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1
McPhee, Joseph B., Manjeet Bains, Geoff Winsor, Shawn Lewenza, Agnieszka Kwasnicka, Michelle D. Brazas, Fiona S. L. Brinkman, and R. E. W. Hancock. "Contribution of the PhoP-PhoQ and PmrA-PmrB Two-Component Regulatory Systems to Mg2+-Induced Gene Regulation in Pseudomonas aeruginosa." Journal of Bacteriology 188, no. 11 (June 1, 2006): 3995–4006. http://dx.doi.org/10.1128/jb.00053-06.
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ABSTRACT When grown in divalent cation-limited medium, Pseudomonas aeruginosa becomes resistant to cationic antimicrobial peptides and polymyxin B. This resistance is regulated by the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems. To further characterize Mg2+ regulation in P. aeruginosa, microarray transcriptional profiling was conducted to compare wild-type P. aeruginosa grown under Mg2+-limited and Mg2+-replete conditions to isogenic phoP and pmrA mutants grown under Mg2+-limited conditions. Under Mg2+-limited conditions (0.02 mM Mg2+), approximately 3% of the P. aeruginosa genes were differentially expressed compared to the expression in bacteria grown under Mg2+-replete conditions (2 mM Mg2+). Only a modest subset of the Mg2+-regulated genes were regulated through either PhoP or PmrA. To determine which genes were directly regulated, a bioinformatic search for conserved binding motifs was combined with confirmatory reverse transcriptase PCR and gel shift promoter binding assays, and the results indicated that very few genes were directly regulated by these response regulators. It was found that in addition to the previously known oprH-phoP-phoQ operon and the pmrHFIJKLM-ugd operon, the PA0921 and PA1343 genes, encoding small basic proteins, were regulated by Mg2+ in a PhoP-dependent manner. The number of known PmrA-regulated genes was expanded to include the PA1559-PA1560, PA4782-PA4781, and feoAB operons, in addition to the previously known PA4773-PA4775-pmrAB and pmrHFIJKLM-ugd operons.
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Guina, Tina, Eugene C. Yi, Houle Wang, Murray Hackett, and Samuel I. Miller. "A PhoP-Regulated Outer Membrane Protease of Salmonella enterica Serovar Typhimurium Promotes Resistance to Alpha-Helical Antimicrobial Peptides." Journal of Bacteriology 182, no. 14 (July 15, 2000): 4077–86. http://dx.doi.org/10.1128/jb.182.14.4077-4086.2000.
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ABSTRACT The outer membrane protein contents of Salmonella enterica serovar Typhimurium strains with PhoP/PhoQ regulon mutations were compared by two-dimensional gel electrophoresis. At least 26 species of outer membrane proteins (OMPs) were identified as being regulated by PhoP/PhoQ activation. One PhoP/PhoQ-activated OMP was identified by semiautomated tandem mass spectrometry coupled with electronic database searching as PgtE, a member of theEscherichia coli OmpT and Yersinia pestis Pla family of outer membrane proteases. Salmonella PgtE expression promoted resistance to alpha-helical cationic antimicrobial peptides (α-CAMPs). Strains expressing PgtE cleaved C18G, an 18-residue α-CAMP present in culture medium, indicating that protease activity is likely to be the mechanism of OmpT-mediated resistance to α-CAMPs. PhoP/PhoQ did not regulate the transcription or export of PgtE, indicating that another PhoP/PhoQ-dependent mechanism is required for PgtE outer membrane localization. PgtE is a posttranscriptionally regulated component of the PhoP/PhoQ regulon that contributes toSalmonella resistance to innate immunity.
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Eguchi, Yoko, Tadashi Okada, Shu Minagawa, Taku Oshima, Hirotada Mori, Kaneyoshi Yamamoto, Akira Ishihama, and Ryutaro Utsumi. "Signal Transduction Cascade between EvgA/EvgS and PhoP/PhoQ Two-Component Systems of Escherichia coli." Journal of Bacteriology 186, no. 10 (May 15, 2004): 3006–14. http://dx.doi.org/10.1128/jb.186.10.3006-3014.2004.
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ABSTRACT Transcriptional analysis of a constitutively active mutant of the EvgA/EvgS two-component system of Escherichia coli resulted in enhanced expression of 13 PhoP/PhoQ-regulated genes, crcA, hemL, mgtA, ompT, phoP, phoQ, proP, rstA, rstB, slyB, ybjG, yrbL, and mgrB. This regulatory network between the two systems also occurred as a result of overproduction of the EvgA regulator; however, enhanced transcription of the phoPQ genes did not further activate expression of the PhoP/PhoQ-regulated genes. These results demonstrated signal transduction from the EvgA/EvgS system to the PhoP/PhoQ system in E. coli and also identified the genes that required the two systems for enhanced expression. This is one example of the intricate signal transduction networks that are posited to exist in E. coli.
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van Velkinburgh, Jennifer C., and John S. Gunn. "PhoP-PhoQ-Regulated Loci Are Required for Enhanced Bile Resistance in Salmonella spp." Infection and Immunity 67, no. 4 (April 1, 1999): 1614–22. http://dx.doi.org/10.1128/iai.67.4.1614-1622.1999.
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ABSTRACT As enteric pathogens, Salmonella spp. are resistant to the actions of bile. Salmonella typhimurium andSalmonella typhi strains were examined to better define the bile resistance phenotype. The MICs of bile for wild-typeS. typhimurium and S. typhi were 18 and 12%, respectively, and pretreatment of log-phase S. typhimurium with 15% bile dramatically increased bile resistance. Mutant strains of S. typhimurium andS. typhi lacking the virulence regulator PhoP-PhoQ were killed at significantly lower bile concentrations than wild-type strains, while strains with constitutively active PhoP were able to survive prolonged incubation with bile at concentrations of >60%. PhoP-PhoQ was shown to mediate resistance specifically to the bile components deoxycholate and conjugated forms of chenodeoxycholate, and the protective effect was not generalized to other membrane-active agents. Growth of both S. typhimurium and S. typhi in bile and in deoxycholate resulted in the induction or repression of a number of proteins, many of which appeared identical to PhoP-PhoQ-activated or -repressed products. The PhoP-PhoQ regulon was not induced by bile, nor did any of the 21 PhoP-activated or -repressed genes tested play a role in bile resistance. However, of the PhoP-activated or -repressed genes tested, two (prgC andprgH) were transcriptionally repressed by bile in the medium independent of PhoP-PhoQ. These data suggest that salmonellae can sense and respond to bile to increase resistance and that this response likely includes proteins that are members of the PhoP regulon. These bile- and PhoP-PhoQ-regulated products may play an important role in the survival of Salmonella spp. in the intestine or gallbladder.
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Gonzalo-Asensio, Jesús, Carlos Y. Soto, Ainhoa Arbués, Javier Sancho, María del Carmen Menéndez, María J. García, Brigitte Gicquel, and Carlos Martín. "The Mycobacterium tuberculosis phoPR Operon Is Positively Autoregulated in the Virulent Strain H37Rv." Journal of Bacteriology 190, no. 21 (August 29, 2008): 7068–78. http://dx.doi.org/10.1128/jb.00712-08.
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ABSTRACT The attenuated Mycobacterium tuberculosis H37Ra strain is an isogenic counterpart of the virulent paradigm strain H37Rv. Recently, a link between a point mutation in the PhoP transcriptional regulator and avirulence of H37Ra was established. Remarkably, a previous study demonstrated negative autoregulation of the phoP gene in H37Ra. These findings led us to study the transcriptional autoregulation of PhoP in the virulent H37Rv strain. In contrast to the negative autoregulation of PhoP previously published for H37Ra, our experiments using a phoP promoter-lacZ fusion showed that PhoP is positively autoregulated in both H37Rv and H37Ra compared with an H37Rv phoP deletion mutant constructed in this study. Using quantitative reverse transcription-PCR (RT-PCR) analysis, we showed that the phoP gene is transcribed at similar levels in H37Rv and H37Ra. Gel mobility shift and DNase I footprinting assays allowed us to identify the precise binding region of PhoP from H37Rv to the phoP promoter. We also carried out RT-PCR studies to demonstrate that phoP is transcribed together with the adjacent gene phoR, which codes for the cognate histidine kinase of the phoPR two-component system. In addition, quantitative RT-PCR studies showed that phoR is independently transcribed from a promoter possibly regulated by PhoP. Finally, we discuss the possible role in virulence of a single point mutation found in the phoP gene from the attenuated H37Ra strain but not in virulent members of the M. tuberculosis complex.
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Grabenstein, Jens P., Hana S. Fukuto, Lance E. Palmer, and James B. Bliska. "Characterization of Phagosome Trafficking and Identification of PhoP-Regulated Genes Important for Survival of Yersinia pestis in Macrophages." Infection and Immunity 74, no. 7 (July 2006): 3727–41. http://dx.doi.org/10.1128/iai.00255-06.
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ABSTRACT The transcriptional activator PhoP is important for survival of Yersinia pestis in macrophage phagosomes. However, the phagosomes inhabited by Y. pestis have not been well characterized, and the mechanism by which PhoP promotes bacterial survival in these vacuoles is not fully understood. Lysosomal tracers, as well as antibodies to late endosomal or lysosomal proteins, were used in conjunction with confocal or electron microscopy to study the trafficking of phagosomes containing phoP + or phoP mutant Y. pestis strains or latex beads in J774A.1 macrophages. Phagosomes containing phoP + or phoP mutant Y. pestis acquired lysosomal markers to the same degree that phagosomes containing latex beads acquired these markers after 1.5 h of infection, showing that nascent phagosomes containing Y. pestis fuse with lysosomes irrespective of the phoP genotype. Similar results were obtained when phagosomes containing viable or dead phoP + Y. pestis cells or beads were analyzed at 8 h postinfection, indicating that the Y. pestis vacuole does not become secluded from the lysosomal compartment. However, only viable phoP + bacteria induced the formation of spacious phagosomes at 8 h postinfection, suggesting that Y. pestis can actively direct the expansion of its vacuole. PhoP-regulated genes that are important for survival of Y. pestis in phagosomes were identified by Tn5-lacZ mutagenesis and oligonucleotide microarray analysis. Three such genes were identified, and the products of these genes are predicted to promote resistance to antimicrobial peptides (ugd and pmrK) or low-Mg2+ conditions (mgtC) found in phagosomes. Viable count assays carried out with Y. pestis ugd, mgtC, and ugd mgtC mutants revealed that the products of ugd and mgtC function independently to promote early survival of Y. pestis in macrophage phagosomes.
