Research Article

Transcription factor function and promoter architecture govern the evolution of bacterial regulons

J. Christian Perez and Eduardo A. Groisman
PNAS March 17, 2009 106 (11) 4319-4324; https://doi.org/10.1073/pnas.0810343106

  1. Edited by Jeffrey I. Gordon, Washington University School of Medicine, St. Louis, MO, and approved January 16, 2009 (received for review October 14, 2008)

Abstract

Evolutionary changes in ancestral regulatory circuits can bring about phenotypic differences between related organisms. Studies of regulatory circuits in eukaryotes suggest that these modifications result primarily from changes in cis-regulatory elements (as opposed to alterations in the transcription factors that act upon these sequences). It is presently unclear how the evolution of gene regulatory circuits has proceeded in bacteria, given the rampant effects of horizontal gene transfer, which has significantly altered the composition of bacterial regulons. We now demonstrate that the evolution of the regulons governed by the regulatory protein PhoP in the related human pathogens Salmonella enterica and Yersinia pestis has entailed functional changes in the PhoP protein as well as in the architecture of PhoP-dependent promoters. These changes have resulted in orthologous PhoP proteins that differ both in their ability to promote transcription and in their role as virulence regulators. We posit that these changes allow bacterial transcription factors to incorporate newly acquired genes into ancestral regulatory circuits and yet retain control of the core members of a regulon.

Keywords:

Gene regulatory networks undergo major modifications over time, and these modifications provide an important source of phenotypic diversity among closely related organisms (1, 2). In principle, these modifications may result from changes in cis-regulatory sequences and/or from alterations in the deployment and/or activity of the transcription factors (TFs) that act on these sequences. In eukaryotes, in which most studies of the evolution of gene regulation have been carried out, most regulatory changes involve gains and/or losses of TF binding sites (as opposed to modifications in the TF themselves (3–5), although modifications in TFs have also been reported (6–8)). The prevalence of differences in cis-regulatory sites has been ascribed to the fact that changes in the binding site for a particular TF can alter transcription of a single gene selectively without affecting the expression of other genes co-regulated by the same TF (3–5). Consistent with this notion, mutations in cis-regulatory elements underlie several morphological differences among closely related animal species (3, 5, 9, 10).

We hypothesized that the evolution of gene regulation in bacteria may differ from what has been described thus far in eukaryotes for several reasons. First, it has been suggested that TFs are more likely to evolve if they acquire new target genes (7) and, unlike closely related eukaryotic organisms, closely related bacterial species often exhibit substantial differences in gene content, so that orthologous TFs control largely distinct gene sets (11–15). Second, the position and orientation of a TF binding site within a bacterial promoter (i.e., the promoter architecture) are critical for gene transcription (16). By contrast, eukoryotic promoters are characterized by the sparse and uneven distribution of TF binding sites over large DNA regions (17).

To test our hypothesis, we examined the regulatory protein PhoP and its regulated targets in 2 related enteric bacteria: the gastroenteritis-causing Salmonella enterica serovar Typhimurium and the bubonic plague agent Yersinia pestis. The PhoP protein governs virulence and the adaptation to low-Mg2+ environments in these 2 bacterial pathogens (18, 19), and its activity is dictated by the Mg2+-responding sensor PhoQ (20). Although the Salmonella and Yersinia PhoP proteins are 79% identical at the amino acid level, most of the genes directly regulated by PhoP in S. enterica have no homologs in Y. pestis, and vice versa (Perez, et al., unpublished results), probably as a result of horizontal gene transfer.

Here, we show that (i) the Salmonella and Yersinia PhoP proteins differ in their ability to promote transcription of certain species-specific genes although they retain the capacity to transcribe ancestral members of the PhoP regulon, and (ii) the Salmonella and Yersinia genomes each harbor promoter architectures not found in the other genome, but the PhoP protein recognizes a conserved motif in these 2 organisms. Our results suggest that embedding new target genes under the control of an ancestral TF entails changes in TF function and in promoter architecture.

Results

Differential Ability of the Yersinia PhoP Protein to Express Salmonella Genes Absent from Yersinia.

To examine whether the Yersinia PhoP protein was functionally equivalent to the Salmonella PhoP protein, we used the experimental strategy schematized in supporting information (SI) Fig. S1. We began by investigating Yersinia's ability to promote transcription of the S. enterica mgtA and ugtL genes, which have no orthologs in Y. pestis and harbor distinct promoter architectures (Fig. 1A) (21–23). Y. pestis produced fluorescence levels similar to those of S. enterica when transcription of a promoterless gfp gene was driven by the promoter of the mgtA gene (Fig. 1B), which is found in most enteric species (24). By contrast, Yersinia elicited significantly lower fluorescence than Salmonella when gfp was expressed from the promoter of the Salmonella-specific ugtL gene (Fig. 1C). In principle, Yersinia's inability to promote ugtL transcription could be caused by the absence of a co-regulator(s) normally present in Salmonella. To test this possibility, we examined ugtL transcription in an engineered Salmonella strain in which the phoPQ operon was replaced by the Y. pestis phoPQ operon (Fig. 1D). The resulting strain synthesized the Yersinia PhoP protein under the same conditions and at similar levels as the Salmonella PhoP protein in the strain expressing Salmonella's phoPQ operon (Fig. 1E). There was no PhoP-promoted ugtL transcription in the strain expressing the Yersinia phoPQ operon (Fig. 1G), even though the mgtA gene was transcribed at similar levels as in the Salmonella strain harboring its own phoPQ operon (Fig. 1F).

