Related organisms typically rely on orthologous regulatory proteins to respond to a given signal. However, the extent to which (or even if) the targets of shared regulatory proteins are maintained across species has remained largely unknown. This question is of particular significance in bacteria due to the widespread effects of horizontal gene transfer. Here, we address this question by investigating the regulons controlled by the DNA-binding PhoP protein, which governs virulence and Mg2+ homeostasis in several bacterial species. We establish that the ancestral PhoP protein directs largely different gene sets in ten analyzed species of the family Enterobacteriaceae, reflecting both regulation of species-specific targets and transcriptional rewiring of shared genes. The two targets directly activated by PhoP in all ten species (the most distant of which diverged >200 million years ago), and coding for the most conserved proteins are the phoPQ operon itself and the lipoprotein-encoding slyB gene, which decreases PhoP protein activity. The Mg2+-responsive PhoP protein dictates expression of Mg2+ transporters and of enzymes that modify Mg2+-binding sites in the cell envelope in most analyzed species. In contrast to the core PhoP regulon, which determines the amount of active PhoP and copes with the low Mg2+ stress, the variable members of the regulon contribute species-specific traits, a property shared with regulons controlled by dissimilar regulatory proteins and responding to different signals.
Organisms often respond to environmental cues by modifying the patterns of expression of multiple genes. Related species typically rely on orthologous DNA-binding regulatory proteins to orchestrate the response to a given stimulus. However, it is unclear whether different organisms express similar or rather distinct groups of genes in response to the same signal. This is of special interest in bacteria because even closely related species, such as Escherichia coli and Salmonella enterica, have quite distinct lifestyles and display significant differences in gene content. Here, we have addressed this question by investigating the sets of genes that the Mg2+-responding PhoP protein controls in ten enteric species. We established that the majority of targets governed by the PhoP protein are not shared across species. This is due to regulation of species-specific genes and also to transcriptional rewiring of shared genes. The few PhoP-regulated targets retained across species encode the most conserved proteins, which determine the level of active PhoP protein and mediate Mg2+ homeostasis. These findings demonstrate that self-governing the activity of a regulatory protein is critical for its proper functioning, even though it may regulate largely different gene sets across species.
Citation: Perez JC, Shin D, Zwir I, Latifi T, Hadley TJ, et al. (2009) Evolution of a Bacterial Regulon Controlling Virulence and Mg2+ Homeostasis. PLoS Genet 5(3): e1000428. doi:10.1371/journal.pgen.1000428
Editor: Josep Casadesús, Universidad de Sevilla, Spain
Received: September 4, 2008; Accepted: February 17, 2009; Published: March 20, 2009
Copyright: © 2009 Perez et al. This 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.
Funding: This work was supported by grant AI49561 from the National Institutes of Health and funds from the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The ability of an organism to orchestrate responses to environmental changes often depends on transcriptional regulatory proteins that control the expression of multiple genes. Related species typically rely on orthologous regulatory proteins to respond to a given stimulus. However, the extent to which the targets of regulation of such orthologs (i.e., the regulon) are retained across species is not clear. This is of special interest in bacteria due to the rampant effects of horizontal gene transfer (reviewed in ,), which raises questions about how bacterial regulons have been shaped by widespread gains and subsequent losses of genes, and about the role played both by the conserved targets of regulation as well as by those that are species-specific. By contrast, in eukaryotes, where most of the experimental studies on the evolution of gene regulation have been carried out, transcriptional rewiring (i.e. gains and/or losses of interactions between orthologous regulatory proteins and orthologous target genes) appears to be the main source of variability in regulatory networks – as related eukaryotic species have similar gene content. We have addressed the evolution of bacterial gene regulation by investigating the regulons controlled by the DNA-binding regulatory protein PhoP in several enteric species, the most distant of which shared a common ancestor >200 million years ago.
The activity of the DNA-binding regulatory protein PhoP is dictated by its cognate sensor PhoQ, which responds to the extracytoplasmic levels of Mg2+: transcription of PhoP-activated genes is promoted in low Mg2+ and repressed in high Mg2+ . The PhoP regulon has been best characterized in the human pathogen Salmonella enterica serovar Typhimurium, where the PhoP protein regulates ~3% of the genes  both directly by binding to its target promoters, and indirectly by altering the levels and/or activity of other regulatory proteins and systems . These PhoP-activated targets include Mg2+ transporters, enzymes involved in the covalent modification of cell envelope components, as well as virulence proteins whose biochemical activities remain largely undefined.
Despite establishing different interactions with their animal and plant hosts, the bubonic plague agent Yersinia pestis, the diarrhea-causing Shigella flexneri and the plant-pathogen Erwinia carotovora depend on a functional PhoP/PhoQ system to cause disease –, like Salmonella –. The phoPQ genes are also found in the human commensal Escherichia coli as well as in the soil dwelling Klebsiella pneumoniae and in Sodalis glossinidius, a secondary symbiont of the tsetse fly. The presence of the PhoP/PhoQ system in this phenotypically diverse group of bacteria suggests that PhoP may regulate the expression of different sets of genes across species (i.e., each regulon might be suited to the niche in which each organism proliferates.) Alternatively or in addition, PhoP could control cellular functions that are shared among different species in spite of their distinct lifestyles.
Here we examine the evolution of the PhoP regulon across ten species of the family Enterobacteriaceae. We experimentally determine that PhoP has adopted largely different targets of regulation, the result of horizontal gene transfer events that altered gene content even among closely related species. We show that transcriptional rewiring events involving gains and/or losses of interactions between PhoP and shared genes have shaped the PhoP regulon and may contribute to phenotypic differences between organisms. Moreover, we establish that the core members of the PhoP regulon (i.e., those maintained in all analyzed members of the Enterobacteriaceae family) participate in regulatory loops designed to control the level and activity of the PhoP/PhoQ system; and that those PhoP-regulated target genes common to most species ensure Mg2+ homeostasis. This demonstrates that governing the activity of the PhoP/PhoQ regulatory system is critical for its proper functioning independently of how different its regulated targets might be.
Identifying Genes Directly Regulated by the PhoP Protein in Y. pestis
We sought to identify the PhoP-regulated genes in the plague agent Y. pestis because: First, a functional PhoP protein is necessary for virulence in mice and for survival inside mammalian cells , as in Salmonella –. And second, S. enterica and Y. pestis are distantly-related members of the family Enterobacteriaceae, having diverged from their last common ancestor >200 million years ago . To enable the identification of genes regulated by the PhoP protein and to distinguish between genes that are directly and indirectly regulated by PhoP, we used both expression microarray analysis of wild-type vs. phoP mutant strains as well as chromatin immunoprecipitation followed by array hybridization (ChIP-chip) on custom-made whole genome tiling arrays (a Y. pestis strain harboring an epitope-tagged phoP gene in the chromosome was utilized for ChIP and as the wild-type strain in the expression microarrays.) Three biological replicates of each sample (RNA or DNA) were prepared from Y. pestis cells grown in defined medium containing low (50 µM) Mg2+, which are inducing conditions for the PhoP/PhoQ system, and hybridized to custom-designed NimbleGen tiling microarrays (Figure S1). Data from the six expression microarrays (three wild-type and three phoP−) were combined for downstream data processing whereas the three ChIP-chip data sets were analyzed individually.