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Chen, Yinghua, Catherine Birck, Jean-Pierre Samama, and F. Marion Hulett. "Residue R113 Is Essential for PhoP Dimerization and Function: a Residue Buried in the Asymmetric PhoP Dimer Interface Determined in the PhoPN Three-Dimensional Crystal Structure." Journal of Bacteriology 185, no. 1 (January 1, 2003): 262–73. http://dx.doi.org/10.1128/jb.185.1.262-273.2003.
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ABSTRACT Bacillus subtilis PhoP is a member of the OmpR/PhoB family of response regulators that is directly required for transcriptional activation or repression of Pho regulon genes in conditions under which Pi is growth limiting. Characterization of the PhoP protein has established that phosphorylation of the protein is not essential for PhoP dimerization or DNA binding but is essential for transcriptional regulation of Pho regulon genes. DNA footprinting studies of PhoP-regulated promoters showed that there was cooperative binding between PhoP dimers at PhoP-activated promoters and/or extensive PhoP oligomerization 3′ of PhoP-binding consensus repeats in PhoP-repressed promoters. The crystal structure of PhoPN described in the accompanying paper revealed that the dimer interface between two PhoP monomers involves nonidentical surfaces such that each monomer in a dimer retains a second surface that is available for further oligomerization. A salt bridge between R113 on one monomer and D60 on another monomer was judged to be of major importance in the protein-protein interaction. We describe the consequences of mutation of the PhoP R113 codon to a glutamate or alanine codon and mutation of the PhoP D60 codon to a lysine codon. In vivo expression of either PhoPR113E, PhoPR113A, or PhoPD60K resulted in a Pho-negative phenotype. In vitro analysis showed that PhoPR113E was phosphorylated by PhoR (the cognate histidine kinase) but was unable to dimerize. Monomeric PhoPR113E∼P was deficient in DNA binding, contributing to the PhoPR113E in vivo Pho-negative phenotype. While previous studies emphasized that phosphorylation was essential for PhoP function, data reported here indicate that phosphorylation is not sufficient as PhoP dimerization or oligomerization is also essential. Our data support the physiological relevance of the residues of the asymmetric dimer interface in PhoP dimerization and function.
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Yang, Baopeng, Chang Liu, Xiaolei Pan, Weixin Fu, Zheng Fan, Yongxin Jin, Fang Bai, Zhihui Cheng, and Weihui Wu. "Identification of Novel phoP-phoQ Regulated Genes that Contribute to Polymyxin B Tolerance in Pseudomonas aeruginosa." Microorganisms 9, no. 2 (February 9, 2021): 344. http://dx.doi.org/10.3390/microorganisms9020344.
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Polymyxin B and E (colistin) are the last resorts to treat multidrug-resistant Gram-negative pathogens. Pseudomonas aeruginosa is intrinsically resistant to a variety of antibiotics. The PhoP-PhoQ two-component regulatory system contributes to the resistance to polymyxins by regulating an arnBCADTEF-pmrE operon that encodes lipopolysaccharide modification enzymes. To identify additional PhoP-regulated genes that contribute to the tolerance to polymyxin B, we performed a chromatin immunoprecipitation sequencing (ChIP-Seq) assay and found novel PhoP binding sites on the chromosome. We further verified that PhoP directly controls the expression of PA14_46900, PA14_50740 and PA14_52340, and the operons of PA14_11970-PA14_11960 and PA14_52350-PA14_52370. Our results demonstrated that mutation of PA14_46900 increased the bacterial binding and susceptibility to polymyxin B. Meanwhile, mutation of PA14_11960 (papP), PA14_11970 (mpl), PA14_50740 (slyB), PA14_52350 (ppgS), and PA14_52370 (ppgH) reduced the bacterial survival rates and increased ethidium bromide influx under polymyxin B or Sodium dodecyl sulfate (SDS) treatment, indicating roles of these genes in maintaining membrane integrity in response to the stresses. By 1-N-phenylnaphthylamine (NPN) and propidium iodide (PI) staining assay, we found that papP and slyB are involved in maintaining outer membrane integrity, and mpl and ppgS-ppgH are involved in maintaining inner membrane integrity. Overall, our results reveal novel PhoP-PhoQ regulated genes that contribute to polymyxin B tolerance.
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Fiedler, Tomas, Maren Mix, Uta Meyer, Stefan Mikkat, Michael O. Glocker, Hubert Bahl, and Ralf-Jörg Fischer. "The Two-Component System PhoPR of Clostridium acetobutylicum Is Involved in Phosphate-Dependent Gene Regulation." Journal of Bacteriology 190, no. 20 (August 8, 2008): 6559–67. http://dx.doi.org/10.1128/jb.00574-08.
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ABSTRACT The phoPR gene locus of Clostridium acetobutylicum ATCC 824 comprises two genes, phoP and phoR. Deduced proteins are predicted to represent a response regulator and sensor kinase of a phosphate-dependent two-component regulatory system. We analyzed the expression patterns of phoPR in Pi-limited chemostat cultures and in response to Pi pulses. A basic transcription level under high-phosphate conditions was shown, and a significant increase in mRNA transcript levels was found when external Pi concentrations dropped below 0.3 mM. In two-dimensional gel electrophoresis experiments, a 2.5-fold increase in PhoP was observed under Pi-limiting growth conditions compared to growth with an excess of Pi. At least three different transcription start points for phoP were determined by primer extension analyses. Proteins PhoP and an N-terminally truncated *PhoR were individually expressed heterologously in Escherichia coli and purified. Autophosphorylation of *PhoR and phosphorylation of PhoP were shown in vitro. Electromobility shift assays proved that there was a specific binding of PhoP to the promoter region of the phosphate-regulated pst operon of C. acetobutylicum.
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Gunn, John S., Robert K. Ernst, Andrea J. McCoy, and Samuel I. Miller. "Constitutive Mutations of the Salmonella entericaSerovar Typhimurium Transcriptional Virulence RegulatorphoP." Infection and Immunity 68, no. 6 (June 1, 2000): 3758–62. http://dx.doi.org/10.1128/iai.68.6.3758-3762.2000.
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ABSTRACT The PhoP-PhoQ two-component system is necessary for the virulence of Salmonella spp. and is responsible for regulating several modifications of the lipopolysaccharide (LPS). Mutagenesis of the transcriptional regulator phoP resulted in the identification of a mutant able to activate transcription of regulated genes ∼100-fold in the absence of PhoQ. Sequence analysis showed two single-base alterations resulting in amino acid changes at positions 93 (S93N) and 203 (Q203R). These mutations were individually created, and although each resulted in a constitutive phenotype, the double mutant displayed a synergistic effect both in the induction of PhoP-activated gene expression and in resistance to antimicrobial peptides. The constitutive phoP gene was placed under the control of an arabinose-inducible promoter to examine the kinetics of PhoP-activated gene induction and the resultant modifications of LPS. Gene induction and 2-hydroxymyristate modification of the lipid A were shown to occur within minutes of the addition of arabinose and to peak at 4 h. As the first constitutive mutant of phoP identified, this allele will be invaluable to future genetic and biochemical studies of this and likely other regulatory systems.
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Ghorbel, Sofiane, Jan Kormanec, Alexandra Artus, and Marie-Joelle Virolle. "Transcriptional Studies and Regulatory Interactions between the phoR-phoP Operon and the phoU, mtpA, and ppk Genes of Streptomyces lividans TK24." Journal of Bacteriology 188, no. 2 (January 15, 2006): 677–86. http://dx.doi.org/10.1128/jb.188.2.677-686.2006.
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ABSTRACT The PhoR/PhoP two-component system of Streptomyces lividans was previously shown to allow the growth of the bacteria at low Pi concentrations and to negatively control antibiotic production. The present study focuses on the transcriptional analysis of phoR and phoP, along with the phoU and mtpA genes that are transcribed divergently from the phoRP operon in S. lividans. The effect of phoR, phoP, phoU, and ppk mutations on transcription of these genes was examined under phosphate-replete and phosphate-limited conditions. We demonstrated that phoR and phoP were cotranscribed as a leaderless bicistronic transcript cleaved at discrete sites toward the 3′ end of phoR. In addition, phoP could also be transcribed alone from a promoter located at the 3′ end of phoR. The phoU and mtpA genes, predicted to encode metal binding proteins, were shown to be transcribed as monocistronic transcripts. The expression of phoR-phoP, phoP, and phoU was found to be induced under conditions of Pi limitation in S. lividans TK24. This induction, requiring both PhoR and PhoP, was significantly weaker in the phoU mutant but much stronger in the ppk mutant than in the parental strain. The expression of mtpA was also shown to be up-regulated when Pi was limiting but independently of PhoR/PhoP. The induction of mtpA expression was much stronger in the phoU mutant strain than in the other strains. This study revealed interesting regulatory interactions between the different genes and allowed us to propose putative roles for PhoU and MtpA in the adaptation to phosphate scarcity.
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Yan, Qing, Wei Gao, Xiao-Gang Wu, and Li-Qun Zhang. "Regulation of the PcoI/PcoR quorum-sensing system in Pseudomonas fluorescens 2P24 by the PhoP/PhoQ two-component system." Microbiology 155, no. 1 (January 1, 2009): 124–33. http://dx.doi.org/10.1099/mic.0.020750-0.
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A quorum-sensing locus, pcoI/pcoR, which is involved in the regulation of root colonization and plant disease-suppressive ability, was previously identified in Pseudomonas fluorescens 2P24. In this study, we performed random mutagenesis using mini-Tn5 in order to screen the upstream transcriptional regulators of pcoI, a biosynthase gene responsible for the synthesis of N-acylhomoserine lactone signal molecules. Two mutants, PM400 and PM410, with elevated pcoI gene promoter activity, were identified from ∼10 000 insertion clones. The amino acid sequences of the interrupted genes in these two mutants were highly similar to PhoQ, a sensor protein of the two-component regulatory system PhoP/PhoQ, which responds to environmental Mg2+ starvation and regulates virulence in Salmonella typhimurium and antimicrobial peptide resistance in Pseudomonas aeruginosa. The promoter activity of pcoI was also induced under low-Mg2+ conditions in the 2P24 strain of P. fluorescens. Deletion mutagenesis and complementation experiments demonstrated that the transcription of pcoI was negatively regulated by the sensor PhoQ but positively regulated by the response regulator PhoP. Genetic evidence also indicated that transcription of the outer-membrane protein gene oprH was induced by Mg2+ starvation through regulation of the wild-type PhoP/PhoQ system. Additionally, PhoQ was involved in biofilm formation by 2P24 under low-Mg2+ conditions through a PhoP-independent pathway.