Fig. 1.
Fig. 1.

The Salmonella and Yersinia PhoP proteins differ in their ability to promote gene transcription. (A) The Salmonella mgtA and ugtL promoters differ both in the orientation and the distance of the PhoP box to the promoter −10 region. The PhoP box is located in the place that corresponds to the promoter −35 region in the mgtA promoter but is 20 bp further upstream in the ugtL promoter. (B and C) GFP expression driven by the Salmonella mgtA and ugtL promoters in Salmonella and Yersinia. Approximately 150-nt DNA fragments corresponding to the mgtA and ugtL promoter regions (covering ≈130 nucleotides upstream and ≈20 nucleotides downstream of the transcription start sites) were cloned in front of a promoterless gfp gene in the low-copy plasmid pMS201. Organisms were grown in defined medium containing 50 μM MgSO4, inducing conditions for the PhoP/PhoQ system. GFP expression was normalized to cell density. Shown are the ratios of the normalized GFP values between wild-type Salmonella (14028s) or Yersinia (KIM6) and their respective isogenic phoP mutant strains (EG15598 and EG14737, respectively). Values shown are mean plus SD of at least 3 independent experiments. (D) Schematic of the genomic context of the phoPQ locus in Salmonella strain EG13918 coding for the Salmonella PhoP-HA and PhoQ proteins and its derivative harboring the Yersinia phoP-HA and phoQ genes (in blue) (EG17569). (E) Expression of the Salmonella and Yersinia PhoP proteins in the Salmonella strains EG13918 and EG17569 depicted in D. Western blot analysis was performed with anti-HA and anti-RpoB antibodies (to detect the PhoP-HA and RpoB proteins, respectively) on cell extracts prepared from bacteria grown as described in B and C in medium containing 10 mM (H) or 50 μM (L) MgSO4. (F and G) mgtA and ugtL expression in the Salmonella strains depicted in D. Cells were grown as described in B and C. Transcript levels were determined by quantitative real-time PCR and normalized to ribosomal RNA levels. Shown are the ratios of the normalized transcript levels present in the strains described in D relative to those produced by the phoPQ mutant EG15598. Values shown are mean plus SD of at least 3 independent experiments. (H–M) Single-round in vitro transcription assays with linear templates corresponding to the mgtA (H–J) and ugtL (K–M) promoters, E. coli RNA polymerase, and increasing amounts of phosphorylated Salmonella or Yersinia PhoP proteins. The upper band in H–I corresponds to treR, a PhoP-repressed transcript going in the reverse orientation (22). The upper band in K and L corresponds to a spurious transcript observed in vitro but not in vivo (21, 45). Quantification of the in vitro transcription assays is shown in J for mgtA and in M for ugtL.

We reasoned that if the observed differences in ugtL expression were caused by intrinsic properties of the PhoP proteins, it should be possible to recapitulate the observed behavior (Fig. 1 B, C, F, and G) in an in vitro transcription system where the only variable was the PhoP protein. Consistent with this notion, ugtL transcription in vitro was significantly higher with the Salmonella PhoP protein than with the Yersinia PhoP protein (Fig. 1 K–M). By contrast, the Salmonella and Yersinia PhoP proteins promoted similar levels of mgtA transcription in vitro (Fig. 1 H–J). Cumulatively, these results indicate that the function of the PhoP protein has been conserved only partially since Salmonella and Yersinia diverged from their last common ancestor > 200 million years ago (25).

The Yersinia PhoP Protein Binds to the Salmonella ugtL Promoter but Does Not Recruit RNA Polymerase.

The inability of the Yersinia PhoP protein to transcribe the ugtL gene could be caused by the absence of binding to the ugtL promoter and/or could result from a defect in a subsequent step in transcription such as recruitment of RNA polymerase. We ruled out the first possibility for 2 reasons. First, the Yersinia PhoP protein bound to both the ugtL and mgtA promoter regions in vivo just like the Salmonella PhoP protein (Fig. 2 A and B). Second, the purified PhoP proteins from Yersinia and Salmonella displayed similar affinities toward a fragment harboring the ugtL promoter in vitro (Fig. S2). As expected, the PhoP protein occupied the ugtL and mgtA promoters only when grown under low Mg2+-inducing conditions for the PhoP/PhoQ system (Fig. 2 A and B).

