The Rcs phosphorelay system is specific to enteric pathogens/commensals and activates ydeI, a gene important for persistent Salmonella infection of mice.
ABSTRACT: Bacteria utilize phosphorelay systems to respond to environmental or intracellular stimuli. Salmonella enterica encodes a four-step phosphorelay system that involves two sensor kinase proteins, RcsC and RcsD, and a response regulator, RcsB. The physiological stimulus for Rcs phosphorelay activation is unknown; however, Rcs-regulated genes can be induced in vitro by osmotic shock, low temperature and antimicrobial peptide exposure. In this report we investigate the role of the Rcs pathway using phylogenetic analysis and experimental techniques. Phylogenetic analysis determined that full-length RcsC- and RcsD-like proteins are generally restricted to Enterobacteriaceae species that have an enteric pathogenic or commensal relationship with the host. Experimental data show that RcsD and RcsB, in addition to RcsC, are important for systemic infection in mice and polymyxin B resistance in vitro. To identify Rcs-regulated genes that confer these phenotypes, we took advantage of our observation that RcsA, a transcription factor and binding partner of RcsB, is not required for polymyxin B resistance or survival in mice. S. enterica serovar Typhimurium oligonucleotide microarrays were used to identify 18 loci that are activated by RcsC, RcsD and RcsB but not RcsA. Five of the 18 loci encode genes that contribute to polymyxin B resistance. One of these genes, ydeI, was shown by quantitative real-time PCR to be regulated by the Rcs pathway independently of RcsA. Additionally, the stationary-phase sigma factor, RpoS (sigmaS), regulates ydeI transcription. In vivo infections show that ydeI mutants are out-competed by wild type 10- to 100-fold after oral inoculation, but are only modestly attenuated after intraperitoneal inoculation. These data indicate that ydeI is an Rcs-activated gene that plays an important role in persistent infection of mice, possibly by increasing bacterial resistance to antimicrobial peptides.
Project description:The rcs phosphorelay pathway components were originally identified as regulators of capsule synthesis. In addition to the transmembrane sensor kinase RcsC, the RcsA coregulator, and the response regulator RcsB, two new components have been characterized, RcsD and RcsF. RcsD, the product of the yojN gene, now renamed rcsD, acts as a phosphorelay between RcsC and RcsB. Transcription of genes for capsule synthesis (cps) requires both RcsA and RcsB; transcription of other promoters, including that for the small RNA RprA, requires only RcsB. RcsF was described as an alternative sensor kinase for RcsB. We have examined the role of RcsF in the activation of both the rprA and cps promoters. We find that a number of signals that lead to activation of the phosphorelay require both RcsF and RcsC; epistasis experiments place RcsF upstream of RcsC. The RcsF sequence is characteristic of lipoproteins, consistent with a role in sensing cell surface perturbation and transmitting this signal to RcsC. Activation of RcsF does not require increased transcription of the gene, suggesting that modification of the RcsF protein may act as an activating signal. Signals from RcsC require RcsD to activate RcsB. Sequencing of an rcsC allele, rcsC137, that leads to high-level constitutive expression of both cps and rprA suggests that the response regulator domain of RcsC plays a role in negatively regulating the kinase activity of RcsC. The phosphorelay and the variation in the activation mechanism (dependent upon or independent of RcsA) provide multiple steps for modulating the output from this system.
Project description:The Rcs phosphorelay system is a modified two-component signal transduction system found exclusively in Enterobacteriaceae. In this study, we characterized the roles of the Rcs system in Erwinia amylovora, a highly virulent and necrogenic enterobacterium causing fire blight disease on rosaceous plants. Our results showed that rcsB, rcsC, rcsD and rcsBD mutants were non-pathogenic on immature pear fruit. The bacterial growth of these mutants was also greatly reduced compared with that of the wild-type strain in immature pear fruit. In an in vitro amylovoran assay, rcsB and rcsD mutants were deficient in amylovoran production, whereas the rcsC mutant exhibited higher amylovoran production than that of the wild-type. Consistent with amylovoran production, expression of the amylovoran biosynthetic gene amsG, using green fluorescent protein as a reporter, was not detectable in rcsB, rcsD and rcsBD mutants both in vitro and in vivo. The expression of amsG in vitro was higher in the rcsC mutant than in the wild-type, whereas its expression in vivo was higher in the wild-type than in the rcsC mutant. In addition, rcs mutants were more susceptible to polymyxin B treatment than the wild-type, suggesting that the Rcs system conferred some level of resistance to polymyxin B. Furthermore, rcs mutants showed irregular and slightly reduced motility on swarming plates. Together, these results indicate that the Rcs system plays a major role in virulence and survival of E. amylovora in immature pear fruit.