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Choi, Jeongjoon, and Eduardo A. Groisman. "Horizontally acquired regulatory gene activates ancestral regulatory system to promote Salmonella virulence." Nucleic Acids Research 48, no. 19 (October 12, 2020): 10832–47. http://dx.doi.org/10.1093/nar/gkaa813.
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Abstract Horizontally acquired genes are typically regulated by ancestral regulators. This regulation enables expression of horizontally acquired genes to be coordinated with that of preexisting genes. Here, we report a singular example of the opposite regulation: a horizontally acquired gene that controls an ancestral regulator, thereby promoting bacterial virulence. We establish that the horizontally acquired regulatory gene ssrB is necessary to activate the ancestral regulatory system PhoP/PhoQ of Salmonella enterica serovar Typhimurium (S. Typhimurium) in mildly acidic pH, which S. Typhimurium experiences inside macrophages. SsrB promotes phoP transcription by binding upstream of the phoP promoter. SsrB also increases ugtL transcription by binding to the ugtL promoter region, where it overcomes gene silencing by the heat-stable nucleoid structuring protein H-NS, enhancing virulence. The largely non-pathogenic species S. bongori failed to activate PhoP/PhoQ in mildly acidic pH because it lacks both the ssrB gene and the SsrB binding site in the target promoter. Low Mg2+ activated PhoP/PhoQ in both S. bongori and ssrB-lacking S. Typhimurium, indicating that the SsrB requirement for PhoP/PhoQ activation is signal-dependent. By controlling the ancestral genome, horizontally acquired genes are responsible for more crucial abilities, including virulence, than currently thought.
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Martin-Orozco, Natalia, Nicolas Touret, Michael L. Zaharik, Edwin Park, Raoul Kopelman, Samuel Miller, B. Brett Finlay, Philippe Gros, and Sergio Grinstein. "Visualization of Vacuolar Acidification-induced Transcription of Genes of Pathogens inside Macrophages." Molecular Biology of the Cell 17, no. 1 (January 2006): 498–510. http://dx.doi.org/10.1091/mbc.e04-12-1096.
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The objective of these studies was to analyze the role of the ionic environment of phagosomal vacuoles in the control of pathogens by macrophages. Digital imaging and flow cytometry were used to follow the induction of the phoP promoter of Salmonella enterica Typhimurium within live macrophages. Manipulating the Mg2+concentration within the Salmonella-containing vacuole (SCV) was without effect on the early induction of PhoPQ. Moreover, direct measurement of [Mg2+] within the SCV using nanosensor particles showed that, during this initial period of phoP activation, the concentration of the divalent cation is rapidly regulated and stabilizes around 1 mm. Extrusion of other divalent cations via the Nramp1 efflux pump was similarly ruled out as an important contributor to the activation of the regulon. By contrast, induction of PhoP was greatly attenuated when the pH gradient across the SCV membrane was dissipated. A second, more modest pH-independent component of PhoP induction was unmasked by inhibition of the vacuolar proton pump. This second component was eliminated by pretreatment of cells with IFNγ, even though the cytokine augmented the overall PhoP response. These findings demonstrate the existence of at least three separate activators of phoP transcription: resting and IFNγ-stimulated pH-sensitive components, plus a pH-independent component.
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Sanowar, Sarah, and Hervé Le Moual. "Functional reconstitution of the Salmonella typhimurium PhoQ histidine kinase sensor in proteoliposomes." Biochemical Journal 390, no. 3 (September 5, 2005): 769–76. http://dx.doi.org/10.1042/bj20050060.
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Two-component signal-transduction systems are widespread in bacteria. They are usually composed of a transmembrane histidine kinase sensor and a cytoplasmic response regulator. The PhoP/PhoQ two-component system of Salmonella typhimurium contributes to virulence by co-ordinating the adaptation to low concentrations of environmental Mg2+. Limiting concentrations of extracellular Mg2+ activate the PhoP/PhoQ phosphorylation cascade modulating the transcription of PhoP-regulated genes. In contrast, high concentrations of extracellular Mg2+ stimulate the dephosphorylation of the response regulator PhoP by the PhoQ kinase sensor. In the present study, we report the purification and functional reconstitution of PhoQHis, a PhoQ variant with a C-terminal His tag, into Escherichia coli liposomes. The functionality of PhoQHis was essentially similar to that of PhoQ as shown in vivo and in vitro. Purified PhoQHis was inserted into liposomes in a unidirectional orientation, with the sensory domain facing the lumen and the catalytic domain facing the extraluminal environment. Reconstituted PhoQHis exhibited all the catalytic activities that have been described for histidine kinase sensors. Reconstituted PhoQHis was capable of autokinase activity when incubated in the presence of Mg2+-ATP. The phosphoryl group could be transferred from reconstituted PhoQHis to PhoP. Reconstituted PhoQHis catalysed the dephosphorylation of phospho-PhoP and this activity was stimulated by the addition of extraluminal ADP.
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Lin, Quei Yen, Yi-Lin Tsai, Ming-Che Liu, Wei-Cheng Lin, Po-Ren Hsueh, and Shwu-Jen Liaw. "Serratia marcescensarn, a PhoP-Regulated Locus Necessary for Polymyxin B Resistance." Antimicrobial Agents and Chemotherapy 58, no. 9 (June 23, 2014): 5181–90. http://dx.doi.org/10.1128/aac.00013-14.
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ABSTRACTPolymyxins, which are increasingly being used to treat infections caused by multidrug-resistant bacteria, perform poorly againstSerratia marcescens. To investigate the underlying mechanisms, Tn5mutagenesis was performed and two mutants exhibiting increased polymyxin B (PB) susceptibility were isolated. The mutants were found to have Tn5inserted into thearnBandarnCgenes. In other bacteria,arnBandarnCbelong to the seven-genearnoperon, which is involved in lipopolysaccharide (LPS) modification. LPSs ofarnmutants had greater PB-binding abilities than that of wild-type LPS. Further, we identified PhoP, a bacterial two-component response regulator, as a regulator of PB susceptibility inS. marcescens. By the reporter assay, we found PB- and low-Mg2+-induced expression ofphoPandarnin the wild-type strain but not in thephoPmutant. Complementation of thephoPmutant with the full-lengthphoPgene restored the PB MIC and induction by PB and low Mg2+levels, as in the wild type. An electrophoretic mobility shift assay (EMSA) further demonstrated that PhoP bound directly to thearnpromoter. The PB challenge test confirmed that pretreatment with PB and low Mg2+levels protectedS. marcescensfrom a PB challenge in the wild-type strain but not in thephoPmutant. Real-time reverse transcriptase-PCR also indicated that PB serves as a signal to regulate expression ofugd, a gene required for LPS modification, inS. marcescensthrough a PhoP-dependent pathway. Finally, we found that PB-resistant clinical isolates displayed greater expression ofarnAupon exposure to PB than did susceptible isolates. This is the first report to describe the role ofS. marcescensarnin PB resistance and its modulation by PB and Mg2+through the PhoP protein.
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Minagawa, Shu, Hiroshi Ogasawara, Akinori Kato, Kaneyoshi Yamamoto, Yoko Eguchi, Taku Oshima, Hirotada Mori, Akira Ishihama, and Ryutaro Utsumi. "Identification and Molecular Characterization of the Mg2+ Stimulon of Escherichia coli." Journal of Bacteriology 185, no. 13 (July 1, 2003): 3696–702. http://dx.doi.org/10.1128/jb.185.13.3696-3702.2003.
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ABSTRACT Transcription profile microarray analysis in Escherichia coli was performed to identify the member genes of the Mg2+ stimulon that respond to the availability of external Mg2+ in a PhoP/PhoQ two-component system-dependent manner. The mRNA levels of W3110 in the presence of 30 mM MgCl2, WP3022 (phoP defective), and WQ3007 (phoQ defective) were compared with those of W3110 in the absence of MgCl2. The expression ratios of a total of 232 genes were <0.75 in all three strains (the supplemental data are shown at http://www.nara.kindai.ac.jp/nogei/seiken/array.html ), suggesting that the PhoP/PhoQ system is involved directly or indirectly in the transcription of these genes. Of those, 26 contained the PhoP box-like sequences with the direct repeats of (T/G)GTTTA within 500 bp upstream of the initiation codon. Furthermore, S1 nuclease assays of 26 promoters were performed to verify six new Mg2+ stimulon genes, hemL, nagA, rstAB, slyB, vboR, and yrbL, in addition to the phoPQ, mgrB, and mgtA genes reported previously. In gel shift and DNase I footprinting assays, all of these genes were found to be regulated directly by PhoP. Thus, we concluded that the phoPQ, mgrB, mgtA, hemL, nagA, rstAB, slyB, vboR, and yrbL genes make up the Mg2+ stimulon in E. coli.
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Newcombe, J., J. C. Jeynes, E. Mendoza, J. Hinds, G. L. Marsden, R. A. Stabler, M. Marti, and J. J. McFadden. "Phenotypic and Transcriptional Characterization of the Meningococcal PhoPQ System, a Magnesium-Sensing Two-Component Regulatory System That Controls Genes Involved in Remodeling the Meningococcal Cell Surface." Journal of Bacteriology 187, no. 14 (July 2005): 4967–75. http://dx.doi.org/10.1128/jb.187.14.4967-4975.2005.
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ABSTRACT We previously identified and characterized a two-component regulatory system in the meningococcus with homology to the phoP-phoQ system in salmonella and showed that allele replacement of the NMB0595 regulator gene led to loss of virulence, sensitivity to antimicrobial peptides, perturbed protein expression, and magnesium-sensitive growth. On the basis of these findings we proposed that the system should be designated the meningococcal PhoPQ system. Here we further characterized the NMB0595 mutant and demonstrated that it had increased membrane permeability and was unable to form colonies on solid media with low magnesium concentrations, features that are consistent with disruption of PhoPQ-mediated modifications to the lipooligosaccharide structure. We examined the transcriptional profiles of wild-type and NMB0595 mutant strains and found that magnesium-regulated changes in gene expression are completely abrogated in the mutant, indicating that, similar to the salmonella PhoPQ system, the meningococcal PhoPQ system is regulated by magnesium. Transcriptional profiling of the mutant indicated that, also similar to the salmonella PhoPQ system, the meningococcal system is involved in control of virulence and remodeling of the bacterial cell surface in response to the host environment. The results are consistent with the hypothesis that the PhoP homologue plays a role in the meningococcus similar to the role played by PhoP in salmonella. Elucidating the role that the PhoPQ system and PhoPQ-regulated genes play in the response of the meningococcus to the host environment may provide new insights into the pathogenic process.