Fig. 2.
Fig. 2.

The Yersinia PhoP protein binds to the ugtL promoter but cannot recruit RNA polymerase. (A–D) Promoter occupancy by PhoP (A and B) and RNA polymerase (C and D) determined by ChIP in isogenic Salmonella strains expressing the Salmonella (EG13918) or the Yersinia (EG17569) phoPQ operon. Cells were grown in N-minimal medium containing 10 mM (H) or 10 μM (L) MgCl2. Shown are the mean plus SD of at least 3 independent experiments.

We then tested the ability of the Salmonella and Yersinia PhoP proteins to recruit RNA polymerase to the ugtL and mgtA promoters in vivo using ChIP with an antibody directed to the β subunit of the enzyme. RNA polymerase bound to both the mgtA and ugtL promoter regions when the Salmonella strain expressing its own phoP gene was grown in the presence of the low Mg2+-inducing signal (Fig. 2 C and D). By contrast, RNA polymerase could be recruited to the mgtA promoter but not to the ugtL promoter in the Salmonella strain expressing the Yersinia phoP gene (Fig. 2 C and D). As expected, there was no RNA polymerase recruitment to either promoter in either strain under high Mg2+-repressing conditions (Fig. 2 C and D). Taken together, these findings indicate that the Yersinia PhoP protein fails to promote ugtL transcription because of inefficient RNA polymerase recruitment rather than because of defective binding to the ugtL promoter.

Promoter Architecture Dictates the Ability of the Salmonella and Yersinia PhoP Proteins to Transcribe Target Genes.

The orientation and/or location of the PhoP box seem to be responsible (at least in part) for the distinct abilities of the Salmonella and Yersinia PhoP proteins to transcribe the mgtA and ugtL genes, because the corresponding promoters differ in both of these properties (Fig. 1A). In agreement with this notion, the Salmonella strain expressing the Yersinia phoPQ operon transcribed the ancestral slyB (Fig. S3) and phoP (Fig. 1E) genes, whose promoters share the location and orientation of the PhoP box with the mgtA promoter (Fig. 3A) but not the horizontally acquired pagK, mgtC, pagC, and mig-14 genes (Fig. S3 and data not shown), the promoters of which contain the PhoP box in the same orientation as in the ugtL promoter (Fig. 3A). This finding suggested to us that the architecture of natural PhoP-activated promoters in Yersinia would resemble the Salmonella mgtA promoter architecture and not conform to the ugtL promoter structure.

Fig. 3.
Fig. 3.

Architectures of PhoP-activated promoters in Salmonella and Yersinia. (A and B) Sequences of the promoter regions of the transcripts directly activated by PhoP in Salmonella (A) and Yersinia (B). The transcription start sites are indicated in blue, the PhoP boxes are in red, and the putative −10 sequences are underlined. Boxes and arrows indicate the location and orientation of the PhoP binding sites across promoters. The Salmonella PhoP-activated transcripts have been described previously (23, 26). The Yersinia PhoP-dependent transcripts were uncovered as described in Materials and Methods (Fig. S4). PhoP binding to all promoters was determined in vivo by ChIP-chip and/or ChIP-real-time PCR and in several cases in vitro by DNase I footprinting (Fig. S4). (C) Summary of the distribution of promoter architectures and the differential ability of the Salmonella and Yersinia PhoP proteins to activate transcription. Although the mgtA-like promoter architecture is shared by several Salmonella and Yersinia PhoP-activated genes, other members of the regulon harbor promoter structures that are absent from the other organism. The Salmonella and Yersinia PhoP proteins differ in their ability to activate expression from genes of the latter group.

To determine the architecture of PhoP-activated promoters in Yersinia, we identified the transcription start site (by conducting S1 mapping or primer extension experiments) and defined the PhoP binding site (by DNase I footprinting experiments with the Yersinia PhoP protein) for 14 Y. pestis genes directly controlled by the PhoP protein (Figs. 3B and S4) (see Materials and Methods for a description of how these genes were uncovered). Analysis of the sequence, location, and orientation of the PhoP box revealed interesting similarities and differences between PhoP-activated promoters in Salmonella and Yersinia. First, a comparison with previously reported Salmonella PhoP-regulated promoters (Fig. 3A) (23, 26) revealed that the sequence of the PhoP-binding motif is similar in Salmonella and Yersinia (Fig. S5). Second, 9 of 14 Yersinia promoters contain a PhoP box in the same orientation and at the same distance from the −10 region as in the Salmonella mgtA promoter. And third, the remaining 5 Yersinia promoters also harbor the PhoP box in the same orientation as in the Salmonella mgtA promoter but further upstream: for 4 of these promoters the PhoP box is located at the same distance from the −10 region as in a group of Salmonella PhoP-activated promoters (i.e., rstA, pagP, and ompX). Therefore, none of the Yersinia PhoP-activated promoters has the PhoP box in the orientation found in the Salmonella ugtL promoter.

