PhoP Regulator Research Articles

PhoPQ Regulates Quinolone and Cephalosporin Resistance Formation in Salmonella Enteritidis at the Transcriptional Level.

The two-component system (TCS) PhoPQ has been demonstrated to be crucial for the formation of resistance to quinolones and cephalosporins in Salmonella Enteritidis (S. Enteritidis). However, the mechanism underlying PhoPQ-mediated antibiotic resistance formation remains poorly understood. Here, it was shown that PhoP transcriptionally regulated an assortment of genes associated with envelope homeostasis, the osmotic stress response, and the redox balance to confer resistance to quinolones and cephalosporins in S. Enteritidis. Specifically, cells lacking the PhoP regulator, under nalidixic acid and ceftazidime stress, bore a severely compromised membrane on the aspects of integrity, fluidity, and permeability, with deficiency to withstand osmolarity stress, an increased accumulation of intracellular reactive oxygen species, and dysregulated redox homeostasis, which are unfavorable for bacterial survival. The phosphorylated PhoP elicited transcriptional alterations of resistance-associated genes, including the outer membrane porin ompF and the aconitate hydratase acnA, by directly binding to their promoters, leading to a limited influx of antibiotics and a well-maintained intracellular metabolism. Importantly, it was demonstrated that the cavity of the PhoQ sensor domain bound to and sensed quinolones/cephalosporins via the crucial surrounding residues, as their mutations abrogated the binding and PhoQ autophosphorylation. This recognition mode promoted signal transduction that activated PhoP, thereby modulating the transcription of downstream genes to accommodate cells to antibiotic stress. These findings have revealed how bacteria employ a specific TCS to sense antibiotics and combat them, suggesting PhoPQ as a potential drug target with which to surmount S. Enteritidis. IMPORTANCE The prevalence of quinolone and cephalosporin-resistant S. Enteritidis is of increasing clinical concern. Thus, it is imperative to identify novel therapeutic targets with which to treat S. Enteritidis-associated infections. The PhoPQ two-component system is conserved across a variety of Gram-negative pathogens, by which bacteria adapt to a range of environmental stimuli. Our earlier work has demonstrated the importance of PhoPQ in the resistance formation in S. Enteritidis to quinolones and cephalosporins. In the current work, we identified a global profile of genes that are regulated by PhoP under antibiotic stresses, with a focus on how PhoP regulated downstream genes, either positively or negatively. Additionally, we established that PhoQ sensed quinolones and cephalosporins in a manner of directly binding to them. These identified genes and pathways that are mediated by PhoPQ represent promising targets for the development of a drug potentiator with which to neutralize antibiotic resistance in S. Enteritidis.

Molecular Mechanisms of Phosphate Sensing, Transport and Signalling in Streptomyces and Related Actinobacteria.

Phosphorous, in the form of phosphate, is a key element in the nutrition of all living beings. In nature, it is present in the form of phosphate salts, organophosphates, and phosphonates. Bacteria transport inorganic phosphate by the high affinity phosphate transport system PstSCAB, and the low affinity PitH transporters. The PstSCAB system consists of four components. PstS is the phosphate binding protein and discriminates between arsenate and phosphate. In the Streptomyces species, the PstS protein, attached to the outer side of the cell membrane, is glycosylated and released as a soluble protein that lacks its phosphate binding ability. Transport of phosphate by the PstSCAB system is drastically regulated by the inorganic phosphate concentration and mediated by binding of phosphorylated PhoP to the promoter of the PstSCAB operon. In Mycobacterium smegmatis, an additional high affinity transport system, PhnCDE, is also under PhoP regulation. Additionally, Streptomyces have a duplicated low affinity phosphate transport system encoded by the pitH1–pitH2 genes. In this system phosphate is transported as a metal-phosphate complex in simport with protons. Expression of pitH2, but not that of pitH1 in Streptomyces coelicolor, is regulated by PhoP. Interestingly, in many Streptomyces species, three gene clusters pitH1–pstSCAB–ppk (for a polyphosphate kinase), are linked in a supercluster formed by nine genes related to phosphate metabolism. Glycerol-3-phosphate may be transported by the actinobacteria Corynebacterium glutamicum that contains a ugp gene cluster for glycerol-3-P uptake, but the ugp cluster is not present in Streptomyces genomes. Sugar phosphates and nucleotides are used as phosphate source by the Streptomyces species, but there is no evidence of the uhp gene involved in the transport of sugar phosphates. Sugar phosphates and nucleotides are dephosphorylated by extracellular phosphatases and nucleotidases. An isolated uhpT gene for a hexose phosphate antiporter is present in several pathogenic corynebacteria, such as Corynebacterium diphtheriae, but not in non-pathogenic ones. Phosphonates are molecules that contains phosphate linked covalently to a carbon atom through a very stable C–P bond. Their utilization requires the phnCDE genes for phosphonates/phosphate transport and genes for degradation, including those for the subunits of the C–P lyase. Strains of the Arthrobacter and Streptomyces genera were reported to degrade simple phosphonates, but bioinformatic analysis reveals that whole sets of genes for putative phosphonate degradation are present only in three Arthrobacter species and a few Streptomyces species. Genes encoding the C–P lyase subunits occur in several Streptomyces species associated with plant roots or with mangroves, but not in the laboratory model Streptomyces species; however, the phnCDE genes that encode phosphonates/phosphate transport systems are frequent in Streptomyces species, suggesting that these genes, in the absence of C–P lyase genes, might be used as surrogate phosphate transporters. In summary, Streptomyces and related actinobacteria seem to be less versatile in phosphate transport systems than Enterobacteria.