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Tierrez, Alberto, and Francisco García-del Portillo. "The Salmonella Membrane Protein IgaA Modulates the Activity of the RcsC-YojN-RcsB and PhoP-PhoQ Regulons." Journal of Bacteriology 186, no. 22 (November 15, 2004): 7481–89. http://dx.doi.org/10.1128/jb.186.22.7481-7489.2004.
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ABSTRACT The Salmonella enterica serovar Typhimurium membrane protein IgaA and the PhoP-PhoQ two-component system are used by this pathogen to attenuate the intracellular growth rate within fibroblasts. IgaA has also recently been shown to contribute to virulence by exerting tight repression of the RcsC-YojN-RcsB phosphorelay in host tissues. Here we show that loss of repression of the RcsC-YojN-RcsB system, linked to an R188H mutation in the IgaA protein (igaA1 allele), is accompanied by altered expression of PhoP-PhoQ-activated (pag) genes. The changes in gene expression were different depending on the specific pag gene analyzed. Thus, transcription of ugd, which is required for lipopolysaccharide modification and colanic acid capsule synthesis, was enhanced in the igaA1 mutant. RcsB and its coregulator RcsA promoted this alteration in a PhoP-PmrA-independent manner. Unlike ugd, activation of the RcsC-YojN-RcsB phosphorelay negatively affected the expression of all other pag genes tested. In this case, RcsB alone was responsible for this effect. We also found that PhoP, but not PmrA, negatively modulates the expression of gmm, a gene required for colanic acid synthesis that is regulated positively by RcsC-YojN-RcsB. Finally, it was observed that the fine regulation of pag genes exerted by RcsB requires the RpoS protein and that an active RcsB, but not RcsA, diminishes expression of the phoP gene. These data support the hypothesis that in Salmonella there is an intimate regulatory circuit between the PhoP-PhoQ and RcsC-YojN-RcsB phosphorelays, which is revealed only when the RcsC-YojN-RcsB signaling route is derepressed. Consistent with the phenotypes observed in fibroblast cells, IgaA is predicted to favor expression of the entire PhoP-PhoQ regulon based on its repression of the RcsC-YojN-RcsB phosphorelay.
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Prágai, Zoltán, Nicholas E. E. Allenby, Nicola O'Connor, Sarah Dubrac, Georges Rapoport, Tarek Msadek, and Colin R. Harwood. "Transcriptional Regulation of the phoPR Operon in Bacillus subtilis." Journal of Bacteriology 186, no. 4 (February 15, 2004): 1182–90. http://dx.doi.org/10.1128/jb.186.4.1182-1190.2004.
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ABSTRACT When Bacillus subtilis is subjected to phosphate starvation, the Pho regulon is activated by the PhoP-PhoR two-component signal transduction system to elicit specific responses to this nutrient limitation. The response regulator, PhoP, and its cognate histidine sensor kinase, PhoR, are encoded by the phoPR operon that is transcribed as a 2.7-kb bicistronic mRNA. The phoPR operon is transcribed from two σA-dependent promoters, P1 and P2. Under conditions where the Pho regulon was not induced (i.e., phosphate-replete conditions or phoR-null mutant), a low level of phoPR transcription was detected only from promoter P1. During phosphate starvation-induced transition from exponential to stationary phase, the expression of the phoPR operon was up-regulated in a phosphorylated PhoP (PhoP∼P)-dependent manner; in addition to P1, the P2 promoter becomes active. In vitro gel shift assays and DNase I footprinting experiments showed that both PhoP and PhoP∼P could bind to the control region of the phoPR operon. The data indicate that while low-level constitutive expression of phoPR is required under phosphate-replete conditions for signal perception and transduction, autoinduction is required to provide sufficient PhoP∼P to induce other members of the Pho regulon. The extent to which promoters P1 and P2 are activated appears to be influenced by the presence of other sigma factors, possibly the result of sigma factor competition. For example, phoPR is hyperinduced in a sigB mutant and, later in stationary phase, in sigH, sigF, and sigE mutants. The data point to a complex regulatory network in which other stress responses and post-exponential-phase processes influence the expression of phoPR and, thereby, the magnitude of the Pho regulon response.
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Wei, Chudan, Tian Ding, Changqing Chang, Chengpeng Yu, Xingwei Li, and Qiongguang Liu. "Global Regulator PhoP is Necessary for Motility, Biofilm Formation, Exoenzyme Production, and Virulence of Xanthomonas citri Subsp. citri on Citrus Plants." Genes 10, no. 5 (May 6, 2019): 340. http://dx.doi.org/10.3390/genes10050340.
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Citrus canker caused by Xanthomonas citri subsp. citri is one of the most important bacterial diseases of citrus, impacting both plant growth and fruit quality. Identifying and elucidating the roles of genes associated with pathogenesis has aided our understanding of the molecular basis of citrus-bacteria interactions. However, the complex virulence mechanisms of X. citri subsp. citri are still not well understood. In this study, we characterized the role of PhoP in X. citri subsp. citri using a phoP deletion mutant, ΔphoP. Compared with wild-type strain XHG3, ΔphoP showed reduced motility, biofilm formation, as well as decreased production of cellulase, amylase, and polygalacturonase. In addition, the virulence of ΔphoP on citrus leaves was significantly decreased. To further understand the virulence mechanisms of X. citri subsp. citri, high-throughput RNA sequencing technology (RNA-Seq) was used to compare the transcriptomes of the wild-type and mutant strains. Analysis revealed 1017 differentially-expressed genes (DEGs), of which 614 were up-regulated and 403 were down-regulated in ΔphoP. Gene ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses suggested that the DEGs were enriched in flagellar assembly, two-component systems, histidine metabolism, bacterial chemotaxis, ABC transporters, and bacterial secretion systems. Our results showed that PhoP activates the expression of a large set of virulence genes, including 22 type III secretion system genes and 15 type III secretion system effector genes, as well as several genes involved in chemotaxis, and flagellar and histidine biosynthesis. Two-step reverse-transcription polymerase chain reaction analysis targeting 17 genes was used to validate the RNA-seq data, and confirmed that the expression of all 17 genes, except for that of virB1, decreased significantly. Our results suggest that PhoP interacts with a global signaling network to co-ordinate the expression of multiple virulence factors involved in modification and adaption to the host environment during infection.
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Baker, Sarah J., Craig Daniels, and Renato Morona. "PhoP/Q regulated genes inSalmonella typhi: identification of melittin sensitive mutants." Microbial Pathogenesis 22, no. 3 (March 1997): 165–79. http://dx.doi.org/10.1006/mpat.1996.0099.
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Derzelle, Sylviane, Evelyne Turlin, Eric Duchaud, Sylvie Pages, Frank Kunst, Alain Givaudan, and Antoine Danchin. "The PhoP-PhoQ Two-Component Regulatory System of Photorhabdus luminescens Is Essential for Virulence in Insects." Journal of Bacteriology 186, no. 5 (March 1, 2004): 1270–79. http://dx.doi.org/10.1128/jb.186.5.1270-1279.2004.
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ABSTRACT Photorhabdus luminescens is a symbiont of entomopathogenic nematodes. Analysis of the genome sequence of this organism revealed a homologue of PhoP-PhoQ, a two-component system associated with virulence in intracellular bacterial pathogens. This organism was shown to respond to the availability of environmental magnesium. A mutant with a knockout mutation in the regulatory component of this system (phoP) had no obvious growth defect. It was, however, more motile and more sensitive to antimicrobial peptides than its wild-type parent. Remarkably, the mutation eliminated virulence in an insect model. No insect mortality was observed after injection of a large number of the phoP bacteria, while very small amounts of parental cells killed insect larvae in less than 48 h. At the molecular level, the PhoPQ system mediated Mg2+-dependent modifications in lipopolysaccharides and controlled a locus (pbgPE) required for incorporation of 4-aminoarabinose into lipid A. Mg2+-regulated gene expression of pbgP1 was absent in the mutant and was restored when phoPQ was complemented in trans. This finding highlights the essential role played by PhoPQ in the virulence of an entomopathogen.
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Núñez-Hernández, Cristina, Alberto Tierrez, Álvaro D. Ortega, M. Graciela Pucciarelli, Marta Godoy, Blanca Eisman, Josep Casadesús, and Francisco García-del Portillo. "Genome Expression Analysis of Nonproliferating Intracellular Salmonella enterica Serovar Typhimurium Unravels an Acid pH-Dependent PhoP-PhoQ Response Essential for Dormancy." Infection and Immunity 81, no. 1 (October 22, 2012): 154–65. http://dx.doi.org/10.1128/iai.01080-12.
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Genome-wide expression analyses have provided clues on howSalmonellaproliferates inside cultured macrophages and epithelial cells. However,in vivostudies show thatSalmonelladoes not replicate massively within host cells, leaving the underlying mechanisms of such growth control largely undefined.In vitroinfection models based on fibroblasts or dendritic cells reveal limited proliferation of the pathogen, but it is presently unknown whether these phenomena reflect events occurringin vivo. Fibroblasts are distinctive, since they represent a nonphagocytic cell type in whichS. entericaserovar Typhimurium actively attenuates intracellular growth. Here, we show in the mouse model thatS. Typhimurium restrains intracellular growth within nonphagocytic cells positioned in the intestinal lamina propria. This response requires a functional PhoP-PhoQ system and is reproduced in primary fibroblasts isolated from the mouse intestine. The fibroblast infection model was exploited to generate transcriptome data, which revealed that ∼2% (98 genes) of theS. Typhimurium genome is differentially expressed in nongrowing intracellular bacteria. Changes include metabolic reprogramming to microaerophilic conditions, induction of virulence plasmid genes, upregulation of the pathogenicity islands SPI-1 and SPI-2, and shutdown of flagella production and chemotaxis. Comparison of relative protein levels of several PhoP-PhoQ-regulated functions (PagN, PagP, and VirK) in nongrowing intracellular bacteria and extracellular bacteria exposed to diverse PhoP-PhoQ-inducing signals denoted a regulation responding to acidic pH. These data demonstrate thatS. Typhimurium restrains intracellular growthin vivoand support a model in which dormant intracellular bacteria could sense vacuolar acidification to stimulate the PhoP-PhoQ system for preventing intracellular overgrowth.