The promoter of the Yersinia mgtC gene exhibits an architecture not previously reported for Salmonella PhoP-activated promoters: the PhoP box is at a similar distance from the −10 region as the Salmonella ugtL promoter but is in the opposite orientation (Fig. 3 B and C). Thus, we wondered whether the Salmonella PhoP protein could promote transcription from the Yersinia mgtC promoter (the Salmonella and Yersinia mgtC genes are xenologs rather than orthologs, because they seem to have been acquired independently by the Salmonella and Yersinia lineages) (24, 27). The Salmonella PhoP protein could not promote transcription of the Yersinia mgtC gene (Fig. S6), but it elicited normal transcription levels of the Yersinia y1795 gene (data not shown), which has the Salmonella mgtA-like promoter architecture (Fig. 3 B and C). These results suggest that the ability of the PhoP protein to promote gene transcription is dependent on the orientation of the PhoP box.

The αCTD Subunit of RNA Polymerase Is Necessary for PhoP-Dependent Transcription of ugtL but Not mgtA.

The location of a TF binding site within a bacterial promoter determines the RNA polymerase subunit (e.g., σ or αCTD for the carboxy-terminal domain of the α subunit) with which an activator interacts to promote gene transcription (16). In class I promoters, the activator binds upstream of the −35 element in a promoter, and activation typically relies on interactions with the flexible αCTD. In class II promoters, the activator binds to a target that overlaps the promoter −35 element usually contacting the σ subunit of RNA polymerase (16). Because the αCTD is essential for viability (28), we used in vitro transcription assays with RNA polymerase reconstituted with either wild-type or truncated αCTD (29) to probe the requirement of αCTD for expression of PhoP-regulated promoters. The αCTD was dispensable for PhoP-dependent mgtA transcription (Fig. S7), as expected for a class II promoter and consistent with previous findings reported for the Escherichia coli mgtA promoter and the E. coli PhoP protein (22). By contrast, the αCTD was necessary for PhoP-dependent transcription of the class I ugtL promoter (Fig. S7).

A Salmonella Strain Expressing the Yersinia phoPQ Operon in Lieu of Its Own phoPQ Operon Is Attenuated for Virulence.

A functional PhoP/PhoQ system is required for virulence in both Salmonella and Yersinia (18, 19). However, a Salmonella strain expressing the Yersinia phoPQ operon was strongly attenuated for virulence, because all the mice inoculated with a dose > 1,000 higher than the LD50 survived, as did those inoculated with a phoP null mutant, whereas those inoculated with a Salmonella strain harboring its own phoPQ operon died (data not shown).

Discussion

It is becoming increasingly clear that transcriptional regulatory circuits undergo major modifications over time and that these modifications provide an important source of phenotypic diversity. Although several studies have addressed the evolution of gene regulatory circuits in eukaryotes, little is known about this process in bacteria. Here, we report that the evolution of the regulons controlled by the PhoP protein in the enteric pathogens S. enterica and Y. pestis has entailed functional changes in the PhoP protein as well as in the architecture of PhoP-dependent promoters, with conservation of the DNA motif recognized by the PhoP protein. Several lines of evidence support this notion. First, the Salmonella and Yersinia PhoP proteins differ in their ability to promote transcription of subsets of PhoP-regulated genes from the other species (Figs. 1, S3, and S6). Second, Salmonella and Yersinia harbor PhoP-regulated promoter architectures not found in the other species (Fig. 3). Third, the sequence of the PhoP binding motif is similar in Salmonella and Yersinia (Fig. S5). Fourth, a Salmonella strain expressing the Yersinia phoPQ operon in lieu of its own phoPQ operon is attenuated for virulence (data not shown). Our data provide a singular example of functional differences between orthologous TFs and between the architectures of their target promoters across bacterial species.