Based solely on the expression microarray data (i.e. without including the ChIP-chip results), 31 and 14 transcription units appeared to be activated and repressed by the phoP gene, respectively (Table S2) (see Text S1 for a description of how the tiling microarray data were processed and Table S1 for a list of all the probes that exhibited differential expression (>2-fold)). We note that a single transcription unit may entail several co-transcribed ORFs (which is readily inferred from the expression pattern of contiguous probes in the tiling array) as well as transcripts corresponding to genes not previously annotated. This set of transcripts represents potential direct and indirect targets of the PhoP protein.
We considered a transcription unit to be directly regulated by PhoP if there was a ChIP peak <300 bp from the 5′ end of a transcript that appeared phoP-regulated in the expression microarrays. In most cases there were ChIP peaks with significant scores (FDR≤0.05) in similar positions in all three biological replicates. We used real time PCR to verify whether these transcripts were indeed phoP-regulated and if the promoter regions of these transcripts were enriched in the ChIP samples (data not shown). Then, we identified their transcription start sites by primer extension or S1 mapping, and determined that these start sites were phoP-dependent. PhoP binding sites were experimentally and/or computationally identified within 100 nt upstream of the transcription start sites. The location and orientation of the PhoP boxes in all these promoters are similar to those in well-characterized PhoP-activated promoters in E. coli and S. enterica ,. Sixteen transcripts met all these criteria (Table S3). Thus, in this study we refer to these sixteen transcripts as the Yersinia PhoP regulon. (We found ChIP peaks in front of only two repressed transcripts (Table S2) suggesting that PhoP may repress them directly.)
A list of PhoP regulon members in Y. pestis biovar Microtus has been recently reported . Curiously, only four of the 18 promoters described as directly regulated by PhoP overlap with the data set presented here. The reason(s) for the discrepancy are not entirely clear, but could be due to the following: First, our approach was based on a genome-wide search for in vivo PhoP binding regions located <300 bp away from phoP-dependent transcripts identified in vivo whereas Li. et al.  investigated binding of the purified PhoP protein in vitro to DNA fragments located upstream of ORFs that could be phoP-regulated. Second, Li et al. grew Yersinia under conditions  that are different from those shown to induce the Yersinia PhoP/PhoQ system effectively . And third, the Y. pestis strain utilized by Li et al. is not the same as that examined in this work. Importantly, the structures of all the PhoP-regulated promoters identified in our study resemble those of well-characterized PhoP-activated promoters in other organisms ,,. By contrast, the only promoters described in  that harbor structures reminiscent of previously characterized PhoP-dependent promoters are those overlapping with our data, and the remaining putative PhoP-activated promoters contain PhoP boxes at locations uncharacteristic of a bacterial transcriptional activator (i.e., >100 nt upstream of the transcription start site, overlapping with the −10 sequence, or positioned downstream of the transcription start site.)
The PhoP Regulons of Y. pestis and S. enterica
Based on their occurrence in Y. pestis and S. enterica, we classified the identified PhoP-regulated targets from Yersinia and the 20 transcription units known to be directly activated by PhoP in S. enterica ,, into three groups: (1) genes/operons present in Yersinia but not in Salmonella, or vice versa; (2) genes/operons present in both species but controlled by PhoP in only one of the two species; and (3) orthologous genes/operons controlled by PhoP in both Yersinia and Salmonella (Figure 1). As discussed below, the first group of genes is by far the most abundant and their sporadic distribution in other enteric species suggest that they have been horizontally acquired. The second group of genes (five out of sixteen) represent transcriptional rewiring events, a phenomenon that, to our knowledge, has not been experimentally addressed in bacteria. The third group of genes is the smallest set and, as the results presented in the following sections indicate, they appear to play distinct critical roles in the proper functioning of the PhoP/PhoQ system.
Figure 1. Diagram depicting examples of PhoP-regulated targets based on their occurrence in S. enterica and Y. pestis.
The Salmonella-specific ugtL gene has no homolog in Yersinia, and the converse is true for the Yersinia y4126 gene. As discussed in the main text, this class of genes is by far the most abundant and their sporadic distribution in other enteric species suggest that they may have been horizontally acquired. The rstA and psiE genes are present in both species but are controlled by PhoP in only one of the two species. Genes of this class (five out of sixteen) represent transcriptional rewiring events, a phenomenon that has not been experimentally addressed in bacteria. The slyB gene is controlled by PhoP in both Yersinia and Salmonella, and encodes a product that negatively regulates PhoP activity and is critical for the proper functioning of the PhoP/PhoQ system.doi:10.1371/journal.pgen.1000428.g001
Most genes Directly Regulated by PhoP in Y. pestis or in S. enterica Have No Homologs in Other Enteric Species
The majority of genes directly regulated by PhoP in Y. pestis have no BLAST matches in S. enterica and vice versa (Figure 2 and Table S4). To determine whether the absence of homologs was particular to the Yersinia-Salmonella pairwise comparison, we searched the genomes of eight additional members of the family Enterobacteriaceae for the presence of sequences homologous to the identified PhoP-regulated protein-coding genes of Yersinia and Salmonella. This analysis revealed that over half of the gene products directly controlled by PhoP in Yersinia lack homologs in at least three other species (absence of homologous sequences is represented by white squares in Figure 2A). This finding indicates that these genes have been gained and/or lost during the evolution of the family Enterobacteriaceae. Analysis of the S. enterica PhoP regulon revealed a similar pattern as nearly half of the protein-coding genes directly regulated by PhoP had no homologs in most of the other enteric species (white area in Figure 2B). This is in parallel to the overall differences in gene content that exist between the phylogenetically most distant species in the analyzed group, Escherichia coli and Y. pestis, which share only ~50% of their genes . Consistent with the conclusions from a purely computational comparison of the E. coli and S. enterica PhoP regulons , our data indicate that orthologous PhoP proteins promote expression of largely distinct gene sets in individual enteric species.
Figure 2. Limited distribution of Y. pestis and S. enterica PhoP-activated genes across the Enterobacteriaceae family.
A–B. Matrices of conservation scores (CS) of proteins encoded by the genes directly activated by PhoP in Y. pestis (A) and S. enterica (B). CS is the BlastP score of the closest homologue in a particular species divided by the BlastP score of the protein against itself. CS values (represented by colors) can range from 0 when no homolog or ortholog is detected in another species, to 1 when the closest homolog exhibits 100% amino acid identity. The phylogenetic relationships shown to the left of the figures are based on orthologous housekeeping genes present in all species. Note that branch lengths do not represent phylogenetic distances.doi:10.1371/journal.pgen.1000428.g002
While the biochemical function of the species-specific PhoP-activated gene products remains largely unknown, several of the Salmonella-specific targets have been implicated in survival within host cells, such as mig-14 , or in resistance to host antimicrobial products, such as ugtL . The majority of PhoP-activated Yersinia-specific targets encode uncharacterized proteins.