Project description:Mutants of Salmonella enterica carrying the igaA1 allele, selected as able to overgrow within fibroblast cells in culture, are mucoid and show reduced motility. Mucoidy is caused by derepression of wca genes (necessary for capsule synthesis); these genes are regulated by the RcsC/YojN/RcsB phosphorelay system and by the RcsA coregulator. The induction of wca expression in an igaA1 mutant is suppressed by mutations in rcsA and rcsC. Reduced motility is caused by lowered expression of the flagellar master operon, flhDC, and is suppressed by mutations in rcsB or rcsC, suggesting that mutations in the igaA gene reduce motility by activating the RcsB/C system. A null igaA allele can be maintained only in an igaA(+)/igaA merodiploid, indicating that igaA is an essential gene. Lethality is suppressed by mutations in rcsB, rcsC, and yojN, but not in rcsA, suggesting that the viability defect of an igaA null mutant is mediated by the RcsB/RcsC system, independently of RcsA (and therefore of the wca genes). Because all the defects associated with igaA mutations are suppressed by mutations that block the RcsB/RcsC system, we propose a functional interaction between the igaA gene product and either the Rcs regulatory network or one of its regulated products.
Project description:The Rcs phosphorelay pathway is a complex signaling pathway involved in the regulation of many cell surface structures in enteric bacteria. In response to environmental stimuli, the sensor histidine kinase (RcsC) autophosphorylates and then transfers the phosphate through intermediary steps to the response regulator (RcsB), which, once phosphorylated, regulates gene expression. Here, we show that Salmonella biofilm development depends on the phosphorylation status of RcsB. Thus, unphosphorylated RcsB, hitherto assumed to be inactive, is essential to activate the expression of the biofilm matrix compounds. The prevention of RcsB phosphorylation either by the disruption of the phosphorelay at the RcsC or RcsD level or by the production of a nonphosphorylatable RcsB allele induces biofilm development. On the contrary, the phosphorylation of RcsB by the constitutive activation of the Rcs pathway inhibits biofilm development, an effect that can be counteracted by the introduction of a nonphosphorylatable RcsB allele. The inhibition of biofilm development by phosphorylated RcsB is due to the repression of CsgD expression, through a mechanism dependent on the accumulation of the small noncoding RNA RprA. Our results indicate that unphosphorylated RcsB plays an active role for integrating environmental signals and, more broadly, that RcsB phosphorylation acts as a key switch between planktonic and sessile life-styles in Salmonella enterica serovar Typhimurium.
Project description:Two-component systems and phosphorelays play central roles in the ability of bacteria to rapidly respond to changing environments. In E. coli and related enterobacteria, the complex Rcs phosphorelay is a critical player in the bacterial response to antimicrobial peptides, beta-lactam antibiotics, and other disruptions at the cell surface. The Rcs system is unusual in that an inner membrane protein, IgaA, is essential due to its negative regulation of the RcsC/RcsD/RcsB phosphorelay. While it is known that IgaA transduces signals from the outer membrane lipoprotein RcsF, how it interacts with the phosphorelay has remained unknown. Here we performed in vivo interaction assays and genetic dissection of the critical proteins and found that IgaA interacts with the phosphorelay protein RcsD, and that this interaction is necessary for regulation. Interactions between IgaA and RcsD within their respective periplasmic domains of these two proteins anchor repression of signaling. However, the signaling response depends on a second interaction between cytoplasmic loop 1 of IgaA and a truncated Per-Arndt-Sim (PAS-like) domain in RcsD. A single point mutation in the PAS-like domain increased interactions between the two proteins and blocked induction of the phosphorelay. IgaA may regulate RcsC, the histidine kinase that initiates phosphotransfer through the phosphorelay, indirectly, via its contacts with RcsD. Unlike RcsD, and unlike many other histidine kinases, the periplasmic domain of RcsC is dispensable for the response to signals that induce the Rcs phosphorelay system. The multiple contacts between IgaA and RcsD constitute a poised sensing system, preventing potentially toxic over-activation of this phosphorelay while enabling it to rapidly and quantitatively respond to signals.
