PhoP Protein Research Articles

Structure of the Response Regulator PhoP from Mycobacterium tuberculosis Reveals a Dimer through the Receiver Domain

The PhoP protein from Mycobacterium tuberculosis is a response regulator of the OmpR/PhoB subfamily, whose structure consists of an N-terminal receiver domain and a C-terminal DNA-binding domain. How the DNA-binding activities are regulated by phosphorylation of the receiver domain remains unclear due to a lack of structural information on the full-length proteins. Here we report the crystal structure of the full-length PhoP of M. tuberculosis. Unlike other known structures of full-length proteins of the same subfamily, PhoP forms a dimer through its receiver domain with the dimer interface involving α4-β5-α5, a common interface for activated receiver domain dimers. However, the switch residues, Thr99 and Tyr118, are in a conformation resembling those of nonactivated receiver domains. The Tyr118 side chain is involved in the dimer interface interactions. The receiver domain is tethered to the DNA-binding domain through a flexible linker and does not impose structural constraints on the DNA-binding domain. This structure suggests that phosphorylation likely facilitates/stabilizes receiver domain dimerization, bringing the DNA-binding domains to close proximity, thereby increasing their binding affinity for direct repeat DNA sequences.

The RstB sensor acts on the PhoQ sensor to control expression of PhoP-regulated genes.

The PhoP response regulator and PhoQ sensor, which are encoded by the phoPQ operon, constitute the PhoP/PhoQ two-component system. Genome-wide transcription analysis revealed that heterologous expression of the RstB protein, a sensor of the RstA/RstB two-component system, leads to enhanced transcription of PhoP-activated genes in wild-type Salmonella. We determined that RstB-induction increases the levels of phoP mRNA as well as PhoP protein, while lack of the phoPQ genes abolishes RstB-promoted transcription of the PhoP-regulated genes. This regulatory function of RstB did not require its enzymatic activities, and thus the truncated RstB protein containing only periplasmic and transmembrane regions was able to promote PhoP-activated transcription. The RstB protein appeared to target the PhoQ sensor rather than the PhoP response regulator because RstB-induction failed to enhance transcription of the PhoP-regulated genes in a strain maintaining the normal PhoP function, even without PhoQ.

Defining the plasticity of transcription factor binding sites by Deconstructing DNA consensus sequences: the PhoP-binding sites among gamma/enterobacteria.

Transcriptional regulators recognize specific DNA sequences. Because these sequences are embedded in the background of genomic DNA, it is hard to identify the key cis-regulatory elements that determine disparate patterns of gene expression. The detection of the intra- and inter-species differences among these sequences is crucial for understanding the molecular basis of both differential gene expression and evolution. Here, we address this problem by investigating the target promoters controlled by the DNA-binding PhoP protein, which governs virulence and Mg2+ homeostasis in several bacterial species. PhoP is particularly interesting; it is highly conserved in different gamma/enterobacteria, regulating not only ancestral genes but also governing the expression of dozens of horizontally acquired genes that differ from species to species. Our approach consists of decomposing the DNA binding site sequences for a given regulator into families of motifs (i.e., termed submotifs) using a machine learning method inspired by the “Divide & Conquer” strategy. By partitioning a motif into sub-patterns, computational advantages for classification were produced, resulting in the discovery of new members of a regulon, and alleviating the problem of distinguishing functional sites in chromatin immunoprecipitation and DNA microarray genome-wide analysis. Moreover, we found that certain partitions were useful in revealing biological properties of binding site sequences, including modular gains and losses of PhoP binding sites through evolutionary turnover events, as well as conservation in distant species. The high conservation of PhoP submotifs within gamma/enterobacteria, as well as the regulatory protein that recognizes them, suggests that the major cause of divergence between related species is not due to the binding sites, as was previously suggested for other regulators. Instead, the divergence may be attributed to the fast evolution of orthologous target genes and/or the promoter architectures resulting from the interaction of those binding sites with the RNA polymerase.