PhoP-Regulated Protein-Coding Genes Exhibit Different Levels of Sequence Conservation in Enteric Bacteria
We determined the degree of sequence identity for each pair of homologs by calculating their conservation scores (CS) ,, which represent the BlastP score of the closest homologue in a particular species divided by the BlastP score of the protein against itself. CS values range from 0 when no homolog or ortholog is detected in another species, to 1 when the closest homolog exhibits 100% amino acid identity. This analysis revealed that only three gene products directly controlled by the PhoP protein both in Yersinia and Salmonella – PhoP, PhoQ, and SlyB – are relatively well conserved in the analyzed enteric species (red squares in Figure 2A and 2B). By contrast, the well-conserved psiE, ompX and rstA genes are regulated by PhoP only in one of the two species.
A subset of the genes regulated directly by PhoP in either Yersinia or Salmonella encodes products poorly conserved in other enterics (blue squares in Figures 2A and 2B). These genes are unlikely to be true orthologs because the level of amino acid identity between the corresponding products is considerable lower than the median ~72% identity that is found between E. coli and Y. pestis orthologs ) (blue in Figure 2 represents roughly 40% amino acid identity). For instance, PhoP promotes transcription of the Salmonella pagP gene and the Yersinia y2563 gene by binding to their respective promoter regions (Figure 2). Even though the pagP and y2563 genes are recovered as the best reciprocal hits in a BLAST search, their gene products are only 42% identical and do not seem to carry out the same biochemical function because the PagP-mediated palmitoylation of the lipid A established in S. enterica  has not been detected in Y. pestis . Therefore, even when homologous genes are regulated in a like manner, the activities of their gene products may not be retained across species.
Differences in the PhoP regulons of Y. pestis and S. enterica Resulting from Transcriptional Rewiring
The gains and/or losses of interactions between orthologous regulatory proteins and orthologous target genes (i.e., transcriptional rewiring) account for the majority of differences in the targets of regulation among related eukaryotic species –. Rewiring events have not been considered in previous analyses of bacterial regulons because the purely computational comparisons reported to date have focused on gene content, and thus, they assumed that if a regulatory protein controls a particular target in one species, such regulatory relationship will be conserved in another species –. By combining ChIP-chip and expression data obtained with full genome tiling arrays, we could experimentally determine the contribution that rewiring events make to the composition of the PhoP regulons.
We established that the PhoP protein controls transcription of both the outer membrane-encoding gene ompX and the regulatory gene rstA in Salmonella but not in Yersinia. This may result in significant phenotypic differences between these two species because the rstA gene encodes a regulatory protein that modulates the levels of the alternative sigma factor RpoS  and of a Fur-repressed iron transporter  in Salmonella. Conversely, the phosphate-starvation inducible gene psiE, the putative aminidase encoding gene y1877 (ybjR), and the putative inner membrane protein coding gene y2124 (STM3036) are regulated by PhoP in Yersinia but not in Salmonella. Considering that ~16 transcripts directly regulated by PhoP have homologous sequences in Y. pestis and S. enterica, this means that ~30% of them have undergone transcriptional rewiring events. To our knowledge, this is the first report on the prevalence of transcriptional rewiring in a bacterial regulon based on experimental evidence.
In addition to changes in regulon membership, rewiring events can result in novel interactions between orthologous regulatory proteins and orthologous target genes that are qualitatively similar (i.e., a target gene is turned on in response to the same signal)  but generate quantitatively different outputs. We determined that the Yersinia PhoP protein governs transcription of the pbgP (y1917) operon and the ugd (y2147) gene directly, by binding to their respective promoters (Table S3, see also ), but that it does so indirectly in Salmonella (i.e., by activating a different regulatory protein) –. Though the direct and indirect pathways are qualitatively similar in the two species (i.e., low Mg2+ promotes transcription of the polymyxin B-resistance conferring pbgP and ugd genes), the indirect pathway operating in Salmonella exhibits signal amplification and expression persistence relative to the direct pathway present in Yersinia .
The Virulence Gene mgtC Was Independently Embedded in the PhoP Regulons of Y. pestis and S. enterica
The PhoP-activated mgtC gene encodes an inner membrane protein necessary for virulence in mice, survival within macrophages and growth in low Mg2+ in Salmonella , and for the survival of Y. pestis inside macrophages . However, the low level of amino acid identity between the two MgtC proteins, the sporadic phylogenetic distribution of the mgtC gene within the family Enterobacteriaceae  as well as the presence of mgtC homologs in more distant bacterial species such as Burkholderia cenocepacia  raises the possibility that the Salmonella and Yersinia mgtC genes may be xenologs rather than orthologous (i.e., acquired separately by Salmonella and Yersinia in independent horizontal-gene transfer events) and likely incorporated into the two PhoP regulons independently. This could reflect that the mgtC gene enables survival within mammalian cells and in Mg2+-limiting environments, which are conditions that activate the PhoP/PhoQ system .
Autoregulation of the PhoP/PhoQ System Has Been Maintained in Y. pestis and S. enterica Despite Modifications in the cis-Regulatory Region of Its phoP Locus
The Salmonella PhoP/PhoQ system is transcriptionally autoregulated in a positive fashion by the PhoP protein binding to a promoter located immediately upstream of the phoP coding region ,. We established that the Yersinia phoP and phoQ genes were turned on in low Mg2+ in a PhoP-dependent fashion (Figure 3), just like the Salmonella phoP and phoQ genes . However, a PhoP box could not be identified immediately upstream of the phoP coding region in Yersinia and the PhoP protein did not bind to this region in vivo (Figure 3A and 3B). We determined that positive autoregulation of the Yersinia phoP and phoQ genes is mediated by a promoter located upstream of y1795, the gene located 5′ of phoP and predicted to be transcribed in the same direction as the phoP and phoQ genes (Figure 3A) because: First, the PhoP protein bound to this region in vivo (Figure 3B) and footprinted it in vitro (Figure 3C and 3D). Second, transcription initiated at this promoter was phoP-dependent (Figure 3E) and extended into the phoP gene as determined by reverse transcription-PCR (Figure 3F). Third, point mutations in the predicted PhoP box of the y1795 promoter abolished PhoP-dependent expression from this promoter (Figure 3G). And fourth, Western blot analysis of cell extracts prepared from a Yersinia strain encoding a PhoP-HA protein from its normal chromosomal location and probed with anti-HA antibodies demonstrated higher levels of PhoP-HA protein following growth under inducing (i.e., low) than in repressing (i.e., high) Mg2+ concentrations (Figure 3H).
Figure 3. The phoP and phoQ genes are part of a PhoP-activated three-gene operon in Y. pestis.