SenX3-RegX3, an Important Two-Component System, Regulates Strain Growth and Butenyl-spinosyn Biosynthesis in Saccharopolyspora pogona.

SummaryButenyl-spinosyn produced by Saccharopolyspora pogona exhibits strong insecticidal activity and a broad pesticidal spectrum. Currently, important functional genes involved in butenyl-spinosyn biosynthesis remain unknown, which leads to difficulty in efficient understanding of its regulatory mechanism and improving its production by metabolic engineering. Here, we present data supporting a role of the SenX3-RegX3 system in regulating the butenyl-spinosyn biosynthesis. EMSAs and qRT-PCR demonstrated that RegX3 positively controls butenyl-spinosyn production in an indirect way. Integrated proteomic and metabolomic analysis, regX3 deletion not only strengthens the basal metabolic ability of S. pogona in the mid-growth phase but also promotes the flow of the acetyl-CoA produced via key metabolic pathways into the TCA cycle rather than the butenyl-spinosyn biosynthetic pathway, which ultimately leads to continued growth but reduced butenyl-spinosyn production. The strategy demonstrated here may be valuable for revealing the regulatory role of the SenX3-RegX3 system in the biosynthesis of other natural products.

The transcriptional regulator PhoP mediates the tolC molecular mechanism on APEC biofilm formation and pathogenicity

ABSTRACTAs a transcriptional regulator of the classical binary regulatory system, PhoP plays an important role in the life activities of avian pathogenic Escherichia coli (APEC). In previous experiments, we found that the absence of phoP affects APEC biofilm formation and pathogenicity. To further explore the molecular mechanism of phoP regulation of these phenomena, the differentially expressed gene tolC was screened based on phoP-derived transcriptional data, and the specific sequence identity of the PhoP binding sequence was predicted by bioinformatics and verified by electrophoretic mobility shift assay (EMSA). The results showed that PhoP can directly bind to the tolC promoter. On this basis, tolC deletion and complementary strains were constructed. Biofilm formation was quantified by crystal violet staining and rdar morphology change was observed in these strains. Loss of tolC reduced biofilm formation. We also examined pathological changes in organ paraffin sections by challenging chicks with the strains. After loss of tolC, the clinical signs of pericarditis and liver and spleen enlargement in chicks were alleviated, and pathogenicity to the host cells was decreased. Additionally, quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that tolC deletion downregulated secA/secB/secE/secY transcript levels, which are part of the type II secretion system secreting virulence effector element. These results indicate that tolC contributes to the phoP-mediated effect on APEC biofilm formation and pathogenicity.