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Lee, Sang-Won, Kyu-Sik Jeong, Sang-Wook Han, Seung-Eun Lee, Bong-Kwan Phee, Tae-Ryong Hahn, and Pamela Ronald. "The Xanthomonas oryzae pv. oryzae PhoPQ Two-Component System Is Required for AvrXA21 Activity, hrpG Expression, and Virulence." Journal of Bacteriology 190, no. 6 (January 18, 2008): 2183–97. http://dx.doi.org/10.1128/jb.01406-07.
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ABSTRACT The rice pathogen recognition receptor, XA21, confers resistance to Xanthomonas oryzae pv. oryzae strains producing the type one system-secreted molecule, AvrXA21. X. oryzae pv. oryzae requires a regulatory two-component system (TCS) called RaxRH to regulate expression of eight rax (required for AvrXA21 activity) genes and to sense population cell density. To identify other key components in this critical regulatory circuit, we assayed proteins expressed in a raxR gene knockout strain. This survey led to the identification of the phoP gene encoding a response regulator that is up-regulated in the raxR knockout strain. Next we generated a phoP knockout strain and found it to be impaired in X. oryzae pv. oryzae virulence and no longer able to activate the response regulator HrpG (hypersensitive reaction and pathogenicity G) in response to low levels of Ca2+. The impaired virulence of the phoP knockout strain can be partially complemented by constitutive expression of hrpG, indicating that PhoP controls a key aspect of X. oryzae pv. oryzae virulence through regulation of hrpG. A gene encoding the cognate putative histidine protein kinase, phoQ, was also isolated. Growth curve analysis revealed that AvrXA21 activity is impaired in a phoQ knockout strain as reflected by enhanced growth of this strain in rice lines carrying XA21. These results suggest that the X. oryzae pv. oryzae PhoPQ TCS functions in virulence and in the production of AvrXA21 in partnership with RaxRH.
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Murata, Takeshi, Will Tseng, Tina Guina, Samuel I. Miller, and Hiroshi Nikaido. "PhoPQ-Mediated Regulation Produces a More Robust Permeability Barrier in the Outer Membrane of Salmonella enterica Serovar Typhimurium." Journal of Bacteriology 189, no. 20 (August 10, 2007): 7213–22. http://dx.doi.org/10.1128/jb.00973-07.
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ABSTRACT The PhoPQ two-component system of Salmonella enterica serovar Typhimurium produces a remodeling of the lipid A domain of the lipopolysaccharide, including the PagP-catalyzed addition of palmitoyl residue, the PmrAB-regulated addition of the cationic sugar 4-aminoarabinose and phosphoethanolamine, and the LpxO-catalyzed addition of a 2-OH group onto one of the fatty acids. By using the diffusion rates of the dyes ethidium, Nile red, and eosin Y across the outer membrane, as well as the susceptibility of cells to large, lipophilic agents, we evaluated the function of this membrane as a permeability barrier. We found that the remodeling process in PhoP-constitutive strains produces an outer membrane that serves as a very effective permeability barrier in an environment that is poor in divalent cations or that contains cationic peptides, whereas its absence in phoP null mutants produces an outer membrane severely compromised in its barrier function under these conditions. Removing combinations of the lipid A-remodeling functions from a PhoP-constitutive strain showed that the known modification reactions explain a major part of the PhoPQ-regulated changes in permeability. We believe that the increased barrier property of the remodeled bilayer is important in making the pathogen more resistant to the stresses that it encounters in the host, including attack by the cationic antimicrobial peptides. On the other hand, drug-induced killing assays suggest that the outer membrane containing unmodified lipid A may serve as a better barrier in the presence of high concentrations (e.g., 5 mM) of Mg2+.
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García-Calderón, Clara B., Josep Casadesús, and Francisco Ramos-Morales. "Rcs and PhoPQ Regulatory Overlap in the Control of Salmonella enterica Virulence." Journal of Bacteriology 189, no. 18 (July 6, 2007): 6635–44. http://dx.doi.org/10.1128/jb.00640-07.
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ABSTRACT Genetic screens based on the use of MudJ-generated lac fusions permitted the identification of novel genes regulated by the Rcs signal transduction system in Salmonella enterica serovar Typhimurium. Besides genes that are also found in the Escherichia coli genome, our screens identified Salmonella-specific genes regulated by RcsB, including bapA, siiE, srfA, and srfB. Here we show that the srfABC operon is negatively regulated by RcsB and by PhoP. In vivo studies using mutants with constitutive activation of the Rcs and/or PhoPQ system suggested that there is an overlap between these regulatory systems in the control of Salmonella virulence.
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Robichon, Denis, Maryvonne Arnaud, Rozenn Gardan, Zoltan Pragai, Mary O'Reilly, Georges Rapoport, and Michel Débarbouillé. "Expression of a New Operon from Bacillus subtilis, ykzB-ykoL, under the Control of the TnrA and PhoP-PhoR Global Regulators." Journal of Bacteriology 182, no. 5 (March 1, 2000): 1226–31. http://dx.doi.org/10.1128/jb.182.5.1226-1231.2000.
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ABSTRACT The ykzB and ykoL genes encode two peptides, of 51 and 60 amino acids, the functions of which are unknown. The ykzB and tnrA genes are contiguous and transcribed divergently. Expression of ykzB andykoL is induced by glutamate and is under the control of the TnrA global regulator of nitrogen utilization. TnrA regulated its own synthesis in glutamate minimal medium. Two DNA sequences (TnrAB1 and TnrAB2) homologous to the TnrA binding site are present in the region between tnrA and ykzB. Deletion mapping indicated that the TnrAB2 binding site was involved in activation of the ykzB promoter. In addition, transcription oftnrA depends on the presence of the TnrAB1 binding site. The ykzB and ykoL genes are probably in the same transcriptional unit. A single promoter involved in transcription in the presence of glutamate was mapped by primer extension.ykoL expression was induced by phosphate limitation and depended on the PhoP-PhoR two-component regulatory system. Its promoter was mapped to the region between ykoL and ykzB. Four boxes similar to the PhoP binding site are present upstream from the ykoL promoter. These boxes are probably recognized by PhoP∼P during the activation of transcription in phosphate limitation conditions.
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Bourret, Travis J., Miryoung Song, and Andrés Vázquez-Torres. "Codependent and Independent Effects of Nitric Oxide-Mediated Suppression of PhoPQ and Salmonella Pathogenicity Island 2 on Intracellular Salmonella enterica Serovar Typhimurium Survival." Infection and Immunity 77, no. 11 (September 8, 2009): 5107–15. http://dx.doi.org/10.1128/iai.00759-09.
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ABSTRACT Here we show that the Salmonella enterica serovar Typhimurium PhoQ sensor kinase lessens the cytotoxicity of reactive nitrogen species (RNS) generated by inducible nitric oxide synthase (iNOS) in the innate response of mononuclear phagocytic cells. This observation is consistent with the expression patterns of PhoP-activated genes during moderate nitrosative stress in the innate host response. In contrast, RNS synthesized during high-NO fluxes of gamma interferon (IFN-γ)-activated macrophages repress PhoP-activated lpxO, pagP, and phoP gene transcription. Because PhoP-regulated Salmonella pathogenicity island 2 (SPI2) genes are also repressed by high-order RNS (39), we investigated whether the NO-mediated inhibition of PhoPQ underlies the repression of SPI2. Our studies indicate that a third of the expression of the SPI2 spiC gene recorded in nonactivated macrophages depends on PhoQ. Transcription of spiC is repressed in IFN-γ-primed macrophages in an iNOS-dependent manner, irrespective of the phoQ status of the bacteria. Transcription of spiC is restored in IFN-γ-treated, iNOS-deficient macrophages to levels sustained by a phoQ mutant in nonactivated phagocytes, suggesting that most NO-dependent repression of spiC is due to the inhibition of PhoPQ-independent targets. Comparison of the intracellular fitness of spiC, phoQ, and spiC phoQ mutants revealed that PhoPQ and SPI2 have codependent and independent effects on S. Typhimurium survival during innate nitrosative stress. However, the intracellular survival of most S. Typhimurium bacteria is conferred by the PhoPQ two-component regulator, and the SPI2 type III secretion system is repressed by high-order RNS of IFN-γ-activated macrophages.
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Xu, Ya, Cheng-Heng Liao, Li-Li Yao, Xu Ye, and Bang-Ce Ye. "GlnR and PhoP Directly Regulate the Transcription of Genes Encoding Starch-Degrading, Amylolytic Enzymes in Saccharopolyspora erythraea." Applied and Environmental Microbiology 82, no. 23 (September 16, 2016): 6819–30. http://dx.doi.org/10.1128/aem.02117-16.
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ABSTRACTStarch-degrading enzymes hydrolyze starch- and starch-derived oligosaccharides to yield glucose. We investigated the transcriptional regulation of genes encoding starch-degrading enzymes in the industrial actinobacteriumSaccharopolyspora erythraea. We observed that most genes encoding amylolytic enzymes (one α-amylase, one glucoamylase, and four α-glucosidases) were regulated by GlnR and PhoP, which are global regulators of nitrogen and phosphate metabolism, respectively. Electrophoretic mobility shift assays and reverse transcription-PCR (RT-PCR) analyses demonstrated that GlnR and PhoP directly interact with their promoter regions and collaboratively or competitively activate their transcription. Deletion ofglnRcaused poor growth on starch, maltodextrin, and maltose, whereas overexpression ofglnRandphoPincreased the total activity of α-glucosidase, resulting in enhanced carbohydrate utilization. Additionally, transcript levels of amylolytic genes and total glucosidase activity were induced in response to nitrogen and phosphate limitation. Furthermore, regulatory effects of GlnR and PhoP on starch-degrading enzymes were conserved inStreptomyces coelicolorA3(2). These results demonstrate that GlnR and PhoP are involved in polysaccharide degradation by mediating the interplay among carbon, nitrogen, and phosphate metabolism in response to cellular nutritional states. Our study reveals a novel regulatory mechanism underlying carbohydrate metabolism, and suggests new possibilities for designing genetic engineering approaches to improve the rate of utilization of starch in actinobacteria.IMPORTANCEThe development of efficient strategies for utilization of biomass-derived sugars, such as starch and cellulose, remains a major technical challenge due to the weak activity of associated enzymes. Here, we found that GlnR and PhoP directly regulate the transcription of genes encoding amylolytic enzymes and present insights into the regulatory mechanisms of degradation and utilization of starch in actinobacteria. Two nutrient-sensing regulators may play important roles in creating a direct association between nitrogen/phosphate metabolisms and carbohydrate utilization, as well as modulate the C:N:P balance in response to cellular nutritional states. These findings highlight the interesting possibilities for designing genetic engineering approaches and optimizing the fermentation process to improve the utilization efficiency of sugars in actinobacteria via overexpression of theglnRandphoPgenes and nutrient signal stimulation.
