixation, and
nitrification pathways are shown by colored solid lines with genes names
involved in the pathway. The dashed black line shows possible non-enzymatic
interconversions of nitrogen oxides. The dotted line shows additional
formation of NO and hydroxylamine during nitrite ammonification.
NO is a signaling and defense molecule in animals, but bacteria are sensitive to high
NO concentrations due to its reactivity and membrane permeability [3]. NO and hydroxylamine,
two toxic intermediates in 6-electron reduction of nitrite, could be formed during
nitrite ammonification [4,5]. In
addition to a classical NO reductase (NorB) present in denitrifying species, two
other bacterial NO detoxification enzymes have been characterized: an NO reductase
(flavorubredoxin NorVW in Escherichia coli) [6] and an NO dioxygenase
(flavohemoglobin Hmp or Fhp in E. coli, Bacillus subtilis, Ralstonia
eutropha, and Pseudomonas species) [7–9].
An unusual redox enzyme, called the hybrid cluster protein (Hcp) or
“prismane protein,” has been extensively studied in strictly
anaerobic (Desulfovibrio species) and facultative anaerobic
(E. coli, Salmonella typhimurium, Acidothiobacillus ferrooxidans,
Shewanella oneidensis) bacteria, where it is induced mostly during
conditions of nitrite or nitrate stress, suggesting a role in nitrogen metabolism
[10–14]. In the latter
bacteria, the hcp gene always forms a possible operon with NADH
oxidoreductase hcr, whose product catalyzes reduction of Hcp in the
presence of NADH [11]. Until recently, the in vivo electron-accepting substrate of
Hcp was unknown, and based on the crystal structure, NO was assumed to be a good
candidate for this role [12,15]. In
vitro studies demonstrated oxygen-sensitive hydroxylamine reductase activity of the
E. coli Hcp protein, suggesting its possible role in
detoxification of reactive by-products of nitrite reduction [16]. More recently, the
requirement of the hcp gene for in vivo hydroxylamine reduction was
observed in Rhodobacter capsulatus E1F1 [17].
Expression of the denitrification genes is known to be activated by nitrogen oxides
and low oxygen tension [18]. Both in denitrifying and ammonifying
γ-proteobacteria, the nitrate/nitrite signal is processed by the
two-component sensor-regulator NarX-NarL and its paralog NarQ-NarP in E.
coli that control the respiratory nitrate reductase operon
nar and the nitrite ammonifying loci nir and
nrf [19]. Various transcription factors of the FNR family have been
described as NO-sensing regulators of denitrification: DNR in
Pseudomonas species, NNR in Paracoccus
denitrificans, and NnrR in Rhodobacter sphaeroides and
Bradyrhizobium japonicum [18]. The DNR/NNR and NnrR proteins
cluster phylogenetically in separate subgroups, separately from other family members
including FNR, a global regulator of anaerobiosis in facultative anaerobic bacteria
[20].
Another NO-responsive transcriptional factor, σ54-dependent
NorR, activates expression of the NO reductases norVW in E.
coli and norAB in R. eutropha
[21,22]. Three tandem
upstream activator sites with the core consensus GT-(N7)-AC were
identified as NorR-binding sites observed in both promoter regions. Analysis of the
adjacent regions of additional norR orthologs in bacterial genomes
revealed similar tandem NorR-binding sites upstream of the norA and
norB genes in Ralstonia species,
norVW in Salmonella species, hmp
in Vibrio cholerae and Pseudomonas aeruginosa, and
hcp in V. vulnificus [23].
A nitrite-sensitive transcriptional repressor, named NsrR, has been identified in
lithoautotrophic β-proteobacterium Nitrosomonas europeae,
where it regulates expression of the copper-nitrite reductase nirK
[24].
Co-localization of the nsrR ortholog and the hcp
gene in R. capsulatus E1F1 suggested that NsrR and nitrite could be
involved in the regulation of hydroxylamine assimilation in this
α-proteobacterium [17]. NsrR is a member of the Rrf2 family of transcriptional
regulators, which includes a putative Rrf2 regulator for a redox operon in
D. vulgaris [25], the iron-responsive repressor RirA,
which controls iron uptake in rhizobia [26], and the IscR repressor for the Fe-S
cluster assembly operon in E. coli [27].
Despite this diversity of regulatory systems, our understanding of the regulation of
the nitrogen oxides metabolism in bacteria is very limited. For example, NO- and
nitrite-dependent activation of expression of hmp in E.
coli and B. subtilis, hcp-hcr (nipAB) and dnrN
(nipC) in S. typhimurium, and norB
and aniA (nirK) in Neisseria gonorrhoeae has been
described [28–30], but specific transcriptional factors involved in this control
are not yet known. In this study, we analyzed regulation of the nitrosative stress
and denitrification genes in available bacterial genomes using comparative genomics
approaches [31,32] and predicted a large
number of new regulatory elements for these genes. In addition to a complete
description of the previously known NorR, DNR, and NnrR regulons, we report
identification of a novel FNR-like regulator, named HcpR, for the
hcp and other redox-related genes in anaerobic bacteria. Starting
from very limited data, we were able to identify the NsrR-binding motif and describe
the NsrR regulons in sequenced γ- and β-proteobacteria, as well
as in the Bacillus and Streptomyces species.
Combining published experimental and newly obtained comparative data, we have
reconstructed the NO- and nitrite-dependent transcriptional regulatory network for
dissimilatory metabolism of nitrogen oxides in bacteria.