Adaptation to host-specific bacterial pathogens drive rapid evolution of novel PhoP/PhoQ regulation pathway modulating the virulence

The presence of the PhoP-PhoQ system is usually different in various bacterial groups, suggesting that PhoP can control the expression of different genes in species. However, little is known about the evolution of the PhoP-PhoQ system among bacterial pathogens. Here, we study the evolution of PhoP and PhoQ regulation in 15 species of Enterobacteriaceae family. We have determined that the regulatory objectives adopted by PhoP and PhoQ are mainly different, due to the result of horizontal gene transfer events and even the change in the genetic content between closely related species. We have compared many possibilities tests (M1 vs. M2 and M7 with M8) to determine the positive selection. Estimating parameters at M1 and M2, with positive selection in M2 of the two proteins. The proportions of positive selection sites significant with ω = 4.53076 for PhoP and ω = 4.21041 PhQ. M8 was significant for PhoP and PhQ proteins. To further confirm the positive selection results, we used the Selecton server to confer positive selection on individual sites using the Mechanistic-Empirical Combination model, and we noticed that several sites had been identified under selection pressure during the evolution. There was a strong indication for the positive selection in bacterial genes of PhoP and PhoQ showed the results. By the use of REL and IFEL, the positive selection for PhoP was detected 14 and 11 sites respectively at different codon positions. The positively selected sites of amino acids such as Arginine, Alanine, Lysine, and Leucine are more important for the production of signals. Our results suggest that the positive selection of PhoP-PhoQ genes in host adaptation during evolution raises an intriguing possibility causes subtle variations in actions of PhoP-PhoQ and also increases the opportunities that cause modification in protein structure for the evolution of increasing pathogenicity in bacterial pathogens.

Interplay of PhoP and DevR response regulators defines expression of the dormancy regulon in virulent Mycobacterium tuberculosis

The DevR response regulator of Mycobacterium tuberculosis is an established regulator of the dormancy response in mycobacteria and can also be activated during aerobic growth conditions in avirulent strains, suggesting a complex regulatory system. Previously, we reported culture medium-specific aerobic induction of the DevR regulon genes in avirulent M. tuberculosis H37Ra that was absent in the virulent H37Rv strain. To understand the underlying basis of this differential response, we have investigated aerobic expression of the Rv3134c-devR-devS operon using M. tuberculosis H37Ra and H37Rv devR overexpression strains, designated as LIX48 and LIX50, respectively. Overexpression of DevR led to the up-regulation of a large number of DevR regulon genes in aerobic cultures of LIX48, but not in LIX50. To ascertain the involvement of PhoP response regulator, also known to co-regulate a subset of DevR regulon genes, we complemented the naturally occurring mutant phoPRa gene of LIX48 with the WT phoPRv gene. PhoPRv dampened the induced expression of the DevR regulon by >70-80%, implicating PhoP in the negative regulation of devR expression. Electrophoretic mobility shift assays confirmed phosphorylation-independent binding of PhoPRv to the Rv3134c promoter and further revealed that DevR and PhoPRv proteins exhibit differential DNA binding properties to the target DNA. Through co-incubations with DNA, ELISA, and protein complementation assays, we demonstrate that DevR forms a heterodimer with PhoPRv but not with the mutant PhoPRa protein. The study puts forward a new possible mechanism for coordinated expression of the dormancy regulon, having implications in growth adaptations critical for development of latency.

ATP reduction by MgtC and Mg2+ homeostasis by MgtA and MgtB enables Salmonella to accumulate RpoS upon low cytoplasmic Mg2+ stress

RpoS is one of several alternative sigma factors known to alter gene expression profiles by RpoS-associated RNA polymerase in response to a variety of stresses. The enteric bacteria Salmonella enterica and Escherichia coli accumulate RpoS under low Mg2+ concentrations via a common mechanism in which the PhoP regulator activates expression of antiadaptor proteins that, by sequestering the adaptor RssB, prevent RpoS degradation by the protease ClpXP. Here, we demonstrate that this genetic program alone does not fully support RpoS accumulation when cytoplasmic Mg2+ concentration drops to levels that impair protein synthesis. Under these circumstances, only S. enterica continues RpoS accumulation in a manner dependent on other PhoP-activated programs (i.e. ATP reduction by the MgtC protein and Mg2+ import by the MgtA and MgtB transporters) that maintain translation homeostasis. Moreover, we provide evidence that the mgtC gene, which is present in S. enterica but not in E. coli, is responsible for the differences in RpoS accumulation between these two bacterial species. Our results suggest that bacteria possess a mechanism to control RpoS accumulation responding to cytoplasmic Mg2+ levels, the difference of which causes distinct RpoS accumulation in closely related bacterial species.