Novel Aspects of the Acid Response Network of E. coli K-12 Are Revealed by a Study of Transcriptional Dynamics

Understanding gene regulation and its adaptive significance requires not only a detailed knowledge of individual molecular interactions that give rise to changes in gene expression but also an overview of complete genetic networks and the ways in which components within them interact. Increasingly, such studies are being done using luminescent or fluorescent reporter proteins that enable monitoring of gene expression dynamics in real time, particularly during changes in expression. We show here that such an approach is valid for dissecting the responses of the AR2 or GAD network of Escherichia coli K-12 to changes in pH, which is one of the most complex networks known in E. coli. In addition to confirming several regulatory interactions that have been revealed by previous studies, this approach has identified new components in this system that lead to complex dynamics of gene expression following a drop in pH, including an auto-regulatory loop involving the YdeO activator protein and novel roles for the PhoP protein.

Activated by Different Signals, the PhoP/PhoQ Two-Component System Differentially Regulates Metal Uptake

The PhoP/PhoQ two-component system controls several physiological and virulence functions in Salmonella enterica. This system is activated by low Mg(2+), acidic pH, and antimicrobial peptides, but the biological consequences resulting from sensing multiple signals are presently unclear. Here, we report that the PhoP/PhoQ system regulates different Salmonella genes depending on whether the inducing signal is acidic pH or low Mg(2+). When Salmonella experiences acidic pH, the PhoP/PhoQ system promotes Fe(2+) uptake in a process that requires the response regulator RstA, activating transcription of the Fe(2+) transporter gene feoB. In contrast, the PhoP-induced RstA protein did not promote feoB expression at neutral pH with low Mg(2+). The PhoP/PhoQ system promotes the expression of the Mg(2+) transporter mgtA gene only when activated in bacteria starved for Mg(2+). This is because mgtA transcription promoted at high Mg(2+) concentrations by the acidic-pH-activated PhoP protein failed to reach the mgtA coding region due to the mgtA leader region functioning as a Mg(2+) sensor. Our results show that a single two-component regulatory system can regulate distinct sets of genes in response to different input signals.

Identifying promoter features of co-regulated genes with similar network motifs

BackgroundA large amount of computational and experimental work has been devoted to uncovering network motifs in gene regulatory networks. The leading hypothesis is that evolutionary processes independently selected recurrent architectural relationships among regulators and target genes (motifs) to produce characteristic expression patterns of its members. However, even with the same architecture, the genes may still be differentially expressed. Therefore, to define fully the expression of a group of genes, the strength of the connections in a network motif must be specified, and the cis-promoter features that participate in the regulation must be determined.ResultsWe have developed a model-based approach to analyze proteobacterial genomes for promoter features that is specifically designed to account for the variability in sequence, location and topology intrinsic to differential gene expression. We provide methods for annotating regulatory regions by detecting their subjacent cis-features. This includes identifying binding sites for a transcriptional regulator, distinguishing between activation and repression sites, direct and reverse orientation, and among sequences that weakly reflect a particular pattern; binding sites for the RNA polymerase, characterizing different classes, and locations relative to the transcription factor binding sites; the presence of riboswitches in the 5'UTR, and for other transcription factors. We applied our approach to characterize network motifs controlled by the PhoP/PhoQ regulatory system of Escherichia coli and Salmonella enterica serovar Typhimurium. We identified key features that enable the PhoP protein to control its target genes, and distinct features may produce different expression patterns even within the same network motif.ConclusionGlobal transcriptional regulators control multiple promoters by a variety of network motifs. This is clearly the case for the regulatory protein PhoP. In this work, we studied this regulatory protein and demonstrated that understanding gene expression does not only require identifying a set of connexions or network motif, but also the cis-acting elements participating in each of these connexions.

Evolution of a Bacterial Regulon Controlling Virulence and Mg2+ Homeostasis

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.

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

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.