ResultsHcpR: Recognition Motifs and Core Regulon
A member of the CRP/FNR family of transcription factors, HcpR has been initially
identified as the regulator of the hcp gene encoding the hybrid
cluster protein and the frdX encoding a ferredoxin-like protein
in the Desulfovibrio species, anaerobic metal-reducing
δ-proteobacteria [33]. The consensus of the candidate
HcpR binding sites, wTGTGAnnnnnnTCACAw, is similar to the CRP consensus of
E. coli.
Close hcpR orthologs were detected in other
δ-proteobacteria, namely two Geobacter species,
Desulfotalea psychrophila and
Desulfuromonas. However, the same CRP-like motifs were not
present in these genomes. As the analysis of the regulator multiple alignment
revealed a substitution in the helix-turn-helix motif involved in DNA
recognition that could cause this change (see “Co-evolution of
regulators and their recognition motifs” for details), and since the
considered species have multiple hcp and frdX
paralogs, we applied the motif detection procedure to a set of corresponding
upstream regions and obtained a new FNR-like palindromic motif with consensus
sequence wyTTGACnnnnGTCAArw, which has a notable distinction from the CRP-like
motif in the third position (not G, but T). This recognition motif was observed
upstream of most hcp and frdX paralogs in the
studied δ-proteobacteria, as well as upstream of some additional genes
in Desulfuromonas and Geobacter species (Figure 2 and Table S1).
10.1371/journal.pcbi.0010055.g002
Genomic Organization of Genes Regulated by HcpR
Yellow circles with numbers denote candidate HcpR sites with different
consensus sequences. These numbers correspond to the HcpR profile
numbers in Figure
3.
10.1371/journal.pcbi.0010055.g003
Maximum Likelihood Phylogenetic Tree of the FNR/CRP Family of
Transcriptional Regulators
The third column contains sequences of helix-turn-helix motifs in the
proteins. Two specificity-determining positions correlated with DNA
motifs are colored (R180 and E181 in proteins
correlate with G3 and A6 in DNA sites,
respectively). The fourth column includes sequence logos for presumably
homogeneous and large site sets and sequence consensi for small sets of
DNA sites and for well-established motifs of other factors (FNR, CRP,
CooA, NtcA, ArcR). The last column indicates the name of a search
profile constructed in this study.
Close orthologs of hcpR from δ-proteobacteria are
present in two cyanobacteria, Anabaena variabilis and
Synechocystis sp. (Avar17201 and
slr0449 in Figure 3), where they are divergently transcribed with the
hcp and norB genes, respectively. Both
these genes are preceded by candidate HcpR sites with consensus sequence
TTGACnnnnGTCAA, and no other similar sites were found in the genomes of these
two cyanobacteria (Figure
2).
To analyze possible regulation of the hcp genes in other
taxonomic groups of bacteria, we considered their gene neighborhoods and found
that genes for FNR-like regulators are often co-localized with
hcp in most Clostridium species,
Bacteroides, Thermotogales, and Treponema
denticola (Figure
2). On the phylogenetic tree of the FNR/CRP protein family (Figure 3), all such regulators
form a separate branch, named HcpR2, and additional representatives of this
branch always co-occur with hcp genes in bacterial genomes. By
applying the motif recognition procedure to a set of hcp
upstream regions from HcpR2-containing genomes, we identified a conserved DNA
motif with consensus GTAACnnnnGTTAC.
Other types of DNA motifs were observed upstream of the hcp
genes in Clostridium thermocellum, C. difficile, and
Porphyromonas gingivalis, and upstream of the
hcp gene in Thermoanaerobacter tengcongensis.
In the latter species the hcp gene has a CRP-like regulatory
site and is preceded by the crp2 gene, which is an ortholog of
the B. subtilis fnr gene, making it likely that
crp2 regulates hcp (Figure 2). The predicted HcpR2 regulons in
most Bacteroides species, P. gingivalis, Fusobacterium
nucleatum, T. denticola, and Thermotogales contain only
hcp genes (Figure
2).
DNR and NnrR Core Regulons
In two denitrifying Pseudomonas species, P.
stutzeri and P. aeruginosa, expression of the
nir, nor, and nos genes is regulated by
the NO-responsive FNR-like transcriptional activator DNR that binds to a DNA
motif similar to the consensus FNR box, TTGATnnnnATCAA [34,35]. By a combination of similarity
search and phylogenetic analysis of the CRP/FNR protein family (Figure 3), we identified DNR
orthologs in the genomes of various denitrifying β-proteobacteria,
including three Ralstonia and two Burkholderia
species, C. violaceum and Thiobacillus
denitrificans (Figure
4). To identify the DNR recognition motif in
denitrifying species, we selected the upstream regions of denitrification genes
encoding nitrite, NO, and nitrous oxide reductases from genomes containing DNR
orthologs and applied the motif detection procedure. The resulting FNR box-like
motif with consensus CTTGATnnnnATCAAG was identified upstream of most
denitrification genes (nirS, nirK, norB, nosZ) (Table S2).
10.1371/journal.pcbi.0010055.g004
Genomic Organization of Genes Regulated by NsrR, NorR, and DNR in
β-Proteobacteria
Magenta, green, and blue circles denote candidate NsrR, NorR, and DNR
sites, respectively. Candidate σ54 promoters
associated with NorR sites are shown by angle arrows. Experimentally
known sites of NorR and DNR are marked by “s.”
Additional sites of the NarP and FNR factors are indicated by purple
squares and black triangles, respectively.
No orthologs of the HcpR, DNR, NsrR (below), and NorR (below) regulators were
identified in α-proteobacterial genomes. The only exception seems to
be R. capsulatus E1F1, whose genome contains an
nsrR ortholog close to the hcp gene within the
nitrate assimilation nas gene cluster [17]. However, in
denitrifying species, including R. sphaeroides and B.
japonicum, the FNR-like transcriptional factor NnrR activates
expression of nitrite and NO reductases and of the nnrS gene
[36–38]. Orthologs of nnrR were identified in six
α-proteobacteria, all of which also possess the nir
and nor genes involved in denitrification (Figure 5). The NnrR orthologs form a separate
branch on the phylogenetic tree of the CRP/FNR family (Figure 3). To analyze the NnrR regulon, the
motif detection procedure was applied to a training set of the nir, nor,
nos, and nnrS upstream regions from
α-proteobacteria. The conserved NnrR recognition motif with consensus
ctTTGcgnnnncgCAAag was identified upstream of most denitrification genes (Table S3).