A) Diagram depicting the genomic context of the phoPQ locus in Y. pestis. The bent arrow shows the PhoP-activated transcription start site determined in (E). The red bar indicates the 288 nt transcript identified in (F). Large arrows represent protein-coding sequences. B) In vivo PhoP binding profile to the genomic region shown in (A) as determined by ChIP-chip. C) DNA sequence of the y1795 promoter region. The transcription start site determined in (E) is indicated by the bent arrow; the PhoP binding site is in red; the putative −10 sequence is underlined, and the region protected by PhoP is boxed. Nucleotide substitutions introduced in the PhoP binding site in (G) are indicated below the box. D) DNase I footprinting analysis of the promoter region of the y1795 gene with increasing amounts (0, 2.5, 5, 10 and 20 pmol) of Y. pestis PhoP protein. The bar indicates the protected region. E) Primer extension analysis of the y1795 gene carried out with RNA samples prepared from wild-type (EG19221) and phoP mutant (EG14737) strains following growth under high (H) or low (L) Mg2+ concentrations. The arrow indicates the mapped transcription start site. F) Reverse-transcription-PCR analysis of RNA samples prepared from Y. pestis wild-type cells. Reverse-transcription was carried out with (+) or without (−) reverse-transcriptase (RT) and the products of these reactions used as template to PCR amplify the 288 nt fragment shown in (A). G) GFP expression driven by Y. pestis strains harboring plasmids with transcriptional fusions between the y1795 promoter containing either wild-type sequence or point mutations in the predicted PhoP box and a promoterless gfp gene. Fluorescence was measured in wild-type (KIM6) and phoP (EG14737) strain backgrounds following growth in defined medium supplemented with 50 µM MgCl2. Shown are the mean and standard deviation values of at least three independent experiments performed in triplicates. H) Western blot analysis of cell extracts prepared from Y. pestis cells harboring an epitope tagged phoP gene (EG19221). Cells were grown as described in (E). Anti-RpoB antibodies were used to detect the RpoB protein, which served as a loading control.doi:10.1371/journal.pgen.1000428.g003
The incorporation of the y1795 gene into Yersinia appears to have occurred after the lineage that gave rise to this genus split from the one originating Serratia because y1795 orthologs have not been found outside Yersinia spp. (see phylogeny in Figure 2). The y1795 gene is predicted to encode a 207 amino acid outer membrane protein and/or lipoprotein without significant similarity to proteins of known function. That the y1795 gene is adjacent to the phoP gene and co-transcribed with phoP and phoQ suggests that it may affect the levels and/or activities of the Yersinia PhoP and PhoQ proteins. While this possibility cannot be presently ruled out, chromosomal replacement of the S. enterica phoP and phoQ genes with the Y. pestis orthologs (without y1795) resulted in a strain that retained the normal regulation of the PhoP-activated mgtA gene . Moreover, open reading frames harboring functions unrelated to those of the two-component system proteins sometimes precede two-component system genes. For instance, the phoPQ genes of Pseudomonas aeruginosa are preceded by and form an operon with oprH, which encodes an outer membrane protein . Likewise, the genes for the PmrA/PmrB two-component system of S. enterica are part of a three-gene operon headed by the pmrC gene ,, which encodes an inner membrane protein implicated in the modification of the lipopolysaccharide .
Conserved PhoP-Regulated Targets Mediate Mg2+ Homeostasis
Mg2+ is the most abundant divalent cation in biological systems . It is essential in the cytosol for ATP-mediated reactions and as a stabilizer of ribosomes , and in membranes where it binds to negatively charged molecules, such as the phosphates in the lipopolysaccharide . Consistent with low Mg2+ being a signal that activates the PhoP/PhoQ system , several of the genes regulated by PhoP both in Yersinia and Salmonella and also present in the vast majority of the analyzed enteric species (Figure 2) encode proteins mediating the adaptation to low Mg2+.
On the one hand, the PhoP protein directly promotes transcription of the Salmonella mgtA and mgtB genes , encoding two of the three known Mg2+ transporters of S. enterica and exhibiting 50% identity to each other. PhoP directly regulates transcription of the mgtCB operon in Y. pestis (Figure S2), which lacks an mgtA gene. (The mgtCB operon also encodes the inner membrane protein MgtC discussed above.) Certain enteric species, such as E. coli and Citrobacter koseri, lack mgtCB but harbor mgtA (Figure 2B); other species such as Serratia proteamaculans and S. enterica harbor both mgtA and mgtCB whereas Sodalis glossinidius lacks homologs of all three genes. We identified putative PhoP boxes upstream of the coding regions of most of these Mg2+ transporter genes suggesting that the PhoP protein directly regulates their expression.
On the other hand, the proteins encoded by the PhoP-activated pbgP operon and the ugd gene are responsible for covalently modifying the lipid A phosphates in the lipopolysaccharide with 4-amino-4-deoxy-L-arabinose at sites normally neutralized by Mg2+, whereas the pagP gene product catalyzes the incorporation of a palmitate chain into lipid A . These two modifications confer resistance to different antimicrobial peptides . Except for Citrobacter koseri, all examined enteric species harbor pbgP and pagP homologs (Figure 2); yet, as discussed above, homologs may not be functionally equivalent.
Our analysis indicates that the PhoP regulon includes products that function in Mg2+ homeostasis, consistent with Mg2+ being the signal that regulates the PhoP/PhoQ system . These products need not be highly conserved as long as they fulfill a required function. For example, E. coli and Yersinia harbor only one PhoP-activated Mg2+ transporter each, MgtA and MgtB, respectively, which are only 51% identical at the amino acid level, which is much lower than the 72% median identity that exists between E. coli and Yersinia proteins .
Conserved Feedback Loops Control the Levels and the Activity of the PhoP/PhoQ System
Only two loci are directly regulated by PhoP both in Salmonella and in Yersinia and present in all ten examined enteric species (Figure 2): phoP/phoQ, encoding the PhoP/PhoQ two-component regulatory system, and slyB (Figure 4), encoding an outer membrane lipoprotein with similarity to a lipoprotein implicated in membrane integrity in Burkholderia spp. . Both phoPQ and slyB seem to be regulated by PhoP across the Enterobacteriaceae family because putative PhoP binding sites could be identified in their promoter regions in all analyzed enteric species (Figures S3 and S4). And in E. coli, direct PhoP regulation of phoPQ and slyB has been demonstrated experimentally .
Figure 4. PhoP promotes transcription of the Y. pestis slyB gene.
A) DNA sequence of the Y. pestis slyB promoter region. The transcription start site is indicated by the bent arrow; the PhoP binding site is in red; the putative −10 sequence is underlined, and the region protected by PhoP in (B) is boxed. B) DNase I footprinting analysis of the promoter region of the Y. pestis slyB gene with increasing amounts of Y. pestis PhoP protein (0, 5, 15, 30 and 60 pmol). The bars indicate protected regions. C) Primer extension analysis of the Y. pestis slyB gene carried out with RNA samples prepared from wild-type (EG19221) or phoP mutant (EG14737) strains following growth in low Mg2+. The arrow indicates the mapped transcription start site.doi:10.1371/journal.pgen.1000428.g004
Mutants lacking a functional phoP gene are defective for growth in low Mg2+ , consistent with PhoP's role in governing the adaptation to low Mg2+ environments. We determined that a Salmonella mutant deleted for the PhoP box in the phoP promoter exhibited an identical phenotype (Figure S5). This demonstrates that positive autoregulation of the phoPQ operon is required to generate sufficient amounts of active PhoP protein to promote expression of the PhoP-regulated gene products mediating growth in low Mg2+ ,.