A Point Mutation in the Two-Component Regulator PhoP-PhoR Accounts for the Absence of Polyketide-Derived Acyltrehaloses but Not That of Phthiocerol Dimycocerosates in Mycobacterium tuberculosis H37Ra

Similarities between Mycobacterium tuberculosis phoP-phoR mutants and the attenuated laboratory strain M. tuberculosis H37Ra in terms of morphological and cytochemical properties, lipid content, gene expression and virulence attenuation prompted us to analyze the functionality of this two-component regulator in the latter strain. Sequence analysis revealed a base substitution resulting in a one-amino-acid change in the likely DNA-binding region of PhoP in H37Ra relative to H37Rv. Using gel-shift assays, we show that this mutation abrogates the ability of the H37Ra PhoP protein to bind to a 40-bp segment of its own promoter. Consistent with this result, the phoP gene from H37Rv but not that from H37Ra was able to restore the synthesis of sulfolipids, diacyltrehaloses and polyacyltrehaloses in an isogenic phoP-phoR knock-out mutant of M. tuberculosis Moreover, complementation of H37Ra with phoP from H37Rv fully restored sulfolipid, diacyltrehalose and polyacyltrehalose synthesis, clearly indicating that the lack of production of these lipids in H37Ra is solely due to the point mutation in phoP. Using a pks2-3/4 knock-out mutant of M. tuberculosis H37Rv, evidence is further provided that the above-mentioned polyketide-derived acyltrehaloses do not significantly contribute to the virulence of the tubercle bacillus in a mouse model of infection. Reasons for the attenuation of H37Ra thus most likely stand in other virulence factors, many of which are expected to belong to the PhoP regulon and another of which, unrelated to PhoP, appears to be the lack of production of phthiocerol dimycocerosates in this strain.

A Positive Feedback Loop Promotes Transcription Surge That Jump-Starts Salmonella Virulence Circuit

The PhoP/PhoQ two-component system is a master regulator of Salmonella pathogenicity. Here we report that induction of the PhoP/PhoQ system results in an initial surge of PhoP phosphorylation; the occupancy of target promoters by the PhoP protein; and the transcription of PhoP-activated genes, which then subsides to reach new steady-state levels. This surge in PhoP activity is due to PhoP positively activating its own transcription, because a strain constitutively expressing the PhoP protein attained steady-state levels of activation asymptotically, without the surge. The strain constitutively expressing the PhoP protein was attenuated for virulence in mice, demonstrating that the surge conferred by PhoP's positive feedback loop is necessary to jump-start Salmonella's virulence program.

Functional genomics of stress response in Pseudomonas putida KT2440.

The metabolically versatile soil bacterium Pseudomonas putida has to cope with numerous abiotic stresses in its habitats. The stress responses of P. putida KT2440 to 4 degrees C, pH 4.5, 0.8 M urea, and 45 mM sodium benzoate were analyzed by determining the global mRNA expression profiles and screening for stress-intolerant nonauxotrophic Tn5 transposon mutants. In 392 regulated genes or operons, 36 gene regions were differentially expressed by more than 2.5-fold, and 32 genes in 23 operons were found to be indispensable for growth during exposure to one of the abiotic stresses. The transcriptomes of the responses to urea, benzoate, and 4 degrees C correlated positively with each other but negatively with the transcriptome of the mineral acid response. The CbrAB sensor kinase, the cysteine synthase CysM, PcnB and VacB, which control mRNA stability, and BipA, which exerts transcript-specific translational control, were essential to cope with cold stress. The cyo operon was required to cope with acid stress. A functional PhoP, PtsP, RelA/SpoT modulon, and adhesion protein LapA were necessary for growth in the presence of urea, and the outer membrane proteins OmlA and FepA and the phosphate transporter PstBACS were indispensable for growth in the presence of benzoate. A lipid A acyltransferase (PP0063) was a mandatory component of the stress responses to cold, mineral acid, and benzoate. Adaptation of the membrane barrier, uptake of phosphate, maintenance of the intracellular pH and redox status, and translational control of metabolism are key mechanisms of the response of P. putida to abiotic stresses.