The same candidate NnrR sites have been previously identified in R.
sphaeroides by a comparison of the nir, nor, and
nnrS upstream regions and then confirmed for the latter
gene by site-directed mutagenesis [36].
10.1371/journal.pcbi.0010055.g005
Genomic Organization of Genes Regulated by NnrR and NsrR in
α-Proteobacteria
Orange and magenta circles denote candidate NnrR and NsrR sites,
respectively. Experimentally known NnrR sites are marked by
“s.”
Co-Evolution of Regulators of the CRP/FNR Family and Their Recognition Motifs
The HcpR recognition motifs identified in several bacteria demonstrated some
diversity, which could be correlated with changes in the regulator DNA-binding
helix-turn-helix domain. In particular, the CRP-like motif wTGTGAnnnnnnTCACAw of
Desulfovibrio species differs from the FNR-like motif
wyTTGACnnnnGTCAArw in other δ-proteobacteria in the third position
(not T, but G). Examination of multiple alignment of the CRP/FNR-like proteins
revealed one specific amino acid (R180) in the HTH motif involved in
DNA recognition, which has changed from arginine (like in E.
coli CRP and Desulfovibrio HcpR) to Ser or Pro in
other δ-proteobacteria (see the HcpR1 branch of the phylogenetic tree
in Figure 3). Similarly, the
difference between the HcpR2 motif GTAACnnnnGTTAC and the motifs of
δ-proteobacteria is consistent with substitution of Glu-181 in the DNA
recognizing HTH domain to Pro in the HcpR2 proteins (Figure 3).
The structure of CRP in complex with its DNA operator has been determined
[39].
Three positions (1ber, chain A residues 180, 181, and 185) interact with the DNA
target site, and mutagenesis studies have shown that point mutations at these
positions alter the specificity of the protein [40]. We systematically analyzed HcpR,
HcpR2, DNR, and NnrR sites identified here, as well as several known consensus
sequences for other CRP/FNR-family regulators and observed a correlation of two
specificity-determining positions, R180 and E181, and
contacting bases in a DNA recognition motif, G3 and A6,
respectively (Figure 3).
A different substitution is observed in three bacterial species, where position
181 in the HcpR2 protein is filled by either Ser (Clostridium
thermocellum and C. difficile), or Gln
(Porphyromonas gingivalis). In agreement with these
replacements, the candidate HcpR2 motifs in these species differ from the common
recognition motif detected for most HcpR2-containing genomes (Figure 3). Finally, the Crp2
regulator in T. tengcongensis has CRP-like regulatory motif and
is orthologous to the FNR regulator of B. subtilis.
The phylogenetic tree of the CRP/FNR regulators (Figure 3) represents four main groups of
proteins analyzed in this study: DNR, NnrR, HcpR1, and HcpR2. It also includes
several well-studied family members with established DNA-binding consensuses.
All respective branches on the tree are deeply rooted, and thus their
phylogenetic relationships to each other are not well resolved and differ from
results of a more extensive phylogenetic analysis of the CRP/FNR-like
transcriptional regulators [41]. Nevertheless, in both trees the
HcpR1 and DNR branches cluster together, whereas HcpR1 and HcpR2 form two quite
distinct branches on the phylogenetic tree (Figure 3). All these proteins lack the
canonical FNR-type cysteine motif, thus excluding their binding of the
oxygen-labile Fe-S cluster [41,42].
NsrR: Recognition Motifs and Core Regulon
The above analysis suggests that HcpR controls the hcp genes in
strictly anaerobic bacteria. However, a large number of facultative anaerobic
bacteria possessing the hcp gene lack hcpR
orthologs. In E. coli, S. typhimurium, and S.
oneidensis, hcp is expressed only under anaerobic conditions in the
presence of nitrite or nitrate [10–12]. In an attempt to explain a
possible molecular mechanism of this induction, we first aligned the upstream
regions of the hcp genes from eight enterobacteria and
identified two highly conserved regions (Figure S1). The upstream potential
recognition motif resembles the consensus sequence of the FNR-binding site and
thus most likely is involved in the anaerobic induction of the
hcp-hcr operon by FNR [12]. The second potential DNA motif,
an imperfect inverted repeat with consensus gATGyAT-(N5)-ATrCATc
located downstream of the FNR site, is likely the binding site for a regulatory
protein that responds to nitrogen oxides.
Construction of a recognition rule and search in complete E.
coli genome identified similar sites in upstream regions of the
hypothetical gene dnrN (ytfE) and the hmp gene
encoding NO-detoxifying flavohemoglobin. Importantly, both these candidate sites
are highly conserved in multiple alignments of dnrN and
hmp upstream regions from related enterobacteria (Figures S2
and S3).
Search in the S. oneidensis genome identified the same DNA
motif upstream of hcp-hcr, SO4302 (dnrN), and
SO2805 (nnrS), the latter encoding a hypothetical
heme-copper-containing membrane protein [36]. The hmp gene is
absent in this genome (Figure
6).
10.1371/journal.pcbi.0010055.g006
Genomic Organization of Genes Regulated by NsrR, NorR, and DNR in
γ-Proteobacteria
Magenta, green, and blue circles denote candidate NsrR, NorR, and DNR
sites, respectively. Candidate σ54 promoters
associated with NorR sites are shown by angle arrows. Experimentally
known sites of NorR and DNR are marked by “s.”
Additional sites of the NarP and FNR factors are indicated by purple
squares and black triangles, respectively.