To explore the slyB function, we examined the behavior of strains lacking or overexpressing the slyB gene and experiencing low Mg2+, which is a condition in which Salmonella requires a functional phoP gene ,. Whereas a slyB-deleted strain grew like wild-type Salmonella, the strain overexpressing slyB was defective for growth, especially in low Mg2+ (Figure 5A–B). To test whether the growth defect of the slyB-overexpressing strain was due to inhibition of PhoP protein activity, we examined transcription of four different PhoP-dependent promoters. We measured GFP expression levels in strains harboring transcriptional fusions to a promoterless gfp gene driven by either of four different PhoP-activated promoters in wild-type and slyB Salmonella strains grown under inducing conditions for the PhoP/PhoQ system. All promoters were expressed to higher levels in the slyB mutant compared to the isogenic wild-type strain (Figure 5C). The slyB gene was responsible for this phenotype because the production of the SlyB protein from a plasmid restored the wild-type levels of expression of the PhoP-activated ugtL gene to a slyB mutant strain harboring a chromosomal lacZYA transcriptional fusion to ugtL (Figure S6). Together, these results indicate that slyB negatively regulates the activity of the PhoP protein. This effect appears to be specific to slyB because transcription of PhoP-dependent targets was not altered when eight other PhoP-activated genes were mutated (data not shown).
Figure 5. SlyB negatively regulates the activity of the PhoP/PhoQ regulatory system.
A–B) Growth curves of wild-type Salmonella cells harboring plasmid pAHE-slyB over-expressing the slyB gene (A) or plasmid vector pAHE (B). Cells were grown in medium containing the Mg2+ concentrations indicated in the figure. 1 mM IPTG was added to all cultures to induce slyB expression. C) GFP expression driven by Salmonella strains harboring plasmids with transcriptional fusions between the promoters for the PhoP-activated genes phoP, pmrD, mig-14, and yobG, and a promoterless gfp gene. Fluorescence was measured in wild-type (14028 s) and slyB (DS292) strain backgrounds following growth in N-minimal medium supplemented with 50 µM MgCl2. Shown are the mean and standard deviation values of at least three independent experiments performed in triplicates.doi:10.1371/journal.pgen.1000428.g005
The ability of the PhoP/PhoQ system to modulate its own activity through positive and negative feedback loops has been conserved over hundreds of millions of years. The key role that these regulatory loops play in the proper functioning of the PhoP/PhoQ system is underscored by the fact that altering the activity of this system by either overexpressing the negative regulator slyB or abolishing phoPQ positive autoregulation render S. enterica unable to grow in low Mg2+ (Figures 5A and S5). The use of regulatory feedbacks to control the output of two-component systems is not exclusive to PhoP/PhoQ because other two-component regulatory systems positively regulate their own expression  and because the CseB/CseC two-component system of Streptomyces coelicolor controls the expression of a lipoprotein that negatively regulates CseB/CseC activity . (Note that even though the activity of the two-component systems CpxR/CpxA and RcsB/RcsC/RcsD is affected by the lipoproteins NlpE and RcsF, respectively, neither nlpE nor rcsF are controlled by the regulatory systems that they affect ,.)
The growing number of available bacterial genome sequences has revealed the existence of large extents of genomic variability even among closely related bacterial species. Despite these differences, closely related organisms typically rely on orthologous regulatory proteins to respond to a given stimulus. Here, we have explored how the content of bacterial regulons is shaped in an environment of widespread gains and losses of genes.
A multi-pronged approach to define regulons.
Experimental studies aimed at uncovering regulons genome-wide have typically relied on only one of two approaches: expression microarray analysis or ChIP-chip. However, transcript analyses do not distinguish between direct and indirect targets and many in vivo binding sites for a regulatory protein do not direct transcription . By utilizing in vivo binding data of a regulatory protein, genome-wide expression data of wild-type and mutant strains, and in silico analyses to define regulon membership, we could obtain an accurate estimate of rewiring in a bacterial regulon and avoid overestimating rewiring prevalence, which results when ChIP-chip data is solely considered (e.g., see –).
Roles of core and variable members of bacterial regulons.
Four aspects have shaped the PhoP regulon during the evolution of enteric bacteria: First, the acquisition and the subsequent loss of target genes; second, transcriptional rewiring of shared genes; third, differences in sequence conservation of the various targets of regulation; and fourth, retention of a small number of target genes. The latter defines a core regulon that controls the level of active PhoP protein and mediates the adaptation to low Mg2+, a condition activating the PhoP/PhoQ system . By contrast, the members of the variable regulon aid bacterial proliferation in the particular niches preferred by individual species.
The notion that bacterial regulons consist of core and variable portions, as demonstrated in this study, is further supported by recent (largely computational) analyses of the RpoE regulon of enteric bacteria , where core regulon members determine the amount of the alternative sigma factor RpoE and maintain cell envelope integrity, the disruption of which increases the level of RpoE; and variable members of the regulon participate in virulence functions. Likewise, the targets of the alternative sigma factor PvdS of Pseudomonas spp. have been divided into a core group involved in the production of an iron chelator and an accessory group that is species-specific . Finally, the DNA damage-activated LexA protein controls its own levels in most investigated organisms both by repressing its own transcription as well as that of the recA gene, which encodes a protein that promotes the autocatalytic cleavage (and resulting inactivation) of the LexA protein (reviewed in ). Conserved LexA regulon members participate in DNA repair and translesion synthesis and there is evidence that the broadly distributed LexA protein governs expression of a variable target set of genes , perhaps selected to respond in the specific habitats of individual species. Cumulatively, these findings suggest that the distinct roles played by core and variable portions of bacterial regulons are shared across structurally different regulatory proteins that operate by dissimilar mechanisms and respond to distinct signals.
Materials and Methods
Bacterial Strains, Plasmids, and Growth Conditions
Bacterial strains and plasmids used in this study are listed in Table S5. Primers are listed in Table S6. All S. enterica serovar Typhimurium strains were derived from wild-type strain 14028 s, and grown at 37°C in N-minimal medium  buffered in 50 mM Bis-Tris, pH 7.7, supplemented with 0.1% casamino acids, 38 mM glycerol and 50 µM or 10 mM MgCl2. Y. pestis strains were derived from wild-type strain KIM6 , and grown at the optimal growth temperature of 28°C in defined medium , pH 7.0, supplemented with 0.1% casamino acids, 10 mM (D)-glucosamine, and 50 µM or 10 mM MgSO4. E. coli strain DH5α was used as the host for the preparation of plasmid DNA. Ampicillin and kanamycin were used at 50 µg/ml and chloramphenicol at 20 µg/ml.