The PhoP proteins from Salmonella and Yersinia are fully competent to express genes harboring the conserved promoter architecture driving transcription of the ancestral members of the PhoP regulon but differ in their ability to transcribe species-specific genes harboring promoter architectures not found in the other species (Figs. 1 and 3). PhoP-regulated promoters can be divided into 3 groups based on the location of the PhoP box (Fig. 3 A and B): (i) the prototypical class II promoter (16), exemplified by mgtA and the ancestral members of the regulon phoP and slyB (Perez, et al., unpublished results), in which the PhoP box is ≈12 nucleotides from the −10 region; (ii) promoters in which the PhoP box is located further upstream but in the same orientation as in the mgtA promoter (e.g., the Salmonella rstA, pagP, and ompX promoters and the Yersinia y4126, y3808, y0447, and y2563 promoters); and (iii), the typical class I promoters that contain the PhoP box even further upstream but at a variable distance from the −10 region (e.g., the Salmonella ugtL and mgtC promoters and the Yersinia mgtC promoter). The first 2 promoter architectures are present both in Salmonella and Yersinia, and the PhoP proteins from these 2 species can transcribe these promoters regardless of the species of origin (Figs. 1 B, F, H, and I, S3, and data not shown). The orientation of the PhoP box in the third group of promoters is different in Salmonella and in Yersinia (Fig. 3C). Strikingly, this difference seems to be critical for PhoP function, because the Yersinia PhoP protein is impaired in its ability to elicit transcription from the Salmonella promoters of this group, and vice versa (Figs. 1 C, G, and L, 3C, and S6). The Salmonella genes harboring the latter promoters have no close homologs outside Salmonella spp., suggesting that they were horizontally acquired by the lineage that gave rise to S. enterica.

The surface of a TF that contacts RNA polymerase is determined by the orientation and location of a TF binding site (30). For instance, the transcriptional activator cyclic APM receptor protein from E. coli interacts with αCTD through more than 1 surface, depending on the position of its binding site (31). The fact that αCTD is dispensable for transcription of PhoP-activated class II promoters (e.g., mgtA) but is necessary to transcribe the 2 groups of class I promoters (e.g., ugtL, rstA, and Yersinia mgtC) (Fig. S7 and data not shown) suggests that the Salmonella and Yersinia PhoP proteins interact with both σ and with αCTD, depending on the location of the PhoP box. The contact(s) that PhoP establishes with σ in the first group of promoters (e.g., mgtA) seems to be conserved between the 2 PhoP proteins, because Salmonella and Yersinia can promote transcription from this type of promoter. Similarly, the potential interaction(s) of PhoP with αCTD (and perhaps with σ as well) when transcribing from the second group of promoters (e.g., the Salmonella rstA, pagP, and ompX promoters and the Yersinia y4126, y3808, y0447, and y2563 promoters) is likely to be conserved between Salmonella and Yersinia because these promoters share the position and orientation of the PhoP box relative to the −10 region. By contrast, the Salmonella and Yersinia PhoP proteins seem to establish productive interactions with αCTD in different manners when transcribing the third group of promoters (e.g., the Salmonella ugtL and mgtC promoter and the Yersinia mgtC promoter) that differ in the orientation of the PhoP box. Therefore, although the known flexibility of αCTD (31) enables the PhoP protein to transcribe promoters harboring the PhoP box at a variety of positions (green box in Fig. 3A), certain promoter architectures seem to be species specific and function only with the PhoP protein corresponding to that species. Because the αCTD subunit of E. coli, S. enterica, and Y. pestis are 100% identical at the amino acid level, the functional differences between the Salmonella and Yersinia PhoP proteins cannot be ascribed to differences in the transcription machinery.

The evolution of certain transcriptional regulators in yeast has been ascribed to changes in protein–protein interactions caused by a few amino acid substitutions (1, 2, 32), whereas the reported cases in higher eukaryotes typically involve whole-domain gains or losses (6). (However, some examples in which a few amino acid substitutions seem to be responsible for functional changes have also been reported (7, 33).) The Salmonella and Yersinia PhoP proteins have 2 domains: a 120-residue N-terminal domain joined by a 5-residue linker to a 98-residue C-terminal domain (34). (The N- and C-terminal domains are 80.8% and 75.5% identical, respectively.) The C-terminal region is predicted to make contacts with RNA polymerase through unidentified residues and has a helix-turn-helix DNA binding domain. The helix-turn-helix portion is identical in Salmonella and Yersinia proteins, in agreement with the conservation of the DNA motif that PhoP recognizes (Fig. S5). Moreover, the predicted secondary structures of the entire Salmonella and Yersinia PhoP proteins are superimposable (Fig. S8), suggesting that the functional differences between these orthologous PhoP proteins result from relatively minor changes in the overall structure of the proteins altering the interaction with the transcriptional machinery.

A Salmonella strain expressing the Yersinia phoPQ operon was strongly attenuated for virulence, behaving similarly to a phoP null mutant (data not shown). Given the degree of sequence identity between the sensing domains of the Salmonella and Yersinia PhoQ proteins (35), the virulence attenuation of the Salmonella strain expressing the Yersinia phoPQ operon is unlikely to result from a defect in the sensing ability of the Yersinia PhoQ protein. Rather, it may reflect that Salmonella virulence demands the expression of horizontally acquired PhoP-dependent genes, such as mig-14 (36) and mgtC (24), whose promoters share the location and orientation of the PhoP box with the ugtL promoter (Fig. 3A) and thus are not efficiently transcribed by the Y. pestis PhoP protein (data not shown).