Identification of the conserved palindromic motif suggests that some common
transcription factor co-regulates the hcp-hcr, dnrN, hmp, and
nnrS genes in enterobacteria and
Shewanella species. Since bacterial transcription factors often
directly regulate adjacent genes [43], we analyzed gene neighborhoods
of genes preceded by the predicted sites (Figures 4 and 6). In many proteobacteria, including
Vibrionales, Acinetobacter sp., Chromobacterium
violaceum, Ralstonia, and Bordetella species, as
well as in Gram-positive bacilli and actinobacteria, the flavohemoglobin gene
hmp is positionally clustered with a hypothetical
transcriptional factor from the Rrf2 protein family. The characterized members
of the PF02082 family are the Rrf2 repressor for the electron transport operon
hmc in Desulfovibrio vulgaris
[25],
the iron-sulfur cluster repressor IscR in E. coli
[27],
the iron-responsive regulator RirA in rhizobia [26], and the nitrite-sensitive
repressor NsrR for the nitrite reductase operon nirK in
Nitrosomonas europeae [24]. Phylogenetic analysis (DAR,
unpublished data) demonstrated that all representatives of the Rrf2 protein
family associated with hmp genes appear to be orthologs of the
N. europeae NsrR protein, and thus we tentatively assign
this name to the entire subfamily. Orthologs of the nitrite-sensitive repressor
NsrR were identified in all β- and most γ-proteobacteria,
being absent only in Pasteurellaceae, Pseudomonadales, and V.
cholerae. We predict that this transcriptional factor actually binds
the identified DNA motif upstream of nitrite/NO-induced genes in enterobacteria
and Shewanella.
To further analyze the NsrR regulon, we constructed a recognition rule for the
NsrR sites and used it to scan the genomes of γ- and
β-proteobacteria (Table S4; Figures 4 and 6). The flavohemoglobin gene
hmp has an upstream NsrR site in most of these genomes,
excluding Pseudomonadales, V. cholerae, and
Polaromonas sp., where it is a member of the NO-responsive
regulon NorR (see below). The nnrS gene, another well-conserved
member of the NsrR regulon, was found in some genomes within a possible operon
with nsrR or hmp (Figures 4 and 6). The norB gene encoding
an NO reductase in denitrifying bacteria is preceded by NsrR sites in the
Neisseria species, C. violaceum,
Polaromonas sp., Ralstonia solanacearum, and two
Burkholderia species, B mallei and
B. pseudomallei. Another key enzyme of the denitrification,
the copper-containing nitrite reductase NirK, is predicted to be a member of the
NsrR regulon in the Neisseria species, C.
violaceum, and N. europeae (Figure 4), and in the latter bacterium it was
recently shown to be a target of this nitrite-sensitive repressor
[24].
In addition to γ-proteobacteria, the hcp gene was
found under NsrR regulation in a β-proteobacterium (B.
cepacia), and an α-proteobacterium (R.
capsulatus E1F1).
Orthologs of nsrR have been also found in the complete genomes
of most Bacillus and Streptomyces species,
where they are clustered with the flavohemoglobin gene hmp. The
only exception is B. subtilis, which has a stand-alone
nsrR ortholog, yhdE. The predicted
NsrR-binding motif appears to be well conserved in these Gram-positive bacteria,
and candidate sites were observed only upstream the hmp genes.
Multiple experimental studies in B. subtilis showed nitrite- or
NO-dependent induction of expression of hmp; however, the
mechanism of this control was not known [9,29]. The experimentally mapped hmp
promoter in B. subtilis significantly overlaps with the
predicted tandem NsrR sites [44].
The obtained data suggest that the nitrite-responsive NsrR regulon has a wide
phylogenetic distribution. Its most conserved member is the NO-detoxifying
flavohemoglobin Hmp, which is present both in Gram-negative and Gram-positive
bacteria. Most other regulon members are involved in the nitrosative stress and
denitrification. The identified NsrR recognition motif, a palindrome with
consensus gATGyAT-(N5)-ATrCATc, is well conserved in most analyzed
bacteria (Figure 7). The
only exception is the NsrR recognition motif in Neisseria
species, where symmetrical positions G4 and C16 are
replaced by T and A, respectively.
10.1371/journal.pcbi.0010055.g007
Sequence Logos for the Identified NsrR-Binding Sites in Various
Bacterial Taxa
NorR Regulon
In Vibrionales, the hcp-hcr operon is preceded by a gene that
encodes a homolog of the NO-responsive regulator NorR, named NorR2. NorR is a
σ54-dependent transcriptional activator that regulates
expression of the NO reductase operons, norVW in E.
coli and norAB in R. eutropha
[21,22]. NorR binds to
three tandem operator regions, inverted repeats with degenerate consensus
GT-(N7)-AC, which are localized upstream of the
σ54 promoter site [23]. By applying the motif detection
procedure to the hcp promoter regions from four Vibrionales
genomes, we identified two tandem palindromic sites with consensus
GATGT-(N7)-ACATC (Figure S4). These likely binding sites for
the NorR2 protein are localized immediately upstream of candidate
σ54 promoters well conforming to the consensus, and
thus could be involved in the NO-dependent activation of the
hcp-hcr operon (Table S5). In addition, the
norR2-hcp-hcr gene loci in Vibrionales contain a single
NorR site without an associated σ54 promoter located
upstream of the norR2 gene. This site could be involved in the
negative autoregulation of the NorR2 expression (Figure 6).
To analyze analogous NO-responsive regulons in other species, we performed
exhaustive similarity search and identified norR-like genes in
only a limited number of β- and γ-proteobacteria (Figures 4 and 6). An E.
coli-like arrangement of the divergently transcribed
norR and norVW genes with conserved tandem
NorR-binding sites and a σ54 promoter was found in
S. typhimurium, two Erwinia species, and
two Vibrio species. Other NO-detoxification genes possibly
regulated by candidate NorR sites are the NO-reductase norB in
Ralstonia spp. and Shewanella
putrefaciens, and the NO dioxygenase hmp gene in
V. cholerae, Pseudomonas spp., Polaromonas
sp., and B. fungorum. In all these cases except P.
stutzeri, the norR gene is clustered with the
target genes on the chromosome. In the unfinished genome of P.
stutzeri, the candidate tandem NorR sites followed by candidate
σ54 promoters were found upstream of the
hmp and dnrN genes, but the
norR gene was not found in the sequenced portion of the genome.