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van Velkinburgh, Jennifer C., and John S. Gunn. "PhoP-PhoQ-Regulated Loci Are Required for Enhanced Bile Resistance in Salmonella spp." Infection and Immunity 67, no. 4 (1999): 1614–22. http://dx.doi.org/10.1128/.67.4.1614-1622.1999.
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Shi, Yixin, Michael J. Cromie, Fong-Fu Hsu, John Turk, and Eduardo A. Groisman. "PhoP-regulated Salmonella resistance to the antimicrobial peptides magainin 2 and polymyxin B." Molecular Microbiology 53, no. 1 (May 14, 2004): 229–41. http://dx.doi.org/10.1111/j.1365-2958.2004.04107.x.
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Golubeva, Yekaterina A., and James M. Slauch. "Salmonella enterica Serovar Typhimurium Periplasmic Superoxide Dismutase SodCI Is a Member of the PhoPQ Regulon and Is Induced in Macrophages." Journal of Bacteriology 188, no. 22 (September 15, 2006): 7853–61. http://dx.doi.org/10.1128/jb.00706-06.
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ABSTRACT Salmonella enterica serovar Typhimurium replicates within host macrophages during the systemic stage of infection. In the macrophage, the bacteria must survive the respiratory burst that produces superoxide. Serovar Typhimurium strain 14028 produces two periplasmic superoxide dismutases, SodCI and SodCII, but only SodCI contributes to virulence. Although we have shown that this is primarily due to differences in the two proteins, evidence suggests differential regulation of the two genes. Using transcriptional sodCI- and sodCII-lac fusions, we show that sodCII is under the control of the RpoS sigma factor, as was known for the Escherichia coli ortholog, sodC. In contrast, we show that sodCI is transcriptionally controlled by the PhoPQ two-component regulatory system, which regulates an array of virulence genes required for macrophage survival. Introduction of a phoP-null mutation into the sodCI fusion strain resulted in a decrease in transcription and loss of regulation. The sodCI-lac fusion showed high-level expression in a background containing a phoQ constitutive allele. The sodCI gene is induced 15-fold in bacteria recovered from either the tissue culture macrophages or the spleens of infected mice. Induction in macrophages is dependent on PhoP. The sodCII fusion was induced three- to fourfold in macrophages and animals; this induction was unaffected by loss of PhoP. Thus, sodCI, which is horizontally transferred by the Gifsy-2 phage, is regulated by PhoPQ such that it is induced at the appropriate time and place to combat phagocytic superoxide.
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Cordero-Alba, M., J. Bernal-Bayard, and F. Ramos-Morales. "SrfJ, a Salmonella Type III Secretion System Effector Regulated by PhoP, RcsB, and IolR." Journal of Bacteriology 194, no. 16 (June 1, 2012): 4226–36. http://dx.doi.org/10.1128/jb.00173-12.
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Makarewicz, Oliwia, Svetlana Neubauer, Corinna Preusse, and Rainer Borriss. "Transition State Regulator AbrB Inhibits Transcription of Bacillus amyloliquefaciens FZB45 Phytase through Binding at Two Distinct Sites Located within the Extended phyC Promoter Region." Journal of Bacteriology 190, no. 19 (August 1, 2008): 6467–74. http://dx.doi.org/10.1128/jb.00430-08.
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ABSTRACT We have previously identified the phyC gene of Bacillus amyloliquefaciens FZB45, encoding extracellular phytase, as a member of the PhoP regulon, which is expressed only during phosphate starvation. Its σA-dependent promoter is positively and negatively regulated by the phosphorylated PhoP response regulator in a phosphate-dependent manner (O. Makarewicz, S. Dubrac, T. Msadek, and R. Borriss, J. Bacteriol. 188:6953-6965, 2006). Here, we provide experimental evidence that the transcription of phyC underlies a second control mechanism exerted by the global transient-phase regulator protein, AbrB, which hinders its expression during exponential growth. Gel mobility shift and DNase I footprinting experiments demonstrated that AbrB binds to two different regions in the phyC promoter region that are separated by about 200 bp. One binding site is near the divergently orientated yodU gene, and the second site is located downstream of the phyC promoter and extends into the coding region of the phyC gene. Cooperative binding to the two distant binding regions is necessary for the AbrB-directed repression of phyC transcription. AbrB does not affect the transcription of the neighboring yodU gene.
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Prouty, Angela M., Jennifer C. Van Velkinburgh, and John S. Gunn. "Salmonella enterica Serovar Typhimurium Resistance to Bile: Identification and Characterization of the tolQRA Cluster." Journal of Bacteriology 184, no. 5 (March 1, 2002): 1270–76. http://dx.doi.org/10.1128/jb.184.5.1270-1276.2002.
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ABSTRACT Salmonella enterica serovar Typhimurium is resistant to the action of bile salts, and resistance to bile is enhanced in strains in which the PhoP-PhoQ (PhoPQ) two-component regulatory system has been activated. To identify genes necessary for bile resistance, MudJ transposon mutagenesis was performed on a strain containing a phoP mutation that results in constitutive expression of PhoP-activated genes. After screening >10,000 mutants for the loss of growth on Luria-Bertani broth-bile plates, 14 bile-sensitive mutants were identified. Of these 14 mutants, 3 were found to retain the bile sensitivity phenotype upon P22 transduction, to possess wild-type growth characteristics, and to contain a smooth lipopolysaccharide. Southern hybridization experiments showed that all three strains contained unique insertions. DNA sequencing of the transposon-chromosomal-DNA fusion junctions of these strains showed all to be linked to the putative Salmonella orf1-tolQRA operon, with insertions in tolQ, orf1, and a gene upstream of the orf1-tolQRA operon not previously associated with Tol function (orfX). Through the use of transcriptional fusions, none of the putative tol (or tol-associated) genes were shown to be regulated by PhoPQ, bile, or the RcsC-RcsB two-component system; however, all of the genes (orfX, orf1, tolQRA) are predicted to be cotranscribed. This is the first identification of Salmonella serovar Typhimurium Tol homologs and the first demonstration of their role in bile resistance in this organism. In addition, the observed regulation, operon arrangement, and phenotypes associated with these tol genes demonstrate significant differences from their Escherichia coli homologs.
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Soncini, F. C., E. García Véscovi, F. Solomon, and E. A. Groisman. "Molecular basis of the magnesium deprivation response in Salmonella typhimurium: identification of PhoP-regulated genes." Journal of bacteriology 178, no. 17 (1996): 5092–99. http://dx.doi.org/10.1128/jb.178.17.5092-5099.1996.
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Nam, Daesil, Eunna Choi, Dae-Hyuk Kweon, and Dongwoo Shin. "The RstB sensor acts on the PhoQ sensor to control expression of PhoP-regulated genes." Molecules and Cells 30, no. 4 (August 27, 2010): 363–68. http://dx.doi.org/10.1007/s10059-010-0126-8.
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Baud, David, Jalil Benyacoub, Véronique Revaz, Menno Kok, Françoise Ponci, Martine Bobst, Roy Curtiss, Pierre De Grandi, and Denise Nardelli-Haefliger. "Immunogenicity against Human Papillomavirus Type 16 Virus-Like Particles Is Strongly Enhanced by the PhoPc Phenotype in Salmonella enterica Serovar Typhimurium." Infection and Immunity 72, no. 2 (February 2004): 750–56. http://dx.doi.org/10.1128/iai.72.2.750-756.2004.
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ABSTRACT Recombinant Salmonella strains have been widely used to deliver heterologous antigens and induce immune responses in vaccinated animals and humans. It remains to be established, however, how these bacteria mount an immune response; this has prevented the rational design of vaccines. Here we report for the first time that a particular genetic program, PhoPc, is necessary for recombinant Salmonella strains to induce an antibody response to a heterologous antigen, the human papillomaviruses type 16 (HPV16) virus-like particle (VLP). The PhoPc phenotype results from a point mutation in phoQ, the gene encoding the sensor component of a two-component regulatory system (PhoP-PhoQ) that controls the expression of a number of virulence factors in Salmonellae. To demonstrate that immunogenicity of the viral antigen expressed by the bacterial vector was dependent on the PhoPc phenotype, we have expressed the phoQ mutant gene (phoQ24) in two differently attenuated Salmonella enterica serovar Typhimurium strains. Our data show extrachromosomal phoQ24 to be dominant over the chromosomal copy of the phoQ gene, conferring the PhoPc phenotype on the recipient strains. In addition, activation of PhoPQ-regulated genes by the plasmid-encoded PhoQ24 did not alter bacterial survival and conferred immunogenicity to the HPV16 VLP expressed in the two S. enterica serovar Typhimurium backgrounds, inducing the production of HPV-specific antibodies in mice. This strongly suggests that at least one of the PhoP-regulated genes is necessary for mounting an efficient antibody response to HPV16 VLP. This finding sets the stage for further development of a Salmonella-based vaccine against HPV infection and cervical cancer.
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Main-Hester, Kara L., Katherine M. Colpitts, Gracie A. Thomas, Ferric C. Fang, and Stephen J. Libby. "Coordinate Regulation of Salmonella Pathogenicity Island 1 (SPI1) and SPI4 in Salmonella enterica Serovar Typhimurium." Infection and Immunity 76, no. 3 (December 26, 2007): 1024–35. http://dx.doi.org/10.1128/iai.01224-07.