In sum, the evolution of a bacterial regulon entails modifications in a TF and restructuring of its target promoters, which enable transcription of horizontally acquired targets while retaining control of ancestral genes. These changes are in addition to the well-established transcriptional rewiring events whereby the promoters of conserved genes in related species differ in the presence/absence of TF binding site(s) (1, 37, 38). While there are a few reports of evolutionary changes in eukaryotic TFs contributing to the modification of gene-regulatory networks (6–8, 33), alterations in gene regulation and phenotypic differences in eukaryotes have been ascribed primarily to gains and/or losses of TF binding sites (3, 5, 10). Our results (Fig. 2) and those of others (39) indicate that, in bacteria, TF binding to a DNA sequence in vivo does not necessarily result in gene transcription, thereby highlighting the need to experimentally verify the function of identified TF binding sites.

Materials and Methods

Bacterial Strains, Plasmids, and Growth Conditions.

Bacterial strains and plasmids used in this study are listed in Table S1 and primers are listed in Table S2. All Salmonella enterica serovar Typhimurium strains were derived from wild-type strain 14028s and were grown at 37 °C in N-minimal medium (40) buffered in 50 mM Bis-Tris, pH 7.7, supplemented with 0.1% casamino acids, 38 mM glycerol, and 10 μM or 10 mM MgCl2, unless otherwise indicated. Yersinia pestis strains were derived from wild-type strain KIM6 (41) and were grown at the optimal growth temperature of 28 °C in a modified defined medium (42), pH 7.0, supplemented with 0.1% casamino acids, 10 mM (D)-glucosamine, and 50 μM or 10 mM MgSO4. Escherichia 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. A detailed description of the construction of bacterial strains and plasmids as well as other molecular biology procedures is provided in the SI Text.

Identification of PhoP-Regulated Promoters in Y. pestis.

To uncover the genes directly regulated by PhoP in Y. pestis, we used a combination of expression microarray analysis (wild-type vs. phoP mutant strains) and ChIP followed by array hybridization (ChIP-chip). S1 mapping (43), primer extension (44), and DNase I footprinting (43) experiments were used to validate the microarray and ChIP-chip results and to determine the structure of the PhoP-activated promoters described in Fig. 3B. A comprehensive description of the procedures and the uncovered genes will be published elsewhere.

GFP Expression Analysis.

After overnight culture in defined medium containing 10 mM MgSO4, the Salmonella and Yersinia strains were washed twice with Mg2+-free medium, inoculated (1:50 dilution) in medium containing 50 μM MgSO4, and grown to mid-exponential phase with vigorous shaking. Fluorescence values were normalized to cell density, both of which were measured in a Victor3 1420 Multilabel counter (Perkin Elmer).

RNA Isolation and Real-Time PCR to Determine Transcript Levels.

Salmonella cells were grown as described in the previous paragraphs for the GFP expression analysis. Total RNA was prepared using the SV Total RNA Isolation System (Promega). cDNA was synthesized using TaqMan (Applied Biosystems) and random hexamers following the manufacturer's instructions. Quantification of transcripts was performed by real-time PCR using SYBR Green PCR Master Mix (Applied Biosystems) in an ABI 7000 Sequence Detection System (Applied Biosystems). Results were normalized to the levels of 16S ribosomal RNA.

Acknowledgments

We thank Richard L. Gourse for providing the mutant RNA polymerase enzyme; S. Busby, B. Cohen, R.L. Gourse, and G. Wagner for critically reading the manuscript; and Tammy Latifi for technical assistance. The work presented here was supported, in part, by Grant AI49561 from the National Institutes of Health to E.A.G, who is an Investigator of the Howard Hughes Medical Institute.

Footnotes

  • 2To whom correspondence should be addressed. E-mail: groisman{at}borcim.wustl.edu

  • Author contributions: J.C.P. and E.A.G. designed research; J.C.P. performed research; J.C.P. and E.A.G. analyzed data; and J.C.P. and E.A.G. wrote the paper.

  • ↵1Present address: Department of Microbiology and Immunology, University of California, 600 16th St., Genentech Hall Room N374, San Francisco, CA 94158.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/cgi/content/full/0810343106/DCSupplemental.

  • Freely available online through the PNAS open access option.