Analysis and validation of the pho regulon in the tacrolimus-producer strain Streptomyces tsukubaensis: differences with the model organism Streptomyces coelicolor.

Inorganic and organic phosphate controls both primary and secondary metabolism in Streptomyces genus. Metabolism regulation by phosphate in Streptomyces species is mediated by the PhoR-PhoP two-component system. Response regulator PhoP binds to conserved sequences of 11 nucleotides called direct repeat units (DRus), whose organization and conservation determine the binding of PhoP to distinct promoters. Streptomyces tsukubaensis is the industrial producer of the clinical immunosuppressant tacrolimus (FK506). A bioinformatic genome analysis detected several genes with conserved PHO boxes involved in phosphate scavenging and transport, nitrogen regulation, and secondary metabolite production. In this article, the PhoP regulation has been confirmed by electrophoretic mobility shift assays (EMSA) of the most relevant members of the traditional pho regulon such as the two-component system PhoR-P or genes involved in high-affinity phosphate transport (pstSCAB) and low-affinity phosphate transport (pit). However, the PhoP control over phosphatase genes in S. tsukubaensis is significantly different from the pattern reported in the model bacteria Streptomyces coelicolor. Thus, neither the alkaline phosphatase PhoA nor PhoD is regulated by PhoP. On the contrary, the binding of PhoP to the promoter of a novel putative phosphatase PhoX was confirmed. A crosstalk of the PhoP and GlnR regulators, which balances phosphate and nitrogen utilization, also occurs in S. tsukubaensis but slightly modified. Finally, PhoP regulates genes, like afsS, that link phosphate control and secondary metabolite production in S. tsukubaensis. In summary, there are notable differences between the regulation of specific genes of the pho regulon in S. tsukubaensis and the model organism S. coelicolor.

Global proteomic profiling of Yersinia ruckeri strains

Yersinia ruckeri is the causative agent of enteric redmouth disease (ERM) of salmonids. There is little information regarding the proteomics of Y. ruckeri. Herein, we perform whole protein identification and quantification of biotype 1 and biotype 2 strains of Y. ruckeri grown under standard culture conditions using a shotgun proteomic approach. Proteins were extracted, digested and peptides were separated by a nano liquid chromatography system and analyzed with a high-resolution hybrid triple quadrupole time of flight mass spectrometer coupled via a nano ESI interface. SWATH-MS technology and sophisticated statistical analyses were used to identify proteome differences among virulent and avirulent strains. GO annotation, subcellular localization, virulence proteins and antibiotic resistance ontology were predicted using bioinformatic tools. A total of 1395 proteins were identified in the whole cell of Y. ruckeri. These included proteases, chaperones, cell division proteins, outer membrane proteins, lipoproteins, receptors, ion binding proteins, transporters and catalytic proteins. In virulent strains, a total of 16 proteins were upregulated including anti-sigma regulatory factor, arginine deiminase, phosphate-binding protein PstS and superoxide dismutase Cu–Zu. Additionally, several virulence proteins were predicted such as Clp and Lon pro-teases, TolB, PPIases, PstS, PhoP and LuxR family transcriptional regulators. These putative virulence proteins might be used for development of novel targets for treatment of ERM in fish. Our study represents one of the first global proteomic reference profiles of Y. ruckeri and this data can be accessed via ProteomeXchange with identifier PXD005439. These proteomic profiles elucidate proteomic mechanisms, pathogenicity, host-interactions, antibiotic resistance ontology and localization of Y. ruckeri proteins.

EspR-dependent ESAT-6 Protein Secretion of Mycobacterium tuberculosis Requires the Presence of Virulence Regulator PhoP

Attenuation of Mycobacterium bovis BCG strain is related to the loss of the RD1-encoded ESX-1 secretion system. The ESX-1 system secretes virulence factor ESAT-6 that plays a critical role in modulation of the host immune system, which is essential for establishment of a productive infection. Previous studies suggest that among the reasons for attenuation of Mycobacterium tuberculosis H37Ra is a mutation in the phoP gene that interferes with the ESX-1 secretion system and inhibits secretion of ESAT-6. Here, we identify a totally different and distinct regulatory mechanism involving PhoP and transcription regulator EspR on transcriptional control of the espACD operon, which is required for ESX-1-dependent ESAT-6 secretion. Although both of these regulators are capable of influencing espACD expression, we show that activation of espACD requires direct recruitment of both PhoP and EspR at the espACD promoter. The most fundamental insights are derived from the inhibition of EspR binding at the espACD regulatory region of the phoP mutant strain because of PhoP-EspR protein-protein interactions. Based on these results, a model is proposed suggesting how PhoP and EspR protein-protein interactions contribute to activation of espACD expression and, in turn, control ESAT-6 secretion, an essential pathogenic determinant of M. tuberculosis Together, these results have significant implications on the mechanism of virulence regulation of M. tuberculosis.