In addition, we found that V. cholerae has a second target for
NorR in the genome, the hypothetical gene nnrS, which was
identified as a member of various NO/nitrite-responsive regulons in other
proteobacteria (NsrR, DNR, NnrR, see below).
The consensus sequences of NorR and NorR2 recognition motifs identified in
various taxonomic groups have only a limited number of universally conserved
positions (Figure 8).
Positions G5 and T6 and complementary positions
A14 and C15 are the most conserved ones throughout the
NorR family, being only partially replaced in Polaromonas sp.
(A5). Noteworthy, in some Vibrio species, two
norR paralogs are present, norR1 and
norR2, which are associated with the norVW
and hcp-hcr operons, respectively. The NorR1 and NorR2
consensus sequences differ significantly in four positions (C7,
G13 for NorR1 and G2, C18 for NorR2),
allowing for discrimination of the target sites by the NorR paralogs in these
species.
10.1371/journal.pcbi.0010055.g008
Sequence Logos for the Identified NorR-Binding Sites in Various
Species of Proteobacteria
Complex Regulation of Hybrid Cluster Protein Genes
Differences in the predicted mode of regulation of the hybrid cluster proteins
(Table 1), which are
present in diverse bacterial and archaeal species, are well traced on the
phylogenetic tree of this protein family (Figure 9). Indeed, the hcp
gene is regulated by HcpR (highlighted in yellow) in many anaerobic bacteria, by
NsrR in facultative anaerobic enterobacteria, and some β- and
α-proteobacteria (in magenta), by NorR in most Vibrionales (in green),
and by DNR in A. ferrooxidans and Thermochromatium
tepidum (in blue). It often forms operons with either the NADH
oxidoreductase hcr (in γ-proteobacteria) or the
ferredoxin-like gene frdX (in δ-proteobacteria and
Clostridium spp.), suggesting functional linkage between
the Hcp and Hcr/FrdX proteins. In addition to predicted NsrR sites, the
hcp-hcr operons in enterobacteria are also preceded by
candidate binding sites of the anaerobic activator FNR, suggesting their
induction during anaerobiosis (Figure S1). We also investigated the
regulatory regions of hcp in two Pasteurellaceae lacking all
above-mentioned nitrogen oxides regulators (Actinobacillus
pleuropneumoniae and Mannheimia
succiniciproducens) and found a strong candidate binding site of NarP,
a response regulator from the nitrate/nitrite-specific two-component regulatory
system NarQ-NarP. The NarP regulon in E. coli contains mainly
genes from the nitrate/nitrite respiration pathway [18], whereas in the
above two Pasteurellaceae, the NarP regulon is extended to include the
detoxification genes hcp-hcr, dnrN, and norB.
Additional analysis using the NarP recognition rule revealed candidate NarP
sites upstream of some NsrR-regulated genes in γ-proteobacteria and
Neisseria species (Figure 6). In agreement with these findings,
the nitrate- and nitrite-induced transcription from the hcp
promoter in E. coli was found to depend on the response
regulator proteins NarL and NarP [45]. These observations show that the
nitrite induction of the NO-detoxification genes in different genomes can be
achieved by multiple transcriptional factors.
10.1371/journal.pcbi.0010055.t101
Predicted Members of Regulatory and Metabolic Networks of the Nitrogen
Oxides Dissimilatory Metabolism
10.1371/journal.pcbi.0010055.t102
Continued
10.1371/journal.pcbi.0010055.g009
Maximum Likelihood Phylogenetic Tree of the Hybrid Cluster (Prismane)
Proteins
Genes predicted to be regulated by the nitrogen oxides–related
factors are highlighted by respective colors. Additionally, predicted
FNR-regulated genes are denoted by black circles. Genes positionally
linked to the NADH oxidoreductase hcr and the
hypothetical ferredoxin frdX genes are shown by red and
blue lines, respectively. Archaeal genes are shown by pointed lines.
Additional Members of the Regulons
The main regulatory interactions analyzed in this study are shown in Figure 10 and Table 1. The core regulon
members, that is, genes regulated by the nitrogen-oxide responsive factors NsrR,
HcpR, DNR, NnrR, and NorR in many genomes, are the hybrid cluster protein gene
hcp, the NO-detoxifying flavohemoglobin
hmp, two hypothetical genes dnrN and
nnrS, NO reductase operon norVW, and
multiple denitrification genes, nir, nor, and
nos, encoding nitrite, NO, and nitrous oxide reductases,
respectively. The core regulon members can be regulated by different regulators
in various genomes. Further, some genes may be regulated by several regulators
simultaneously. All considered regulons also contain a large number of
additional members, which are summarized in Table 1.
10.1371/journal.pcbi.0010055.g010
Regulatory Interactions between Genes for Dissimilatory Nitrogen
Oxides Metabolism in Bacteria
(A) Distribution of regulators and regulated genes. The number of cases
when a gene is regulated by a specific transcription factor is indicated
by the length of a colored bar in the histogram. The white bar in the
histogram shows the cases when the gene is present in a genome
possessing at least one of the studied regulons, but is not regulated by
any of them.
(B) Combined regulatory network. Arrows denote regulatory interactions,
with the thickness reflecting the frequency of the interaction in the
analyzed genomes. Experimentally established (for DNR, NnrR, NsrR, and
NorR) and predicted based on the regulon content (for HcpR) signal
molecules are shown in filled ovals, and the protein family for each
transcription factor is shown below.