References

  1. ↵
    1. Tuch BB,
    2. Galgoczy DJ,
    3. Hernday AD,
    4. Li H,
    5. Johnson AD
    (2008) The evolution of combinatorial gene regulation in fungi. PLoS Biol 6:e38.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Tuch BB,
    2. Li H,
    3. Johnson AD
    (2008) Evolution of eukaryotic transcription circuits. Science 319:1797–1799.
    OpenUrlAbstract/FREE Full Text
  3. ↵
    1. Carroll SB
    (2005) Evolution at two levels: On genes and form. PLoS Biol 3:e245.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Stern DL
    (2000) Evolutionary developmental biology and the problem of variation. Evolutio (Lawrence, Kans) 54:1079–1091.
    OpenUrl
  5. ↵
    1. Wray GA
    (2007) The evolutionary significance of cis-regulatory mutations. Nat Rev Genet 8:206–216.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Hsia CC,
    2. McGinnis W
    (2003) Evolution of transcription factor function. Curr Opin Genet Dev 13:199–206.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Wagner GP,
    2. Lynch VJ
    (2008) The gene regulatory logic of transcription factor evolution. Trends Ecol Evol 23:377–385.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Lynch VJ,
    2. Wagner GP
    (2008) Resurrecting the role of transcription factor change in developmental evolution. Evolution 62:2131–2154.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Jeong S,
    2. et al.
    (2008) The evolution of gene regulation underlies a morphological difference between two Drosophila sister species. Cell 132:783–793.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Carroll SB
    (2008) Evo-devo and an expanding evolutionary synthesis: A genetic theory of morphological evolution. Cell 134:25–36.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Lozada-Chavez I,
    2. Janga SC,
    3. Collado-Vides J
    (2006) Bacterial regulatory networks are extremely flexible in evolution. Nucleic Acids Res 34:3434–3445.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Madan Babu M,
    2. Teichmann SA,
    3. Aravind L
    (2006) Evolutionary dynamics of prokaryotic transcriptional regulatory networks. J Mol Biol 358:614–633.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Price MN,
    2. Dehal PS,
    3. Arkin AP
    (2007) Orthologous transcription factors in bacteria have different functions and regulate different genes. PLOS Comput Biol 3:1739–1750.
    OpenUrlPubMed
  14. ↵
    1. Rhodius VA,
    2. Suh WC,
    3. Nonaka G,
    4. West J,
    5. Gross CA
    (2006) Conserved and variable functions of the σE stress response in related genomes. PLoS Biol 4:e2.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Swingle B,
    2. 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.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Browning DF,
    2. Busby SJ
    (2004) The regulation of bacterial transcription initiation. Nat Rev Microbiol 2:57–65.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Wray GA,
    2. et al.
    (2003) The evolution of transcriptional regulation in eukaryotes. Mol Biol Evol 20:1377–1419.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Groisman EA
    (2001) The pleiotropic two-component regulatory system PhoP-PhoQ. J Bacteriol 183:1835–1842.
    OpenUrlFREE Full Text
  19. ↵
    1. Oyston PC,
    2. 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.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Garcia Vescovi E,
    2. Soncini FC,
    3. Groisman EA
    (1996) Mg2+ as an extracellular signal: Environmental regulation of Salmonella virulence. Cell 84:165–174.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Shi YX,
    2. Latifi T,
    3. Cromie MJ,
    4. Groisman EA
    (2004) Transcriptional control of the antimicrobial peptide resistance ugtL gene by the Salmonella PhoP and SlyA regulatory proteins. J Biol Chem 279:38618–38625.
    OpenUrlAbstract/FREE Full Text
  22. ↵
    1. Yamamoto K,
    2. Ogasawara H,
    3. Fujita N,
    4. Utsumi R,
    5. 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.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Zwir I,
    2. et al.
    (2005) Dissecting the PhoP regulatory network of Escherichia coli and Salmonella enterica. Proc Natl Acad Sci USA 102:2862–2867.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Blanc-Potard AB,
    2. Groisman EA
    (1997) The Salmonella selC locus contains a pathogenicity island mediating intramacrophage survival. EMBO J 16:5376–5385.
    OpenUrlAbstract
  25. ↵
    1. Deng W,
    2. et al.
    (2002) Genome sequence of Yersinia pestis KIM. J Bacteriol 184:4601–4611.
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Lejona S,
    2. Aguirre A,
    3. Cabeza ML,
    4. Vescovi EG,
    5. Soncini FC
    (2003) Molecular characterization of the Mg2+-responsive PhoP-PhoQ regulon in Salmonella enterica. J Bacteriol 185:6287–6294.
    OpenUrlAbstract/FREE Full Text
  27. ↵
    1. Blanc-Potard AB,
    2. Lafay B
    (2003) MgtC as a horizontally-acquired virulence factor of intracellular bacterial pathogens: Evidence from molecular phylogeny and comparative genomics. J Mol Evol 57:479–486.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Gaal T,
    2. et al.
    (1996) DNA-binding determinants of the α subunit of RNA polymerase: Novel DNA-binding domain architecture. Genes Dev 10:16–26.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Quinones M,
    2. Kimsey HH,