Bifidobacterium thermophilum RBL67 impacts on growth and virulence gene expression of Salmonella enterica subsp. enterica serovar Typhimurium.

BackgroundBifidobacterium thermophilum RBL67 (RBL67), a human fecal isolate and health promoting candidate shows antagonistic and protective effects against Salmonella and Listeria spec. in vitro. However, the underlying mechanisms fostering these effects remain unknown. In this study, the interactions of RBL67 and Salmonella enterica subsp. enterica serovar Typhimurium N-15 (N-15) were explored by global transcriptional analysis.ResultsGrowth experiments were performed in a complex nutritive medium with controlled pH of 6.0 and suitable for balanced growth of both RBL67 and N-15. RBL67 growth was slightly enhanced in presence of N-15. Conversely, N-15 showed reduced growth in the presence of RBL67. Transcriptional analyses revealed higher expression of stress genes and amino acid related function in RBL67 in co-culture with N-15 when compared to mono-culture. Repression of the PhoP regulator was observed in N-15 in presence of RBL67. Further, RBL67 activated virulence genes located on the Salmonella pathogenicity islands 1 and 2. Flagellar genes, however, were repressed by RBL67. Sequential expression of flagellar, SPI 1 and fimbrial genes is essential for Salmonella infection. Our data revealed that RBL67 triggers expression of SPI 1 and fimbrial determinants prematurely, potentially leading to redundant energy expenditure. In the competitive environment of the gut such energy expenditure could lead to enhanced clearing of Salmonella.ConclusionOur study provides first insights into probiotic-pathogen interactions on global transcriptional level and suggests that deregulation of virulence gene expression might be an additional protective mechanism of probiotica against infections of the host.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-016-0659-x) contains supplementary material, which is available to authorized users.

Role of the PhoP-PhoQ gene regulatory system in adaptation of Yersinia pestis to environmental stress in the flea digestive tract.

The Yersinia pestis PhoPQ gene regulatory system is induced during infection of the flea digestive tract and is required to produce adherent biofilm in the foregut, which greatly enhances bacterial transmission during a flea bite. To understand the in vivo context of PhoPQ induction and to determine PhoP-regulated targets in the flea, we undertook whole-genome comparative transcriptional profiling of Y. pestis WT and ΔphoP strains isolated from infected fleas and from temperature-matched in vitro planktonic and flow-cell biofilm cultures. In the absence of PhoP regulation, the gene expression program indicated that the bacteria experienced diverse physiological stresses and were in a metabolically less active state. Multiple stress response genes, including several toxin–antitoxin loci and YhcN family genes responsible for increased acid tolerance, were upregulated in the phoP mutant during flea infection. The data implied that PhoPQ was induced by low pH in the flea gut, and that PhoP modulated physiological adaptation to acid and other stresses encountered during infection of the flea. This adaptive response, together with PhoP-dependent modification of the bacterial outer surface that includes repression of pH 6 antigen fimbriae, supports stable biofilm development in the flea foregut.

The PhoP-dependent ncRNA Mcr7 modulates the TAT secretion system in Mycobacterium tuberculosis.

The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects through proteins like EspR, which controls Esx1 function, via regulation of the espACD operon. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in an M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host.

Vancomycin resistance in Streptomyces coelicolor is phosphate-dependent but is not mediated by the PhoP regulator.