Though the main target of the HcpR regulators are genes encoding the hybrid
cluster protein Hcp and the hypothetical ferredoxin-containing protein FrdX, the
HcpR regulons are significantly extended in δ-proteobacteria and
clostridia (Table S1, Figure
2, and Table 1). In
Desulfuromonas and Geobacter species, they
include the nitrite reductase nrfHA, the NADH dehydrogenase
ndh, and the nitrate reductase nar operon.
In Desulfovibrio species, the HcpR regulon is extended to
include the apsBA and sat loci involved in the
sulfate reduction pathway. Among hypothetical genes, the predicted HcpR regulons
often contain ferredoxin-, hemerythrin-, or cytochrome c-like
genes. For instance, the CTC0897-CTC0898 operon of C.
tetani encoding a permease and a ferredoxin-like protein,
respectively, is likely regulated by the divergently transcribed paralog of
HcpR2 (CTC0896).
Additional members of the NsrR regulon were identified in all enterobacteria
(Figure 6). Two
hypothetical transporter genes, that are homologous to the tellurite resistance
gene tehB and the nitrite extrusion gene narK,
could be involved in the protection from the nitrosative stress by excretion of
nitrite from cytoplasm. In support of these observations, the NsrR regulons were
found to include a narK-like transporter gene
(nasA) in β-proteobacterium C.
violaceum and a tehB homolog in Photobacterium
profundum. The V. fischeri NsrR regulon includes a
homolog of the eukaryotic alternative oxidase Aox. In Legionella
pneumophila, a non-denitrifying γ-proteobacterium without
hmp, hcp, dnrN, and nnrS genes, the
glbN-lpg2536 operon encoding a heme-containing cyanoglobin
and a hypothetical ferredoxin reductase was found to be a sole NsrR target. We
suggest that both these genes could be involved in the detoxyfication process by
mediating NO oxidation (similar to the flavohemoglobin Hmp).
Denitrifying bacteria like the Pseudomonas and
Burkholderia species contain additional members of the DNR
and NnrR regulons (Figures 4
and 5). For instance, the
hemN gene encoding an O2-independent
coproporphyrinogen III oxidase involved in the protoheme biosynthesis and
relevant for denitrification [46] is preceded by a strong DNR site
in the Burkholderia species but has candidate NnrR-binding
sites in some α-proteobacteria. In Brucella
melitensis, the NnrR regulon includes the nosA gene,
encoding an outer membrane copper receptor, shown to be required for the
assembly of the copper-containing nitrous oxide reductase in P.
stutzeri [47]. Finally, a gene of unknown function
(COG4309) is probably co-transcribed with the NnrR-regulated
nor genes in three rhizobial species, where as in three
β-proteobacteria this gene is a predicted member of the NsrR regulon
and it is often co-transcribed with norB (Figure 4). These observations indicate that
the COG4309 family could be relevant for function of the NO reductase.
Two close dnr paralogs, most likely resulting from a recent
duplication, were found in A. ferrooxidans. One is divergently
transcribed with the hcp-COG0543 operon, which is preceded by a
strong candidate DNR site. The second paralog clusters with the
cgb-COG0543-COG0446 operon, which also is preceded by a
candidate DNR site. The product of the first gene in this operon is similar to a
single-domain hemoglobin in Campylobacter jejuni, whish is
indispensable for defense against NO and nitrosating agents [48]. Thus we predict
that the recently duplicated DNR paralogs in A. ferrooxidans
regulate two different NO-detoxifying systems.
Discussion
The results of this study demonstrate considerable variability of the metabolic and
regulatory systems for nitrogen oxides (Figure 10). Many genes change the regulators in different genomes (Table 1). However, overall, the
system seems to be quite conserved. Genes involved in denitrification, such as
nir, nor, and nos, are mainly regulated by two
NO-responsive transcriptional activators from the FNR/CRP family, NnrR in
α-proteobacteria and DNR in β-proteobacteria, and
Pseudomonas spp. Three different nitrogen oxides-responsive
transcription factors appear to regulate genes required for defense against the
nitrosative stress: the σ54-dependent activator NorR from the
NtrC family in some γ- and β-proteobacteria (present in at least
18 species), the Rrf2 family NsrR repressor widely distributed in proteobacteria and
firmicutes (39 species), and the FNR-like transcription factor HcpR in diverse
anaerobic bacteria (22 species). The primary targets of the newly identified
regulator HcpR are the hybrid-cluster protein Hcp, which has a protective role in
nitrite stress conditions, and the associated ferredoxin-like proteins. NorR usually
regulates cytoplasmic NO reductase norVW and sometimes another
membrane-bound NO reductase (norB) and the NO dioxygenase
hmp. The NsrR regulon almost always includes the
hmp and hcp genes, as well as one or both genes of
unknown function, dnrN and nnrS.
On the whole, the NsrR, NorR, and DNR regulons are differentially distributed in
γ- and β-proteobacteria, and the former prevails over the other
two. All three regulons are present only in three Ralstonia
species, where NsrR controls the NO-detoxifying flavohemoglobin, whereas NorR and
DNR regulate the denitrification genes (nir, nor, and
nos). In addition, the NorR and NsrR factors co-occur in ten other
non-denitrifying species, complementing each other in the control of the nitrosative
stress genes. Finally, NorR and DNR regulators were found only in P.
aeruginosa, and NsrR and DNR co-occur in four denitrifying
β-proteobacteria.
Some regulatory interactions in the identified core regulatory network seem to be
taxon-specific (thin lines in Figure
10B; see also “Complex regulation of hybrid cluster protein
genes” above). They include NsrR-regulated norVW and
nos in P. profundum, norB and
nirK in various β-proteobacteria; NorR-regulated
dnrN in P. stutzeri and nnrS
in P. aeruginosa; HcpR-regulated dnrN in
B. fragilis and Desulfuromonas,
norB in Synechocystis sp.; and DNR-regulated
nnrS and hmp in P. stutzeri,
hcp in A. ferrooxidans and T. tepidum.