    3. Ross W,
    4. Gourse RL,
    5. Waldor MK
    (2006) LexA represses CTXΦ transcription by blocking access of the α C-terminal domain of RNA polymerase to promoter DNA. J Biol Chem 281:39407–39412.
    OpenUrlAbstract/FREE Full Text
  30. ↵
    1. Martin RG,
    2. Gillette WK,
    3. Rhee S,
    4. Rosner JL
    (1999) Structural requirements for marbox function in transcriptional activation of mar/sox/rob regulon promoters in Escherichia coli: Sequence, orientation and spatial relationship to the core promoter. Mol Microbiol 34:431–441.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Niu W,
    2. Kim Y,
    3. Tau G,
    4. Heyduk T,
    5. Ebright RH
    (1996) Transcription activation at class II CAP-dependent promoters: Two interactions between CAP and RNA polymerase. Cell 87:1123–1134.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Tsong AE,
    2. Tuch BB,
    3. Li H,
    4. Johnson AD
    (2006) Evolution of alternative transcriptional circuits with identical logic. Nature 443:415–420.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Lynch VJ,
    2. et al.
    (2008) Adaptive changes in the transcription factor HoxA-11 are essential for the evolution of pregnancy in mammals. Proc Natl Acad Sci USA 105:14928–14933.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Bachhawat P,
    2. Stock AM
    (2007) Crystal structures of the receiver domain of the response regulator PhoP from Escherichia coli in the absence and presence of the phosphoryl analog beryllofluoride. J Bacteriol 189:5987–5995.
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. Prost LR,
    2. Daley ME,
    3. Bader MW,
    4. Klevit RE,
    5. Miller SI
    (2008) The PhoQ histidine kinases of Salmonella and Pseudomonas spp. are structurally and functionally different: Evidence that pH and antimicrobial peptide sensing contribute to mammalian pathogenesis. Mol Microbiol 69:503–519.
    OpenUrlCrossRefPubMed
  36. ↵
    1. Brodsky IE,
    2. Ghori N,
    3. Falkow S,
    4. 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.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Odom DT,
    2. et al.
    (2007) Tissue-specific transcriptional regulation has diverged significantly between human and mouse. Nat Genet 39:730–732.
    OpenUrlCrossRefPubMed
  38. ↵
    1. Borneman AR,
    2. et al.
    (2007) Divergence of transcription factor binding sites across related yeast species. Science 317:815–819.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Shimada T,
    2. Ishihama A,
    3. Busby SJ,
    4. 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.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Snavely MD,
    2. Gravina SA,
    3. Cheung TT,
    4. Miller CG,
    5. Maguire ME
    (1991) Magnesium transport in Salmonella Typhimurium—regulation of Mgta and Mgtb expression. J Biol Chem 266:824–829.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Staggs TM,
    2. Perry RD
    (1991) Identification and cloning of a fur regulatory gene in Yersinia pestis. J Bacteriol 173:417–425.
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Perry RD,
    2. Brubaker RR
    (1979) Accumulation of iron by Yersiniae. J Bacteriol 137:1290–1298.
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Winfield MD,
    2. Latifi T,
    3. Groisman EA
    (2005) Transcriptional regulation of the 4-amino-4-deoxy-L-arabinose biosynthetic genes in Yersinia pestis. J Biol Chem 280:14765–14772.
    OpenUrlAbstract/FREE Full Text
  44. ↵
    1. Tu X,
    2. Latifi T,
    3. Bougdour A,
    4. Gottesman S,
    5. Groisman EA
    (2006) The PhoP/PhoQ two-component system stabilizes the alternative sigma factor RpoS in Salmonella enterica. Proc Natl Acad Sci USA 103:13503–13508.
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. Perez JC,
    2. Latifi T,
    3. Groisman EA
    (2008) Overcoming H-NS-mediated transcriptional silencing of horizontally acquired genes by the PhoP and SlyA proteins in Salmonella enterica. J Biol Chem 283:10773–10783.
    OpenUrlAbstract/FREE Full Text
View Abstract
Back to top
Article Alerts
Email Article
Citation Tools
Request Permissions
Transcription factor function and promoter architecture govern the evolution of bacterial regulons
J. Christian Perez, Eduardo A. Groisman
Proceedings of the National Academy of Sciences Mar 2009, 106 (11) 4319-4324; DOI: 10.1073/pnas.0810343106
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Proceedings of the National Academy of Sciences: 106 (11)
Table of Contents

Submit

Article Classifications

Jump to section

You May Also be Interested in

Analysis of sedentary behaviors of Hadza hunter-gatherers in Tanzania suggests humans require daily muscle activity from movement and resting postures. Image credit: David A. Raichlen.
Evolution of sedentary behaviors
Analysis of sedentary behaviors of Hadza hunter-gatherers in Tanzania suggests humans require daily muscle activity from movement and resting postures.
Image credit: David A. Raichlen.
Analysis of resting-state fMRI data from 928 people raises the possibility that dietary interventions might help protect against age-related cognitive decline. Image credit: Pixabay/sbtlneet.
Brain aging and diet
Analysis of resting-state fMRI data from 928 people raises the possibility that dietary interventions might help protect against age-related cognitive decline.
Image credit: Pixabay/sbtlneet.

Similar Articles