Vancomycin is an essential antibiotic to treat infections caused by multidrug-resistant bacteria. Several bacteria show resistance to vancomycin, including the model actinomycete Streptomyces coelicolor. In this study, vancomycin disk diffusion tests were performed to determine vancomycin resistance in S. coelicolor M145 under rich (TSA medium) or defined (MMCGT medium) growth conditions. A vancomycin-susceptible phenotype was observed when the TSA rich medium was used, whereas a resistant phenotype was obtained when the low-phosphate MMCGT medium was used. To identify which component was responsible for the vancomycin-resistant phenotype, all the components of the MMCGT medium were added individually to the TSA medium, and vice versa. Addition of phosphate to the MMCGT medium (the phosphate concentration is much higher in TSA than in MMCGT) produced a vancomycin-susceptible phenotype in MMCGT. Phosphate regulation of vancomycin resistance is not PhoP-dependent since the same minimum inhibitory concentrations were obtained in S. coelicolor parental and ΔphoP mutant strains. This phosphate regulation was not observed in the vancomycin-producer Amycolatopsis orientalis NRRL 2452, which was always resistant both in TSA and MMCGT (with or without phosphate addition) media. On the other hand, other Streptomyces spp. were susceptible to vancomycin in all conditions tested, including Streptomyces toyocaensis, the producer of a glycopeptide antibiotic different from vancomycin. In conclusion, the phosphate concentration clearly affects the resistance of S. coelicolor to vancomycin.

Reciprocal Regulation of pH 6 Antigen Gene Loci by PhoP and RovA in Yersinia Pestis Biovar Microtus

To explore the transcriptional regulation of the psaEF and psaABC loci by the RovA and PhoP regulators in Yersinia pestis. Primer extension, LacZ fusion, gel mobility shift and DNase I footprinting assays were conducted in combination for this gene regulation study. It was determined that PhoP and RovA recognized the promoter-proximal regions of psaEF and psaABC in order to repress and stimulate their transcription, respectively. The translation/transcription start sites, Shine-Dalgarno sequences (ribosomal binding site), core promoter -10 and -35 elements, PhoP and RovA sites and PhoP/RovA consensus-like sequences were identified to determine the structural organization of PhoP/RovA-dependent promoters of psaEF and psaABC. RovA stimulated psaEF and psaABC, while PhoP repressed these two operons involving the direct association between RovA/PhoP and target promoter regions. The reciprocal regulation of psa genes by PhoP and RovA could contribute to the tightly controlled expression of the pH 6 antigen during infection.

Autoregulation of PhoP/PhoQ and Positive Regulation of the Cyclic AMP Receptor Protein-Cyclic AMP Complex by PhoP in Yersinia pestis

Yersinia pestis is one of the most dangerous bacterial pathogens. PhoP and cyclic AMP receptor protein (CRP) are global regulators of Y. pestis, and they control two distinct regulons that contain multiple virulence-related genes. The PhoP regulator and its cognate sensor PhoQ constitute a two-component regulatory system. The regulatory activity of CRP is triggered only by binding to its cofactor cAMP, which is synthesized from ATP by adenylyl cyclase (encoded by cyaA). However, the association between the two regulatory systems PhoP/PhoQ and CRP-cAMP is still not understood for Y. pestis. In the present work, the four consecutive genes YPO1635, phoP, phoQ, and YPO1632 were found to constitute an operon, YPO1635-phoPQ-YPO1632, transcribed as a single primary RNA, whereas the last three genes comprised another operon, phoPQ-YPO1632, transcribed with two adjacent transcriptional starts. Through direct PhoP-target promoter association, the transcription of these two operons was stimulated and repressed by PhoP, respectively; thus, both positive autoregulation and negative autoregulation of PhoP/PhoQ were detected. In addition, PhoP acted as a direct transcriptional activator of crp and cyaA. The translational/transcriptional start sites, promoter -10 and -35 elements, PhoP sites, and PhoP box-like sequences were determined for these PhoP-dependent genes, providing a map of the PhoP-target promoter interaction. The CRP and PhoP regulons have evolved to merge into a single regulatory cascade in Y. pestis because of the direct regulatory association between PhoP/PhoQ and CRP-cAMP.

Competition between the GlnR and PhoP regulators for the glnA and amtB promoters in Streptomyces coelicolor

Interaction of regulatory networks is a subject of great interest in systems biology of bacteria. Phosphate control of metabolism in Streptomyces is mediated by the two-component system PhoR–PhoP. Similarly, the utilization of different nitrogen sources is controlled by the regulator GlnR. Transcriptomic and biochemical analysis revealed that glnA (encoding a glutamine synthetase), glnR and other nitrogen metabolism genes are under PhoP control. DNA-binding experiments showed that PhoP binds to other nitrogen-regulated genes (SCO0255, SCO01863 and ureA). Using the glnA promoter as model, we observed that PhoP and GlnR compete for binding to the same promoter region, showing GlnR a higher affinity. Using a total of 14 GlnR-binding sites (50 direct repeat units) we established two information-based models that describe the GlnR box as consisting of two 11-nt direct repeats each with clear differences to PHO box. DNA-binding studies with different mutant sequences of glnA promoter revealed that the sequence recognized by GlnR is found in the coding strand whereas that recognized by PhoP is overlapping in the non-coding strand. In amtB promoter PhoP and GlnR boxes are not totally overlapping and both proteins bind simultaneously. PhoP control of nitrogen metabolism genes helps to balance the cellular P/N equilibrium.