The extensions of the NsrR regulon include the denitrification genes
nirK and norB in Neisseria
species, Burkholderia spp., and C. violaceum. The
former is of particular interest as, in contrast to the latter two lineages, the
Neisseria species lack the DNR regulator, assuming a
lineage-specific substitution of both the transcription factor and its binding
sites. Indeed, in the Neisseria species, the complete
denitrification pathway including nitrite, NO, and nitrous oxide reductases, as well
as dnrN and the two-component regulatory system
narQP, seems to be regulated by NsrR. The hypothesis that NsrR
mediates regulation of denitrification genes in Neisseria is
further supported by the observation that in N. gonorrhoeae, the
norB gene is strongly induced by NO independently of FNR and
NarP [30].
Not only genes are shuffled between regulons in different genomes, but there may
exist considerable interaction between regulators. Firstly, in some species the DNR
regulon overlaps with other nitrogen oxide-responsive regulons. The upstream regions
of norB and nirK in C. violaceum,
COG4309-norB in R. solanacearum, and
nnrS2 in Burkholderia species contain two
candidate regulatory sites, a downstream NsrR site and an upstream DNR site (Figure 4), yielding both positive
regulation by the NO-responsive activator DNR and nitrite-induced de-repression by
the NsrR repressor. Secondly, the NO-detoxifying gene hmp in
P. stutzeri is preceded by three candidate NorR sites at
positions −192, −173, and −148 (relative to the
translational start site), a strong DNR site at position −116, and a
putative σ54 promoter at position −91 (Figure 6). This arrangement of
regulatory elements indicates dual positive control of the hmp
expression by different NO-responsive activators, σ54-dependent
NorR and σ70-dependent DNR. Finally, in many cases genes are
regulated by two additional regulators, the oxygen-responsive factor FNR
(hcp-hcr, hmp, and narK in enterobacteria) and
the nitrite/nitrate-sensitive two-component system NarQ/NarP (hcp-hcr,
dnrN, and hmp in enterobacteria, nnrS in
Vibrionales and Shewanella spp.). More complex regulatory
interactions are observed in Neisseria spp., where NsrR regulates
the NarQ/NarP system, whereas the common upstream region of the divergently
transcribed genes norB and nirK contains two
candidate NsrR sites, a candidate NarP site in the middle, and an FNR-binding site
immediately upstream of nirK, the latter being involved in the
anaerobic induction of this gene [49].
Various aspects of the described regulatory network for the nitrogen oxides
metabolism are verified by a large number of independent experimental observations.
Upregulation of the hcp gene in response to growth on nitrate or
nitrite was reported in S. oneidensis, E. coli, S. typhimuruim, and
D. vulgaris [10–12,14]; the same pattern of regulation was observed for
dnrN in S. typhimuruim and P.
stutzeri [12,34]. Our
prediction of positive regulation of hcp-frdX and negative
regulation of the sulfate reduction genes apsBA and
sat [33] was validated in a macroarray hybridization study, where
hcp was upregulated 255-fold with 5 mM nitrite, whereas
aprAB and sat were downregulated nearly
10-fold in the same conditions [14]. In addition, nitrite induced
transcription of thiosufate reductase phsA (a candidate member of
the HcpR regulon) and inhibited the membrane-bound electron transport complex
qmoABC, located just downstream of the apsBA
genes and thus also possibly repressed by HcpR.
The flavohemoglobin gene hmp is induced by NO and nitrite in
E. coli and B. subtilis [28,29], and the mechanism of the
hmp regulation by the nitrite repressor NsrR predicted in this
study is conserved in these diverse bacteria. Another NO-mediated mechanism of
hmp regulation in E. coli by the
O2-responsive repressor FNR was proposed [50]. At that, NO was found to inactivate
FNR anaerobically, restoring the hmp expression. However, our data
indicate that, in contrast to the candidate NsrR site, the FNR binding site is
conserved in only closely related bacteria E. coli and S.
typhimurium (Figure S3). Finally, NO induces the
norB expression in N. gonorrhoeae, but it was
found to be independent of known nitrogen oxide-responsive regulators
[30]. Here
we describe possible co-regulation of all denitrification genes in the
Neisseria species by the nitrite-sensitive repressor NsrR.
Recently, transcriptional regulation of the flavohemoglobin gene
fhp (hmp ortholog) by the NO-responsive regulator
FhpR (NorR ortholog) has been demonstrated in P. aeruginosa
[51].
A recent paper by Elvers et al. [52] describes a new nitrosative
stress-responsive regulon in ɛ-proteobacterium C. jejuni
regulated by a member of the CRP/FNR family. This regulator (named NssR) is
homologous (26% identity) to the HcpR2 factor from T.
maritima described in this study. It has an FNR-like recognition motif
(TTAACnnnnGTTAA) and specificity-determining positions A180 and
Q181, conforming to the correlation between these two positions and
contacting bases in the DNA motif observed in this study. NssR positively regulates
expression of the Campylobacter globin gene cgb,
encoding a single-domain hemoglobin that mediates resistance to NO and nitrosative
stress [48].
Thus the regulation of the cgb gene by HcpR in A.
ferrooxidans predicted in this study is in agreement with verified
NssR-mediated activation of the cgb ortholog in C.
jejuni.
While this study was being completed, we obtained a personal communication from S.
Spiro from Georgia Institute of Technology, Atlanta, Georgia, United States, that
the NsrR ortholog in E. coli (YjeB) is a NO-sensitive
transcriptional repressor of several nitrosative stress responsive genes including
hmp, ytfE (dnrN), and ygbA. Moreover, a common
inverted repeat sequence coincided with the defined here NsrR recognition motif was
shown to be involved in NsrR-mediated repression of the ytfE gene.