Analysis of differentially-regulated genes within a regulatory network by GPS genome navigation

A critical challenge of the post-genomic era is to understand how genes are differentially regulated even when they belong to a given network. Because the fundamental mechanism controlling gene expression operates at the level of transcription initiation, computational techniques have been developed that identify cis regulatory features and map such features into expression patterns to classify genes into distinct networks. However, these methods are not focused on distinguishing between differentially regulated genes within a given network. Here we describe an unsupervised machine learning method, termed GPS for gene promoter scan, that discriminates among co-regulated promoters by simultaneously considering both cis-acting regulatory features and gene expression. GPS is particularly useful for knowledge discovery in environments with reduced datasets and high levels of uncertainty. Application of this method to the enteric bacteria Escherichia coli and Salmonella enterica uncovered novel members, as well as regulatory interactions in the regulon controlled by the PhoP protein that were not discovered using previous approaches. The predictions made by GPS were experimentally validated to establish that the PhoP protein uses multiple mechanisms to control gene transcription, and is a central element in a highly connected network. The scripts and programs used in this work are accessible from the gps-tools.wustl.edu website. Data and predictions are available by request.

The PhoP/PhoQ system controls the intramacrophage type three secretion system of Salmonella enterica

Spi/Ssa is a unique type three secretion system that functions exclusively when Salmonella enterica is inside eukaryotic cells. Expression of the Spi/Ssa system and its secreted effectors is dependent on SsrB/SpiR, a two-component regulatory system encoded in the SPI-2 pathogenicity island that also harbours the spi/ssa genes. Here we determine that the PhoP/PhoQ two-component system controls the intramacrophage expression of spi/ssa genes by regulating the SsrB/SpiR system. We establish that PhoP regulates transcription of the response regulator SsrB and demonstrate binding of the PhoP protein to the ssrB promoter both in vivo using chromatin immunoprecipitation in Salmonella-infected macrophages, and in vitro using purified PhoP protein. We show that PhoP controls the SpiR sensor post-transcriptionally and identify a region in the 5' untranslated region of the spiR message that is required for this effect. The demonstration that the PhoP/PhoQ system is directly involved in the regulation of the SPI-2 pathogenicity island highlights PhoP/PhoQ's central role in Salmonella virulence. We suggest that different regulatory systems convey distinct signals over time to produce the SsrB/SpiR system, which then modulates expression of the Spi/Ssa apparatus and secreted effectors.

Transcriptional Regulation of the 4-Amino-4-deoxy-L-arabinose Biosynthetic Genes in Yersinia pestis

Inducible membrane remodeling is an adaptive mechanism that enables Gram-negative bacteria to resist killing by cationic antimicrobial peptides and to avoid eliciting an immune response. Addition of 4-amino-4-deoxy-l -arabinose (4-aminoarabinose) moieties to the phosphate residues of the lipid A portion of the lipopolysaccharide decreases the net negative charge of the bacterial membrane resulting in protection from the cationic antimicrobial peptide polymyxin B. In Salmonella enterica serovar Typhimurium, the PmrA/PmrB two-component regulatory system governs resistance to polymyxin B by controlling transcription of the 4-aminoarabinose biosynthetic genes. Transcription of PmrA-activated genes is induced by Fe(3+), which is sensed by PmrA cognate sensor PmrB, and by low Mg(2+), in a mechanism that requires not only the PmrA and PmrB proteins but also the Mg(2+)-responding PhoP/PhoQ system and the PhoP-activated PmrD protein, a post-translational activator of the PmrA protein. Surprisingly, Yersinia pestis can promote PhoP-dependent modification of its lipid A with 4-aminoarabinose despite lacking a PmrD protein. Here we report that Yersinia uses different promoters to transcribe the 4-aminoarabinose biosynthetic genes pbgP and ugd depending on the inducing signal. This is accomplished by the presence of distinct binding sites for the PmrA and PhoP proteins in the promoters of the pbgP and ugd genes. Our results demonstrate that closely related bacterial species may use disparate regulatory pathways to control genes encoding conserved proteins.