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ABSTRACT Salmonella enterica serovar Typhimurium harbors five pathogenicity islands (SPI) required for infection in vertebrate hosts. Although the role of SPI1 in promoting epithelial invasion and proinflammatory cell death has been amply documented, SPI4 has only more recently been implicated in Salmonella virulence. SPI4 is a 24-kb pathogenicity island containing six open reading frames, siiA to siiF. Secretion of the 595-kDa SiiE protein requires a type I secretory system encoded by siiC, siiD, and siiF. An operon polarity suppressor (ops) sequence within the 5′ untranslated region upstream of siiA is required for optimal SPI4 expression and predicted to bind the antiterminator RfaH. SiiE concentrations are decreased in a SPI1 mutant strain, suggesting that SPI1 and SPI4 may have common regulatory inputs. SPI1 gene expression is positively regulated by the transcriptional activators HilA, HilC, and HilD, encoded within SPI1, and negatively regulated by the regulators HilE and PhoP. Here, we show that mutations in hilA, hilC, or hilD similarly reduce expression of siiE, and mutations in hilE or phoP enhance siiE expression. Individual overexpression of HilA, HilC, or HilD in the absence of SPI1 cannot activate siiE expression, suggesting that these transcriptional regulators act in concert or in combination with additional SPI1-encoded regulatory loci to activate SPI4. HilA is no longer required for siiE expression in an hns mutant strain, suggesting that HilA promotes SPI4 expression by antagonizing the global transcriptional silencer H-NS. Coordinate regulation suggests that SPI1 and SPI4 play complementary roles in the interaction of S. enterica serovar Typhimurium with the host intestinal mucosa.
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Pilonieta, M. Carolina, Kimberly D. Erickson, Robert K. Ernst, and Corrella S. Detweiler. "A Protein Important for Antimicrobial Peptide Resistance, YdeI/OmdA, Is in the Periplasm and Interacts with OmpD/NmpC." Journal of Bacteriology 191, no. 23 (September 18, 2009): 7243–52. http://dx.doi.org/10.1128/jb.00688-09.
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ABSTRACT Antimicrobial peptides (AMPs) kill or prevent the growth of microbes. AMPs are made by virtually all single and multicellular organisms and are encountered by bacteria in diverse environments, including within a host. Bacteria use sensor-kinase systems to respond to AMPs or damage caused by AMPs. Salmonella enterica deploys at least three different sensor-kinase systems to modify gene expression in the presence of AMPs: PhoP-PhoQ, PmrA-PmrB, and RcsB-RcsC-RcsD. The ydeI gene is regulated by the RcsB-RcsC-RcsD pathway and encodes a 14-kDa predicted oligosaccharide/oligonucleotide binding-fold (OB-fold) protein important for polymyxin B resistance in broth and also for virulence in mice. We report here that ydeI is additionally regulated by the PhoP-PhoQ and PmrA-PmrB sensor-kinase systems, which confer resistance to cationic AMPs by modifying lipopolysaccharide (LPS). ydeI, however, is not important for known LPS modifications. Two independent biochemical methods found that YdeI copurifies with OmpD/NmpC, a member of the trimeric β-barrel outer membrane general porin family. Genetic analysis indicates that ompD contributes to polymyxin B resistance, and both ydeI and ompD are important for resistance to cathelicidin antimicrobial peptide, a mouse AMP produced by multiple cell types and expressed in the gut. YdeI localizes to the periplasm, where it could interact with OmpD. A second predicted periplasmic OB-fold protein, YgiW, and OmpF, another general porin, also contribute to polymyxin B resistance. Collectively, the data suggest that periplasmic OB-fold proteins can interact with porins to increase bacterial resistance to AMPs.
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Mulder, David T., Colin A. Cooper, and Brian K. Coombes. "Type VI Secretion System-Associated Gene Clusters Contribute to Pathogenesis of Salmonella enterica Serovar Typhimurium." Infection and Immunity 80, no. 6 (April 9, 2012): 1996–2007. http://dx.doi.org/10.1128/iai.06205-11.
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ABSTRACTThe enteropathogenSalmonella entericaserovar Typhimurium employs a suite of tightly regulated virulence factors within the intracellular compartment of phagocytic host cells resulting in systemic dissemination in mice. A type VI secretion system (T6SS) withinSalmonellapathogenicity island 6 (SPI-6) has been implicated in this process; however, the regulatory inputs and the roles of noncore genes in this system are not well understood. Here we describe four clusters of noncore T6SS genes in SPI-6 based on a comparative relationship with the T6SS-3 ofBurkholderia malleiand report that the disruption of these genes results in defects in intracellular replication and systemic dissemination in mice. In addition, we show that the expression of the SPI-6-encoded Hcp and VgrG orthologs is enhanced during late stages of macrophage infection. We identify six regions that are transcriptionally active during cell infections and that have regulatory contributions from the regulators of virulence SsrB, PhoP, and SlyA. We show that levels of protein expression are very weak underin vitroconditions and that expression is not enhanced upon the deletion ofssrB,phoP,slyA,qseC,ompR, orhfq, suggesting an unknown activating factor. These data suggest that the SPI-6 T6SS has been integrated into theSalmonellaTyphimurium virulence network and customized for host-pathogen interactions through the action of noncore genes.
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Sola-Landa, Alberto, Antonio Rodríguez-García, Etelvina Franco-Domínguez, and Juan F. Martín. "Binding of PhoP to promoters of phosphate-regulated genes in Streptomyces coelicolor: identification of PHO boxes." Molecular Microbiology 56, no. 5 (March 30, 2005): 1373–85. http://dx.doi.org/10.1111/j.1365-2958.2005.04631.x.
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Solans, Luis, Nacho Aguiló, Sofía Samper, Alexandre Pawlik, Wafa Frigui, Carlos Martín, Roland Brosch, and Jesús Gonzalo-Asensio. "A Specific Polymorphism in Mycobacterium tuberculosis H37Rv Causes Differential ESAT-6 Expression and Identifies WhiB6 as a Novel ESX-1 Component." Infection and Immunity 82, no. 8 (June 2, 2014): 3446–56. http://dx.doi.org/10.1128/iai.01824-14.
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ABSTRACTThe ESX-1 secreted virulence factor ESAT-6 is one of the major and most well-studied virulence factors ofMycobacterium tuberculosis, given that its inactivation severely attenuates virulent mycobacteria. In this work, we show that clinical isolates ofM. tuberculosisproduce and secrete larger amounts of ESAT-6 than the widely usedM. tuberculosisH37Rv laboratory strain. A search for the genetic polymorphisms underlying this observation showed thatwhiB6(rv3862c), a gene upstream of the ESX-1 genetic locus that has not previously been found to be implicated in the regulation of the ESX-1 secretory apparatus, presents a unique single nucleotide insertion in its promoter region in strains H37Rv and H37Ra. This polymorphism is not present in any of the other publicly availableM. tuberculosiscomplex genomes or in any of the 76 clinicalM. tuberculosisisolates analyzed in our laboratory. We demonstrate that in consequence, the virulence master regulator PhoP downregulateswhiB6expression in H37Rv, while it upregulates its expression in clinical strains. Importantly, reintroduction of the wild-type (WT) copy ofwhiB6in H37Rv restored ESAT-6 production and secretion to the level of clinical strains. Hence, we provide clear evidence that inM. tuberculosis—with the exception of the H37Rv strain—ESX-1 expression is regulated by WhiB6 as part of the PhoP regulon, which adds another level of complexity to the regulation of ESAT-6 secretion with a potential role in virulence adaptation.
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Klein, Gracjana, and Satish Raina. "Small regulatory bacterial RNAs regulating the envelope stress response." Biochemical Society Transactions 45, no. 2 (April 13, 2017): 417–25. http://dx.doi.org/10.1042/bst20160367.
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Most bacteria encode a large repertoire of RNA-based regulatory mechanisms. Recent discoveries have revealed that the expression of many genes is controlled by a plethora of base-pairing noncoding small regulatory RNAs (sRNAs), regulatory RNA-binding proteins and RNA-degrading enzymes. Some of these RNA-based regulated processes respond to stress conditions and are involved in the maintenance of cellular homeostasis. They achieve it by either direct posttranscriptional repression of several mRNAs, including blocking access to ribosome and/or directing them to RNA degradation when the synthesis of their cognate proteins is unwanted, or by enhanced translation of some key stress-regulated transcriptional factors. Noncoding RNAs that regulate the gene expression by binding to regulatory proteins/transcriptional factors often act negatively by sequestration, preventing target recognition. Expression of many sRNAs is positively regulated by stress-responsive sigma factors like RpoE and RpoS, and two-component systems like PhoP/Q, Cpx and Rcs. Some of these regulatory RNAs act via a feedback mechanism on their own regulators, which is best reflected by recent discoveries, concerning the regulation of cell membrane composition by sRNAs in Escherichia coli and Salmonella, which are highlighted here.
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Jamet, Anne, Clotilde Rousseau, Jean-Benoît Monfort, Eric Frapy, Xavier Nassif, and Patricia Martin. "A two-component system is required for colonization of host cells by meningococcus." Microbiology 155, no. 7 (July 1, 2009): 2288–95. http://dx.doi.org/10.1099/mic.0.027755-0.
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In order to adapt to changing environments, bacteria have evolved two-component systems (TCSs) that are able to sense and respond to environmental stimuli. The signal perception relies on a sensor protein whose activation allows rapid adaptation through transcriptional regulation achieved by the regulatory protein. The ability to adhere to and grow on the surface of human host cells is an absolute requirement for many pathogens, including Neisseria meningitidis, in order to colonize new hosts and to disseminate inside their host. Among the four TCSs encoded in the meningococcus genome, only the PhoQ (MisS)/PhoP (MisR) system has been shown to constitute a functional signal transduction circuit. To investigate the involvement of this TCS in the adaptation process requisite for host cell colonization, we have tested the ability to grow on host cells of a mutant inactivated for the sensor of the TCS. Our results demonstrate the involvement of the TCS in the adaptation of the meningococcus to growth on host cells. We show that the expression of the PhoQ (MisS)/PhoP (MisR) TCS is cell-contact controlled. Furthermore, this TCS controls the regulation of a group of genes, the REP2 regulon, previously shown to be cell-contact regulated and to encode functions crucial for the adaptation of the bacterium to host cell colonization. Thus, we provide evidence that one of the four TCSs existing in N. meningitidis contributes to the adaptation of the pathogen to growth on host cells.