RNA Isolation and Expression Microarray Analysis
After overnight culture in medium containing 10 mM MgSO4, Y. pestis cells were washed with Mg2+-free medium and grown to A600 ~0.3 in 16 ml of medium containing 50 µM MgSO4 with vigorous shaking. 14 ml of cell culture were collected, mixed with RNAprotect™ Bacteria Reagent (Qiagen) and used to prepare total RNA using RNeasy® Mini Kit (Qiagen). RNA samples were treated with Turbo DNA-free DNase (Ambion) and re-purified with the RNeasy® Mini Kit. Y. pestis KIM tiling arrays were manufactured by NimbleGen Systems Inc (Madison). The array features are illustrated on Figure S1. RNA labeling, array hybridization and data extraction were carried out according to standard operating procedures by NimbleGen Systems Inc (Madison).
Chromatin Immunoprecipitation - Microarray Analysis (ChIP-chip)
After overnight culture in medium containing 10 mM MgSO4, Y. pestis cells were washed with Mg2+-free medium and grown to A600 ~0.3 in 22 ml of medium containing 50 µM MgSO4 with vigorous shaking. ChIP assays were carried out essentially as described . DNA labeling (IP sample with Cy5; input DNA with Cy3), array hybridization, data extraction and analysis were carried out according to standard operating procedures by NimbleGen Systems Inc (Madison). Detailed information about these and other experimental protocols are provided in Text S1.
Tiling array design used for Y. pestis expression microarrays and ChIP-chip analyses. 50 nt oligos overlapping every 25 nt were designed to tile the entire Y. pestis KIM strain genome on both strands. Tiles on the Crick strand are offset 14 bp relative to those on the Watson strand.
(0.2 MB EPS)
PhoP regulates mgtCB transcription in Y. pestis. A) DNA sequence of the Yersinia mgtCB promoter region. The transcription start site identified in (B) is indicated by the bent arrow; the PhoP binding site is in red; the putative −10 sequence is underlined, and the region protected by PhoP (C) is boxed. B) Primer extension analysis of the mgtCB operon carried out with RNA samples prepared from wild-type (EG19221) or phoP mutant (EG14737) strains grown in defined medium containing 10 mM (H) or 50 µM (L) MgSO4 as described under Materials and Methods. The arrow indicates the mapped transcription start site, which is shown in (A). C) DNase I footprinting analysis of the promoter region of the mgtCB operon with a probe for the noncoding strand and increasing amounts of Y. pestis PhoP protein (0, 5, 10, 20 and 40 pmol). The bars indicate the protected regions. D) Occupancy of mgtCB promoter region by PhoP as determined by chromatin immunoprecipitation in a strain expressing PhoP-HA (EG19221) or in wild-type untagged cells (negative control) grown in defined medium containing 50 µM MgSO4 as described under Materials and Methods. Shown are the mean and S.D. values of three independent experiments.
(0.9 MB EPS)
Conservation of PhoP boxes in the phoP promoter across the Enterobacteriaceae family. Shown are the DNA sequences of the promoter regions of the phoP gene in ten enteric species whose genome sequences are available. Predicted PhoP boxes are in red, putative −10 sequences are underlined. The first eight amino acids of the phoP ORF are indicated below the nucleotide sequence. * The Y. pestis sequence corresponds to the y1795 promoter and ORF.
(0.3 MB EPS)
Conservation of PhoP boxes in the slyB promoter across the Enterobacteriaceae family. Shown are the DNA sequences of the promoter regions of the slyB gene in nine enteric species whose genome sequences are available. Predicted PhoP boxes are in red, putative −10 sequences are underlined. The first eight amino acids of the slyB ORF are indicated below the nucleotide sequence.
(0.3 MB EPS)
Elimination of PhoP autoregulation impairs Salmonella growth in low Mg2+. Growth curves of Salmonella wild-type (EG13918 and pphoP::scar), ΔphoP/phoQ (EG15598) and a strain deleted for the PhoP box in the phoP promoter (EG14338). Cells were grown in N-minimal medium containing 10 µM MgCl2 with vigorous shaking at 37°C and A600 was determined every 60 min. The strain pphoP::scar harbors a “scar” sequence upstream of the PhoP box in the phoP promoter which does not influence PhoP expression .
(0.2 MB EPS)
SlyB negatively regulates the activity of the PhoP/PhoQ regulatory system. β-galactosidase activity (Miller units) expressed by Salmonella strains harboring a chromosomal lac transcriptional fusion to ugtL and either plasmid vector pAHE or plasmid pAHE-slyB expressing the slyB gene. Expression was investigated in wild-type (EG11250) and slyB (YS864) strain backgrounds following growth in N-minimal medium supplemented with 10 µM MgCl2. Shown are the mean and standard deviation values of at least three independent experiments performed in duplicates.
(0.2 MB EPS)
PhoP regulated genes.
(1.9 MB XLS)
Transcription units and ORFs whose expression is regulated by PhoP in Y. pestis based on tiling microarray data.
(0.08 MB DOC)
Promoters/Transcripts directly activated by PhoP in Y. pestis. Summary of ChIP-chip data (3 biological replicates) for PhoP-bound regions (peaks) located nearby transcripts whose expression was phoP-activated.
(0.09 MB DOC)
CS is the BlastP score of the closest homologue in a particular species divided by the BlastP score of the protein against itself. This can result in slightly different CS scores when Salmonella or Yersinia proteins are compared because the value in the denominator corresponds to the BlastP score of a protein against itself and Salmonella and Yersinia proteins are rarely identical. In other words, when scoring homologs of the Salmonella SlyB protein, the BlastP score of the Salmonella SlyB protein against itself will be in the denominator, whereas when scoring homologs of the Yersinia SlyB protein, the BlastP score of the Yersinia SlyB protein against itself will be in the denominator.
(0.06 MB DOC)
Bacterial strains and plasmids used in this study.
(0.2 MB DOC)
Primers used in this study.
(0.06 MB DOC)
(0.07 MB DOC)
We thank J. Fay, H. Ochman, and E. O'Shea for critically reading an earlier version of this manuscript.
Conceived and designed the experiments: JCP EAG. Performed the experiments: JCP DS TL TJH. Analyzed the data: JCP IZ EAG. Wrote the paper: JCP EAG.
- 1. Ochman H, Lawrence JG, Groisman EA (2000) Lateral gene transfer and the nature of bacterial innovation. Nature 405: 299–304.
- 2. Pallen MJ, Wren BW (2007) Bacterial pathogenomics. Nature 449: 835–842.
- 3. Borneman AR, Gianoulis TA, Zhang ZD, Yu H, Rozowsky J, et al. (2007) Divergence of transcription factor binding sites across related yeast species. Science 317: 815–819.
- 4. Odom DT, Dowell RD, Jacobsen ES, Gordon W, Danford TW, et al. (2007) Tissue-specific transcriptional regulation has diverged significantly between human and mouse. Nat Genet 39: 730–732.
- 5. Tuch BB, Galgoczy DJ, Hernday AD, Li H, Johnson AD (2008) The evolution of combinatorial gene regulation in fungi. PLoS Biol 6: e38.