The mechanisms of regulation of nitrogen oxides metabolism in bacteria are of great
importance both for ecology and medicine. Nitrifying and denitrifying microorganisms
are significant sources of both nitric and nitrous oxides production in the
atmosphere, and thus have a great impact in the greenhouse effect [53]. Nitrate has become a
pollutant of groundwater and surface water. NO and reactive nitrogen intermediates
are also part of the arsenal of antimicrobial agents produced by macrophages
[54].
Therefore, nitrosative stress tolerance genes, which are inducible after invasion,
provide a strong advantage for pathogenic bacteria that need to resist the host
defense system. Here we tentatively characterized the transcriptional regulatory
network for the genes involved in these significant metabolic processes. Overall,
although each particular prediction made in this study may require experimental
verification, the emerging overall picture seems to be rather consistent and
robust.
Materials and Methods
Complete and partial bacterial genomes were downloaded from GenBank [55]. Preliminary sequence
data were also obtained from the Institute for Genomic Research (http://www.tigr.org), the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/), and the DOE Joint Genome Institute
(http://jgi.doe.gov). The gene identifiers from GenBank are used
throughout. Genome abbreviations are listed in Table 2. Protein similarity search was done using
the Smith-Waterman algorithm implemented in the GenomeExplorer program
[56].
Orthologous proteins were defined by the best bidirectional hits criterion and named
by either a common name of characterized protein or by an identifier in the Clusters
of Orthologous Groups (COG) database for uncharacterized proteins [57]. Distant homologs
were identified using PSI-BLAST [58]. The SEED tool, which combines
protein similarity search, positional gene clustering, and phylogenetic profiling of
genes, was applied for comparative analysis and annotation of multiple microbial
genomes (see the “Nitrosative stress and Denitrification”
subsystems at http://theseed.uchicago.edu/FIG/index.cgi). The phylogenetic trees
were constructed by the maximum likelihood method implemented in PHYLIP
[59] using
multiple sequence alignments of protein sequences produced by CLUSTALX
[60]. In
addition, the InterPro [61], and PFAM [62] databases were used to verify protein
functional and structural annotations.
10.1371/journal.pcbi.0010055.t201
List of Genome Abbreviations Used in This Study
10.1371/journal.pcbi.0010055.t202
Continued
For identification of candidate regulatory motifs, we started from sets of
potentially co-regulated genes (using previous experimental and general functional
considerations). A simple iterative procedure implemented in the program SignalX (as
described previously in [63]) was used for construction of common transcription
factor–binding motifs in sets of upstream gene fragments. Each genome
encoding the studied transcription factor was scanned with the constructed profile
using the GenomeExplorer software (see the detailed description at http://bioinform.genetika.ru/projects/reconstruction/index.htm), and
genes with candidate regulatory sites in the upstream regions were selected. The
threshold for the site search was defined as the lowest score observed in the
training set. Dependent on the DNA motif and the number of sites in the training
set, such threshold could be too strict or too permissive. It seems that the
threshold choice was adequate in our cases, as little clear false positives were
encountered, and, on the other hand, most functionally relevant genes were found to
belong to at least one of the studied regulons (Table 3 and Results and Discussion sections). The
upstream regions of genes that are orthologous to genes containing regulatory sites
of any of studied nitrogen-related factors were examined for candidate sites even if
these were not detected automatically with a given threshold. Among new candidate
members of a regulon, only genes having candidate sites conserved in at least two
other genomes were retained for further analysis. We also included new candidate
regulon members that are functionally related to the nitrogen oxides metabolism.
This procedure allowed us to reject a small number of false positive sites
identified after scanning of microbial genomes (Table 3). Sequence logos for derived regulatory
motifs were drawn using WebLogo package v.2.6 [64] (http://weblogo.berkeley.edu/).
10.1371/journal.pcbi.0010055.t003
Distribution of Predicted Regulatory Sites in Bacterial Genomes
Supporting Information
Multiple Sequence Alignment of the Upstream Regions of the hcp-hcr
Operons from Enterobacteria
(22 KB DOC)
Multiple Sequence Alignment of the Upstream Regions of the dnrN Genes
from Enterobacteria
(20 KB DOC)
Multiple Sequence Alignment of the Upstream Regions of the hmp Genes from
Enterobacteria
(26 KB DOC)
Multiple Sequence Alignment of the Upstream Regions of the hcp-hcr
Operons from Vibrio Species
(24 KB DOC)
Candidate HcpR-Binding Sites
(40 KB XLS)
Candidate DNR-Binding Sites
(34 KB XLS)
Candidate NnrR-Binding Sites
(29 KB XLS)
Candidate NsrR-Binding Sites
(24 KB XLS)
Candidate NorR-Binding Sites and Corresponding σ54
Promoters
(23 KB XLS)
Accession Numbers
The Pfam (http://www.sanger.ac.uk/Software/Pfam/) accession numbers for
products discussed in this paper are: hypothetical transcriptional factor from
Rrf2 protein family (PF02082) and eukaryotic alternative oxidase Aox (PF01786).
Complete and partial bacterial genomes were downloaded from GenBank
[55].
This study was partially supported by grants from the Howard Hughes Medical Institute
(55000309 to MSG), the Russian Fund of Basic Research
(04–04–49361 to DAR), the Russian Science Support Fund (MSG),
and the Russian Academy of Sciences (Programs “Molecular and Cellular
Biology” and “Origin and Evolution of the Biosphere”).
ILD, EJA, and APA were in part supported by a US Department of Energy Genomics: GTL
grant (DE-AC03-76SF00098, to APA). We thank Anna Gerasimova and Dmitry Ravcheev for
the FNR and NarP recognition profiles, and Andrey Mironov and Sergey Stolyar for
useful discussions.
AbbreviationNO
nitric oxide.
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