Transcriptomic studies of phosphate control of primary and secondary metabolism in Streptomyces coelicolor

Phosphate controls the biosynthesis of many classes of secondary metabolites that belong to different biosynthetic groups, indicating that phosphate control is a general mechanism governing secondary metabolism. We refer in this article to the molecular mechanisms of regulation, mediated by the two-component system PhoR-PhoP, of the primary metabolism and the biosynthesis of antibiotics. The two-component PhoR-PhoP system is conserved in all Streptomyces and related actinobacteria sequenced so far, and involves a third component PhoU that modulates the signal transduction cascade. The PhoP DNA-binding sequence is well characterized in Streptomyces coelicolor. It comprises at least two direct repeat units of 11 nt, the first seven of which are highly conserved. Other less conserved direct repeats located adjacent to the core ones can also be bound by PhoP through cooperative protein-protein interactions. The phoR-phoP operon is self-activated and requires phosphorylated PhoP to mediate the full response. About 50 up-regulated PhoP-dependent genes have been identified by comparative transcriptomic studies between the parental S. coelicolor M145 and the ΔphoP mutant strains. The PhoP regulation of several of these genes has been studied in detail using EMSA and DNase I footprinting studies as well as in vivo expression studies with reporter genes and RT-PCR transcriptomic analyses.

Molecular Characterization of Transcriptional Regulation of rovA by PhoP and RovA in Yersinia pestis

Background Yersinia pestis is the causative agent of plague. The two transcriptional regulators, PhoP and RovA, are required for the virulence of Y. pestis through the regulation of various virulence-associated loci. They are the global regulators controlling two distinct large complexes of cellular pathways.Methodology/Principal FindingsBased on the LacZ fusion, primer extension, gel mobility shift, and DNase I footprinting assays, RovA is shown to recognize both of the two promoters of its gene in Y. pestis. The autoregulation of RovA appears to be a conserved mechanism shared by Y. pestis and its closely related progenitor, Y. pseudotuberculosis. In Y. pestis, the PhoP regulator responds to low magnesium signals and then negatively controls only one of the two promoters of rovA through PhoP-promoter DNA association.Conclusions/SignificanceRovA is a direct transcriptional activator for its own gene in Y. pestis, while PhoP recognizes the promoter region of rovA to repress its transcription. The direct regulatory association between PhoP and RovA bridges the PhoP and RovA regulons in Y. pestis.

PhoP and OxyR transcriptional regulators contribute to Yersinia pestis virulence and survival within Galleria mellonella

The virulence of Yersinia pestis KIM6+ was compared with multiple isolates of Yersinia pseudotuberculosis and Yersinia enterocolitica toward larvae of the greater wax moth Galleria mellonella. Although Y. pestis and Y. pseudotuberculosis were able to cause lethal infection in G. mellonella, these species appeared less virulent than the majority of Y. enterocolitica strains tested. Y. pestis survived primarily within hemocytes of G. mellonella, and induced a strong antibacterial peptide response that lasted for at least 3 days in surviving larvae. Immunization with dead bacteria to induce an antibacterial response led to increased survival of the larvae following infection. Mutant strains lacking the either phoP or oxyR, which were less resistant to antibacterial peptides and hydrogen peroxide respectively, were attenuated and restoration of the wild-type genes on plasmids restored virulence. Our results indicate that the Y. pseudotuberculosis-Y. pestis lineage is not as virulent toward G. mellonella as are the majority of Y. enterocolitica isolates. Further, we have shown that G. mellonella is a useful infection model for analyzing Y. pestis host-pathogen interactions, and antibacterial peptide resistance mediated by phoP and reactive oxygen defense mediated by oxyR are important for Y. pestis infection of this insect.