- 6. Garcia Vescovi E, Soncini FC, Groisman EA (1996) Mg2+ as an extracellular signal: Environmental regulation of Salmonella virulence. Cell 84: 165–174.
- 7. Zwir I, Shin D, Kato A, Nishino K, Latifi T, et al. (2005) Dissecting the PhoP regulatory network of Escherichia coli and Salmonella enterica. Proc Natl Acad Sci USA 102: 2862–2867.
- 8. Kato A, Groisman EA (2008) The PhoQ/PhoP regulatory network of Salmonella enterica. Adv Exp Med Biol 631: 7–21.
- 9. Flego D, Marits R, Eriksson AR, Koiv V, Karlsson MB, et al. (2000) A two-component regulatory system, pehR-pehS, controls endopolygalacturonase production and virulence in the plant pathogen Erwinia carotovora subsp. carotovora. Mol Plant Microbe Interact 13: 447–455.
- 10. Grabenstein JP, Fukuto HS, Palmer LE, Bliska JB (2006) Characterization of phagosome trafficking and identification of PhoP-regulated genes important for survival of Yersinia pestis in macrophages. Infect Immun 74: 3727–3741.
- 11. Llama-Palacios A, Lopez-Solanilla E, Poza-Carrion C, Garcia-Olmedo F, Rodriguez-Palenzuela P (2003) The Erwinia chrysanthemi phoP-phoQ operon plays an important role in growth at low pH, virulence and bacterial survival in plant tissue. Mol Microbiol 49: 347–357.
- 12. Llama-Palacios A, Lopez-Solanilla E, Rodriguez-Palenzuela P (2005) Role of the PhoP-PhoQ system in the virulence of Erwinia chrysanthemi strain 3937: involvement in sensitivity to plant antimicrobial peptides, survival at acid pH, and regulation of pectolytic enzymes. J Bacteriol 187: 2157–2162.
- 13. Oyston PC, Dorrell N, Williams K, Li SR, Green M, et al. (2000) The response regulator PhoP is important for survival under conditions of macrophage-induced stress and virulence in Yersinia pestis. Infect Immun 68: 3419–3425.
- 14. Venkatesh B, Babujee L, Liu H, Hedley P, Fujikawa T, et al. (2006) The Erwinia chrysanthemi 3937 PhoQ sensor kinase regulates several virulence determinants. J Bacteriol 188: 3088–3098.
- 15. Fields PI, Groisman EA, Heffron F (1989) A Salmonella locus that controls resistance to microbicidal proteins from phagocytic cells. Science 243: 1059–1062.
- 16. Groisman EA, Chiao E, Lipps CJ, Heffron F (1989) Salmonella typhimurium phoP virulence gene is a transcriptional regulator. Proc Natl Acad Sci USA 86: 7077–7081.
- 17. Miller SI, Kukral AM, Mekalanos JJ (1989) A two-component regulatory system (phoP phoQ) controls Salmonella typhimurium virulence. Proc Natl Acad Sci USA 86: 5054–5058.
- 18. Galan JE, Curtiss R 3rd (1989) Virulence and vaccine potential of phoP mutants of Salmonella typhimurium. Microb Pathog 6: 433–443.
- 19. Deng W, Burland V, Plunkett G 3rd, Boutin A, Mayhew GF, et al. (2002) Genome sequence of Yersinia pestis KIM. J Bacteriol 184: 4601–4611.
- 20. Zwir I, Huang H, Groisman EA (2005) Analysis of differentially-regulated genes within a regulatory network by GPS genome navigation. Bioinformatics 21: 4073–4083.
- 21. Li YL, Gao H, Qin L, Li B, Han YP, et al. (2008) Identification and characterization of PhoP regulon members in Yersinia pestis biovar Microtus. BMC Genomics 9: 143.
- 22. Zhou D, Han Y, Qin L, Chen Z, Qiu J, et al. (2005) Transcriptome analysis of the Mg2+-responsive PhoP regulator in Yersinia pestis. FEMS Microbiol Lett 250: 85–95.
- 23. Winfield MD, Latifi T, Groisman EA (2005) Transcriptional regulation of the 4-Amino-4-deoxy-L-arabinose biosynthetic genes in Yersinia pestis. J Biol Chem 280: 14765–14772.
- 24. Lejona S, Aguirre A, Cabeza ML, Vescovi EG, Soncini FC (2003) Molecular characterization of the Mg2+-responsive PhoP-PhoQ regulon in Salmonella enterica. J Bacteriol 185: 6287–6294.
- 25. Yamamoto K, Ogasawara H, Fujita N, Utsumi R, Ishihama A (2002) Novel mode of transcription regulation of divergently overlapping promoters by PhoP, the regulator of two-component system sensing external magnesium availability. Mol Microbiol 45: 423–238.
- 26. Soncini FC, Vescovi EG, Solomon F, Groisman EA (1996) Molecular basis of the magnesium deprivation response in Salmonella typhimurium: Identification of PhoP-regulated genes. J Bacteriol 178: 5092–5099.
- 27. Monsieurs P, De Keersmaecker S, Navarre WW, Bader MW, De Smet F, et al. (2005) Comparison of the PhoPQ regulon in Escherichia coli and Salmonella typhimurium. J Mol Evol 60: 462–474.
- 28. Brodsky IE, Ghori N, Falkow S, Monack D (2005) Mig-14 is an inner membrane-associated protein that promotes Salmonella typhimurium resistance to CRAMP, survival within activated macrophages and persistent infection. Mol Microbiol 55: 954–972.
- 29. Shi YX, Cromie MJ, Hsu FF, Turk J, Groisman EA (2004) PhoP-regulated Salmonella resistance to the antimicrobial peptides magainin 2 and polymyxin B. Mol Microbiol 53: 229–241.
- 30. Lopez-Bigas N, De S, Teichmann SA (2008) Functional protein divergence in the evolution of Homo sapiens. Genome Biol 9: R33.
- 31. Lopez-Bigas N, Ouzounis CA (2004) Genome-wide identification of genes likely to be involved in human genetic disease. Nucleic Acids Res 32: 3108–3114.
- 32. Guo L, Lim KB, Poduje CM, Daniel M, Gunn JS, et al. (1998) Lipid A acylation and bacterial resistance against vertebrate antimicrobial peptides. Cell 95: 189–198.
- 33. Bishop RE (2005) The lipid A palmitoyltransferase PagP: molecular mechanisms and role in bacterial pathogenesis. Mol Microbiol 57: 900–912.
- 34. Lozada-Chavez I, Janga SC, Collado-Vides J (2006) Bacterial regulatory networks are extremely flexible in evolution. Nucleic Acids Res 34: 3434–3445.
- 35. Madan Babu M, Teichmann SA, Aravind L (2006) Evolutionary dynamics of prokaryotic transcriptional regulatory networks. J Mol Biol 358: 614–633.
- 36. Price MN, Dehal PS, Arkin AP (2007) Orthologous transcription factors in bacteria have different functions and regulate different genes. PLoS Comput Biol 3: 1739–1750.