Binding of PhoP to promoters of phosphate‐regulated genes in Streptomyces coelicolor: identification of PHO boxes

The control of phosphate-regulated genes in Streptomyces coelicolor is mediated by the two-component system PhoR-PhoP. When coupled to the reporter xylE gene the pstS, phoRP and phoU promoters were shown to be very sensitive to phosphate regulation. The transcription start points of the pstS, the phoRP and the phoU promoters were identified by primer extension. phoRP showed a leaderless transcript. The response-regulator (DNA-binding) PhoP protein was overexpressed and purified in Escherichia coli as a GST-PhoP fused protein. The DNA-binding domain (DBD) of PhoP was also obtained in a similar manner. Both PhoP and its truncated DBD domain were found to bind with high affinity to an upstream region of the pstS and phoRP-phoU promoters close to the -35 sequence of each of these promoters. DNase I protection studies revealed a 29 bp protected stretch in the sense strand of the pstS promoter that includes two 11 bp direct repeat units. Footprinting of the bidirectional phoRP-phoU promoter region showed a 51 bp protected sequence that encompasses four direct repeat units, two of them with high similarity to the protected sequences in the pstS promoter. PHO boxes have been identified by alignment of the six direct repeat units found in those promoter regions. Each direct repeat unit adjusts to the consensus G(G/T)TCAYYYR(G/C)G.

Dissecting the PhoP regulatory network of Escherichia coli and Salmonella enterica.

Genetic and genomic approaches have been successfully used to assign genes to distinct regulatory networks. However, the present challenge of distinguishing differentially regulated genes within a network is particularly hard because members of a given network tend to have similar regulatory features. We have addressed this challenge by developing a method, termed Gene Promoter Scan, that discriminates coregulated promoters by simultaneously considering both multiple cis promoter features and gene expression. Here, we apply this method to probe the regulatory networks governed by the PhoP/PhoQ two-component system in the enteric bacteria Escherichia coli and Salmonella enterica. Our analysis uncovered members of the PhoP regulon and interactions with other regulatory systems that were not discovered in previous approaches. The predictions made by Gene Promoter Scan were experimentally validated to establish that the PhoP protein uses multiple mechanisms to control gene transcription, regulates acid resistance determinants, and is a central element in a highly connected network.

Signal-dependent Binding of the Response Regulators PhoP and PmrA to Their Target Promoters in Vivo

Low Mg2+ promotes phosphorylation of the response regulators PhoP and PmrA and transcription of their activated genes in Salmonella enterica. Using chromatin immunoprecipitation, we have now determined that the PhoP and PmrA proteins are recruited to the regulatory region of their target genes when bacteria experience low Mg2+ but not when they are grown in high Mg2+. Even when the PhoP protein was artificially produced at 4-fold higher levels than the wild-type strain, promoter occupancy required the low Mg2+ signal. Substitution of the predicted phosphorylation site Asp-52 with a valine residue abolished phosphorylation of the PhoP protein, resulting in loss of PhoP binding to target promoters and transcription of PhoP-activated genes. Our results indicate that the promoter binding ability of the PhoP and PmrA proteins occurring in low Mg2+ is correlated with phosphorylation of these proteins in vivo.