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Santos-Beneit, Fernando, Mónica Barriuso-Iglesias, Lorena T. Fernández-Martínez, Miriam Martínez-Castro, Alberto Sola-Landa, Antonio Rodríguez-García, and Juan F. Martín. "The RNA Polymerase Omega Factor RpoZ Is Regulated by PhoP and Has an Important Role in Antibiotic Biosynthesis and Morphological Differentiation in Streptomyces coelicolor." Applied and Environmental Microbiology 77, no. 21 (September 9, 2011): 7586–94. http://dx.doi.org/10.1128/aem.00465-11.
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ABSTRACTThe RNA polymerase (RNAP) omega factor (ω) forms a complex with the α2ββ′ core of this enzyme in bacteria. We have characterized therpoZgene ofStreptomyces coelicolor, which encodes a small protein (90 amino acids) identified as the omega factor. Deletion of therpoZgene resulted in strains with a slightly reduced growth rate, although they were still able to sporulate. The biosynthesis of actinorhodin and, particularly, that of undecylprodigiosin were drastically reduced in the ΔrpoZstrain, suggesting that expression of these secondary metabolite biosynthetic genes is dependent upon the presence of RpoZ in the RNAP complex. Complementation of the ΔrpoZmutant with the wild-typerpoZallele restored both phenotype and antibiotic production. Interestingly, therpoZgene contains a PHO box in its promoter region. DNA binding assays showed that the phosphate response regulator PhoP binds to such a region. Since luciferase reporter studies showed thatrpoZpromoter activity was increased in a ΔphoPbackground, it can be concluded thatrpoZis controlled negatively by PhoP, thus connecting phosphate depletion regulation with antibiotic production and morphological differentiation inStreptomyces.
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Weatherspoon-Griffin, Natasha, and Helen J. Wing. "Characterization of SlyA in Shigella flexneri Identifies a Novel Role in Virulence." Infection and Immunity 84, no. 4 (February 1, 2016): 1073–82. http://dx.doi.org/10.1128/iai.00806-15.
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The SlyA transcriptional regulator has important roles in the virulence and pathogenesis of several members of theEnterobacteriaceaefamily, includingSalmonella entericaserovar Typhimurium andEscherichia coli. Despite the identification of theslyAgene inShigella flexnerinearly 2 decades ago, as well as the significant conservation of SlyA among enteric bacteria, the role of SlyA inShigellaremains unknown. The genes regulated by SlyA in closely related organisms often are absent from or mutated inS. flexneri, and consequently many described SlyA-dependent phenotypes are not present. By characterizing the expression ofslyAand determining its ultimate effect in this highly virulent organism, we postulated that novel SlyA-regulated virulence phenotypes would be identified. In this study, we report the first analysis of SlyA inShigellaand show that (i) theslyAgene is transcribed and ultimately translated into protein, (ii)slyApromoter activity is maximal during stationary phase and is negatively autoregulated and positively regulated by the PhoP response regulator, (iii) the exogenous expression ofslyArescues transcription and virulence-associated deficiencies during virulence-repressed conditions, and (iv) the absence ofslyAsignificantly decreases acid resistance, demonstrating a novel and important role inShigellavirulence. Cumulatively, our study illustrates unexpected parallels between the less conservedS. flexneriandS. TyphimuriumslyApromoters as well as a unique role for SlyA inShigellavirulence that has not been described previously in any closely related organism.
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Montero, Manuel, Goizeder Almagro, Gustavo Eydallin, Alejandro M. Viale, Francisco J. Muñoz, Abdellatif Bahaji, Jun Li, Mehdi Rahimpour, Edurne Baroja-Fernández, and Javier Pozueta-Romero. "Escherichia coli glycogen genes are organized in a single glgBXCAP transcriptional unit possessing an alternative suboperonic promoter within glgC that directs glgAP expression." Biochemical Journal 433, no. 1 (December 15, 2010): 107–17. http://dx.doi.org/10.1042/bj20101186.
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Although it is generally accepted that Escherichia coli glycogen genes are organized in two tandemly arranged, differentially regulated glgBX and glgCAP operons, RT (reverse transcriptase)–PCR analyses carried out in the present study showed that E. coli cells possess transcripts comprising the five glgBXCAP genes. glg::lacZY expression analyses in cells lacking the region immediately upstream of the glgB gene revealed an almost total abolishment of glgB, glgX and glgC expression, but only a 50–60% reduction of the wild-type glgA and glgP expression levels. Furthermore, similar analyses showed that glgA and glgP expression was almost totally abolished in cells lacking glgA upstream sequences, including glgC, glgB and the asd–glgB intergenic region upstream of glgB. These results indicate that E. coli glgBXCAP genes are organized in a single transcriptional unit controlled by promoter sequences occurring upstream of glgB, and that an alternative suboperonic promoter is located within glgC, driving expression of the glgA and glgP genes. Computer searches for consensus promoters, and analyses of glgB::lacZY and glgA::lacZY expression in cells containing deletions of glgB and glgA upstream sequences identified regions directing glgBXCAP and glgAP expression. 5′ RACE (rapid amplification of cDNA ends) analyses located a glgBXCAP transcription start site 155 bp upstream of the glgB initiation codon, and a glgAP transcription start site 359 bp upstream of the glgA initiation codon. Finally, glg::lacZY expression analyses on cells lacking the relA or phoP regulatory genes indicated that both the glgBXCAP operon and the suboperonic promoter driving glgAP expression form part of both the RelA and PhoP-PhoQ regulons.
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Rublenko, N. "Molecular genetics of salmonela survival and resistance." Naukovij vìsnik veterinarnoï medicini, no. 2 (144) (December 24, 2018): 6–12. http://dx.doi.org/10.33245/2310-4902-2018-144-2-6-12.
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Salmonella is one of the most common cause of the food borne illness. Salmonella belongs to Enterobacteriacae family and consists of 2 species, which diverge on 6 subspecies.These subspecies consists of 2700 serovars. There are typhoid serovars among them - S. Typhi, Paratyphi A, B, C - which cause typhoid fever in human. The rest of the serovars are non-typhoidal and leads to gastroenteritis both in animal and human. Salmonella enters to a mammal organism as a result of consumption of contaminated food products: meat, eggs, milk and products containing them. The entry of the infection for salmonellosis is the small intestine mucosa. Salmonella attaches to cell walls by fimbria and pili. Salmonella has several systems that are activated in response to adverse conditions such as: high osmolarity, acid or heat shock and nutrient deficiencies. They are based on the principle of a two-component system in which there is a sensor that receives cytoplasmic signals, and a regulator. Regulator (usually DNA-binding protein) initiates the transcription of the virulence genes (Chakraborty, 2015). The sensor is histidine kinase, which phosphorylates the regulatory protein, thereby activating it.During the infectious cycle of salmonella in mammalian organism the formation of specific vacuole SCV takes place (Salmonella-containing vacuole-containing vacuole containing salmonella) in the cytoplasm of the eukaryotic cell (Steele-Mortimer, 2008). SCV is a modified phagosome, which is formed as a result of cytoskeleton rearrangements. The target are usually phagocytic cells : neutrophils, macrophages and epithelial cells of the small intestine mucosa - M-cells (Akhmetova, 2012). Given the specific mechanism of infection, salmonella is considered a facultative intracellular pathogen. Bacterium invades the eukaryotic cell by rearrangement of its cytoskeleton with effector proteins and continue to persistence in a form of SCV. It is well-known nowadays that tolerance to high osmotic pressure is achieved through the EnvZ / OmpR system, which also regulates the expression of the ssrAB operon that is localized on the Salmonella pathogenicity island SPI-2 and triggers the expression of the effector proteins. The ssrAB operon is also regulated by the two-component acid shock response system PhoP / PhoQ (Worley, 2000). The functioning of the PhoP / PhoQ system directly depends on the sigma factor RpoS, which accumulates under low concentrations of magnesium cations (Tu, 2006). According to the researches of transduction between the EnvZ / OmpR components, it is clear that salmonella receives signals from the cytoplasmic environment, and sensory molecules are located on the inner membrane (Kenney, 2019; Wang et al., 2012). The ability to survive under acid shock is provided by the PhoP / PhoQ system, which also operates on the principle of signal transduction. PhoQ is a Histidine Kinase Signal Sensor. Signals are acidic pH, divalent cations and positively charged antimicrobial peptides. An important function of the two-component PhoP / PhoQ system is the control of spi ssa gene expression in a macrophage environment (Bijlsma, 2005). These genes are the main component of the type III secretion systems and are transcribed only when salmonella enters eukaryotic cell. (Bijlsma, 2005). The main regulator of signal transduction systems PhoP/PhoQ and EnvZ/OmpR is sigma-factor RpoS - subunit of bacterial RNA-polymerase - which operates in stationary phase at low pH, high omolarity, heat shock or nutrient deficiency. RpoS protein accumulates in adverse conditions during stationary phase (Mg2+ deficiency, low pH, high osmolarity). Need in magnesium cations is dependent on their ability to act as cofactors in many enzymatic activities. The accumulation begins at exponential (logarithmic) phase of bacterial reproduction. This is the phase of active cell division. Two factors MgtA and MgtB are responsible for Mg2+ transport. Another molecule with the same function is CorA - bivalent cation channel, though its transcriptions doesn’t depent on magnesium concentration in cell. In a case of magnesium deficiency at the stationary phase, RpoS accumulates vigorously an initiates replication of PhoP/PhoQ. PhoP/PhoQ regulates tolerance to inorganic acids. Also, PhoP/PhoQ controls adaptation to magnesium cations deficiency and macrophage activity. Results of many studies on genes coding this system and their mutations led to conclusion the mutation or inactivation of one factor causes decrease in virulence and makes bacterial susceptible to acid environment. To date, the stages of the infectious process for salmonellosis have been studied and described in detail in the literature. Particular attention is paid to signal transduction systems that are common among enterobacteria and help to avoid adverse conditions. Their functioning and regulation are investigated. It is known that salmonella receives signals for the activation of sensors from the cytoplasm, but the nature of these signals is not yet fully understood. Adaptation of the bacteria to adverse conditions and the response to phagocytosis is initiated by the transcription of pathogenic genes and the suppression of the transcription of the operon, which neutralize the conditions in the cytoplasm of salmonella cells. Thus, adapting to the conditions of target cells, salmonella continues to multiply in the body. Key words: salmonella, pH, osmolarity, virulencegenes, operon, signal transduction.