- 37. Cabeza ML, Aguirre A, Soncini FC, Garcia Vescovi E (2007) Induction of RpoS degradation by the two-component system regulator RstA in Salmonella enterica. J Bacteriol 189: 7335–7342.
- 38. Jeong J, Kim H, Yun J, Ryu S, Groisman EA, et al. (2008) RstA-promoted expression of the ferrous iron transporter FeoB under iron-replete conditions enhances Fur activity in Salmonella enterica. J Bacteriol 190: 7326–7334.
- 39. Mitrophanov AY, Jewett MW, Hadley TJ, Groisman EA (2008) Evolution and dynamics of regulatory architectures controlling polymyxin B resistance in enteric bacteria. PLoS Genet 4: e1000233.
- 40. Kato A, Groisman EA (2004) Connecting two-component regulatory systems by a protein that protects a response regulator from dephosphorylation by its cognate sensor. Genes Dev 18: 2302–2313.
- 41. Kox LFF, Wosten MMSM, Groisman EA (2000) A small protein that mediates the activation of a two-component system by another two-component system. EMBO J 19: 1861–1872.
- 42. Mitrophanov AY, Groisman EA (2008) Signal integration in bacterial two-component regulatory systems. Genes Dev 22: 2601–2611.
- 43. Kato A, Mitrophanov AY, Groisman EA (2007) A connector of two-component regulatory systems promotes signal amplification and persistence of expression. Proc Natl Acad Sci USA 104: 12063–12068.
- 44. Blanc-Potard AB, Groisman EA (1997) The Salmonella selC locus contains a pathogenicity island mediating intramacrophage survival. EMBO J 16: 5376–5385.
- 45. Maloney KE, Valvano MA (2006) The mgtC gene of Burkholderia cenocepacia is required for growth under magnesium limitation conditions and intracellular survival in macrophages. Infect Immun 74: 5477–5486.
- 46. Shin D, Lee EJ, Huang H, Groisman EA (2006) A positive feedback loop promotes transcription surge that jump-starts Salmonella virulence circuit. Science 314: 1607–1609.
- 47. Soncini FC, Vescovi EG, Groisman EA (1995) Transcriptional autoregulation of the Salmonella typhimurium phoPQ operon. J Bacteriol 177: 4364–4371.
- 48. Perez JC, Groisman EATranscription factor function promoter architecture govern the evolution of bacterial regulons. Proc Natl Acad Sci USA. In press.
- 49. Macfarlane EL, Kwasnicka A, Ochs MM, Hancock RE (1999) PhoP-PhoQ homologues in Pseudomonas aeruginosa regulate expression of the outer-membrane protein OprH and polymyxin B resistance. Mol Microbiol 34: 305–316.
- 50. Soncini FC, Groisman EA (1996) Two-component regulatory systems can interact to process multiple environmental signals. J Bacteriol 178: 6796–6801.
- 51. Gunn JS, Miller SI (1996) PhoP-PhoQ activates transcription of pmrAB, encoding a two-component regulatory system involved in Salmonella typhimurium antimicrobial peptide resistance. J Bacteriol 178: 6857–6864.
- 52. Lee H, Hsu FF, Turk J, Groisman EA (2004) The PmrA-regulated pmrC gene mediates phosphoethanolamine modification of lipid A and polymyxin resistance in Salmonella enterica. J Bacteriol 186: 4124–4133.
- 53. Romani AM, Scarpa A (2000) Regulation of cellular magnesium. Front Biosci 5: D720–734.
- 54. Reinhart RA (1988) Magnesium metabolism. A review with special reference to the relationship between intracellular content and serum levels. Arch Intern Med 148: 2415–2420.
- 55. Raetz CR, Whitfield C (2002) Lipopolysaccharide endotoxins. Annu Rev Biochem 71: 635–700.
- 56. Bishop RE, Gibbons HS, Guina T, Trent MS, Miller SI, et al. (2000) Transfer of palmitate from phospholipids to lipid A in outer membranes of gram-negative bacteria. EMBO J 19: 5071–5080.
- 57. Groisman EA (2001) The pleiotropic two-component regulatory system PhoP-PhoQ. J Bacteriol 183: 1835–1842.
- 58. Plesa M, Hernalsteens JP, Vandenbussche G, Ruysschaert JM, Cornelis P (2006) The SlyB outer membrane lipoprotein of Burkholderia multivorans contributes to membrane integrity. Res Microbiol 157: 582–592.
- 59. Minagawa S, Ogasawara H, Kato A, Yamamoto K, Eguchi Y, et al. (2003) Identification and molecular characterization of the Mg2+ stimulon of Escherichia coli. J Bacteriol 185: 3696–3702.
- 60. Mitrophanov AY, Groisman EA (2008) Positive feedback in cellular control systems. BioEssays 30: 542–555.
- 61. Hutchings MI, Hong HJ, Leibovitz E, Sutcliffe IC, Buttner MJ (2006) The σE cell envelope stress response of Streptomyces coelicolor is influenced by a novel lipoprotein, CseA. J Bacteriol 188: 7222–7229.
- 62. DiGiuseppe PA, Silhavy TJ (2003) Signal detection and target gene induction by the CpxRA two-component system. J Bacteriol 185: 2432–2440.
- 63. Majdalani N, Heck M, Stout V, Gottesman S (2005) Role of RcsF in signaling to the Rcs phosphorelay pathway in Escherichia coli. J Bacteriol 187: 6770–6778.
- 64. Shimada T, Ishihama A, Busby SJ, Grainger DC (2008) The Escherichia coli RutR transcription factor binds at targets within genes as well as intergenic regions. Nucleic Acids Res 36: 3950–3955.
- 65. Rhodius VA, Suh WC, Nonaka G, West J, Gross CA (2006) Conserved and variable functions of the σE stress response in related genomes. PLoS Biol 4: e2.
- 66. Swingle B, Thete D, Moll M, Myers CR, Schneider DJ, et al. (2008) Characterization of the PvdS-regulated promoter motif in Pseudomonas syringae pv. tomato DC3000 reveals regulon members and insights regarding PvdS function in other pseudomonads. Mol Microbiol 68: 871–889.
- 67. Erill I, Campoy S, Barbe J (2007) Aeons of distress: an evolutionary perspective on the bacterial SOS response. FEMS Microbiol Rev 31: 637–656.
- 68. Snavely MD, Gravina SA, Cheung TT, Miller CG, Maguire ME (1991) Magnesium transport in Salmonella Typhimurium - Regulation of Mgta and Mgtb expression. J Biol Chem 266: 824–829.
- 69. Staggs TM, Perry RD (1991) Identification and cloning of a fur regulatory gene in Yersinia pestis. J Bacteriol 173: 417–425.
- 70. Perry RD, Brubaker RR (1979) Accumulation of iron by yersiniae. J Bacteriol 137: 1290–1298.
- 71. Shin D, Groisman EA (2005) Signal-dependent binding of the response regulators PhoP and PmrA to their target promoters in vivo. J Biol Chem 280: 4089–4094.