Transcriptional Control of the Antimicrobial Peptide Resistance ugtL Gene by the Salmonella PhoP and SlyA Regulatory Proteins

The PhoP/PhoQ two-component system is a master regulator that governs the ability of Salmonella to cause a lethal infection in mice, the adaptation to low Mg(2+) environments, and resistance to a variety of antimicrobial peptides. We have recently established that the PhoP-activated ugtL gene is required for resistance to the antimicrobial peptides magainin 2 and polymyxin B. Here we report that ugtL transcription requires not only the PhoP protein but also the virulence regulatory protein SlyA. The PhoP protein footprinted two regions of the ugtL promoter, mutation of either one of which was sufficient to abolish ugtL transcription. Although the SlyA protein is a transcriptional activator of the ugtL gene, it footprinted the ugtL promoter at a region located downstream of the transcription start site. The PhoP protein footprinted the slyA promoter, indicating that it controls slyA transcription directly. The slyA mutant was hypersensitive to magainin 2 and polymyxin B, suggesting that the virulence attenuation exhibited by slyA mutants may be caused by hypersensitivity to antimicrobial peptides. We propose that the PhoP and SlyA proteins control ugtL transcription using a feed-forward loop design.

Phosphate control of the biosynthesis of antibiotics and other secondary metabolites is mediated by the PhoR-PhoP system: an unfinished story.

The biosynthesis of many different types of antibiotics and other secondary metabolites is regulated by phosphate. Production of these valuable compounds occurs only under phosphate-limiting nutritional conditions. In a few cases, there is evidence showing that the negative phosphate control is exerted at the transcriptional level. Recently, it was shown that phosphate control of antibiotic biosynthesis in Streptomyces lividans and Streptomyces coelicolor is mediated by the two-component PhoR-PhoP system that also controls the alkaline phosphatase gene (phoA). The PhoR protein is a standard membrane sensor kinase, whereas PhoP is a member of the DNA-binding response regulators. In Escherichia coli and Bacillus subtilis, the phosphorylated PhoP protein (PhoP∼P) activates, in response to phosphate starvation, expression of the pho regulon genes by binding to consensus phosphate boxes in the promoter regions (PHO boxes). Expression of phoA in S. lividans is induced by PhoP∼P, and mutants lacking phoP (or phoR and phoP) do not form PhoA. These mutants overproduce large amounts of actinorhodin and undecylprodigiosin. No consensus PHO boxes occur in the upstream region of phosphate-regulated secondary metabolism genes. However, pathway-specific activator proteins (ActII-open reading frame 4 [ORF4], RedD, CcaR, and DnrI) are known to bind to these regions. In S. coelicolor, actII-orf4 is positively regulated by the AfsS protein, which, in turn, is induced by the phosphorylated AfsR protein. It is likely that the PhoR-PhoP system exerts its action on actinorhodin and undecylprodigiosin by a cascade mechanism mediated by AfsR and AfsS. Directed phoR-phoP gene disruption will be very useful for the construction of tailored phosphate-deregulated strains overproducing valuable secondary metabolites.

Signal-dependent Requirement for the Co-activator Protein RcsA in Transcription of the RcsB-regulated ugd Gene

The RcsC/YojN/RcsB phosphorelay system controls gene expression in response to a variety of signals, including changes in temperature, osmolarity, and overproduction of membrane proteins. Transcription of certain RcsB-activated genes, such as the capsule synthesis cps operon, requires the co-activator protein RcsA, whereas expression of other RcsB-activated genes is RcsA-independent. We have established previously that a tolB mutation induces transcription of the Salmonella UDP-glucose dehydrogenase ugd gene in an RcsA- and RcsB-dependent manner. This induction is independent of the two-component systems PhoP/PhoQ and PmrA/PmrB, which are required for ugd expression in response to low Mg2+. We now report that the RcsC/YojN/RcsB system is activated in a pmrA mutant experiencing Fe3+ and low Mg2+, resulting in expression of both cps and ugd genes. However, whereas cps transcription remained RcsA-dependent, ugd transcription became RcsA-independent but dependent on the PhoP protein. S1 mapping experiments demonstrated that RcsA-dependent and -independent transcription of the ugd gene use the same promoter. DNase footprinting analysis identified a PhoP-binding site in the ugd promoter. Yet, PhoP-mediated ugd transcription required either the RcsC/YojN/RcsB or the PmrA/PmrB systems.