Project description:To survive an environmental stress, organisms must detect the stress and mount an appropriate response. One way that bacteria do so is by phosphorelay systems that respond to a stress by activating a regulator that modifies gene expression. To ensure an appropriate response, a given regulator is typically activated solely by its cognate phosphorelay protein(s). However, we now report that the regulator RcsB is activated by both cognate and non-cognate phosphorelay proteins, depending on the condition experienced by the bacterium Salmonella enterica serovar Typhimurium. The RcsC and RcsD proteins form a phosphorelay that activates their cognate regulator RcsB in response to outer membrane stress and cell wall perturbations, conditions Salmonella experiences during infection. Surprisingly, the non-cognate phosphorelay protein BarA activates RcsB during logarithmic growth in Luria-Bertani medium in three ways. That is, BarA's cognate regulator SirA promotes transcription of the rcsDB operon; the SirA-dependent regulatory RNAs CsrB and CsrC further increase RcsB-activated gene transcription; and BarA activates RcsB independently of the RcsC, RcsD, and SirA proteins. Activation of a regulator by multiple sensors broadens the spectrum of environments in which a set of genes is expressed without evolving binding sites for different regulators at each of these genes.
Project description: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.
Project description:Cronobacter species can cause necrotizing enterocolitis and meningitis in neonates and infants, their infection is closely relevant to their responses to extreme growth conditions. In this study, the response of Cronobacter species to amino acid deficiency has been investigated. Four Cronobacter species formed smooth colonies when grown on the solid LB medium, but formed mucoid colonies when grown on the amino acid deficient M9 medium. When the mucoid colonies were stained with tannin mordant, exopolysaccharide around the cells could be discerned. The exopolysaccharide was isolated, analyzed, and identified as colanic acid. When genes wcaD and wcaE relevant to colanic acid biosynthesis were deleted in Cronobacter sakazakii BAA-894, no exopolysaccharide could be produced, confirming the exopolysaccharide formed in C. sakazakii grown in M9 is colanic acid. On the other hand, when genes rcsA, rcsB, rcsC, rcsD, or rcsF relevant to Rcs phosphorelay system was deleted in C. sakazakii BAA-894, colanic acid could not be produced, suggesting that the production of colanic acid in C. sakazakii is regulated by Rcs phosphorelay system. Furthermore, C. sakazakii BAA-894 grown in M9 supplemented with amino acids could not produce exopolysaccharide. Transcriptomes of C. sakazakii BAA-894 grown in M9 or LB were analyzed. A total of 3956 genes were differentially expressed in M9, of which 2339 were up-regulated and 1617 were down-regulated. When C. sakazakii BAA-894 was grown in M9, the genes relevant to the biosynthesis of exopolysaccharide were significantly up-regulated; on the other hand, the genes relevant to the flagellum formation and chemotaxis were significantly down-regulated; in addition, most genes relevant to various amino acid biosynthesis were also significantly regulated. The results demonstrate that amino acid deficiency has a global impact on C. sakazakii cells.
Project description:The Rcs phosphorelay is a multicomponent signaling system that positively regulates colanic acid synthesis and negatively regulates motility and virulence. We have exploited a spontaneously isolated mutant, IgaA(T191P), that is nearly maximally activated for the Rcs system to identify a vast set of genes that respond to the stimulation, and we report new regulatory properties of this signaling system in Salmonella enterica serovar Typhimurium. Microarray data show that the Rcs system normally functions as a positive regulator of SPI-2 and other genes important for the growth of Salmonella in macrophages, although when highly activated the system completely represses the SPI-1/SPI-2 virulence, flagellar, and fimbrial biogenesis pathways. The auxiliary protein RcsA, which works with RcsB to positively regulate colanic acid and other target genes, not only stimulates but also antagonizes the positive regulation of many genes in the igaA mutant. We show that RcsB represses motility through the RcsB box in the promoter region of the master operon flhDC and that RcsA is not required for this regulation. Curiously, RcsB selectively stimulates expression of the flagellar type 3 secretion genes fliPQR; an RcsAB box located downstream of fliR influences this regulation. We show that excess colanic acid impairs swimming and inhibits swarming motility, consistent with the inverse regulation of the two pathways by the Rcs system.
Project description:In response to osmolarity, Salmonella enterica serotype Typhi (S. Typhi) regulates genes required for Vi capsular antigen expression oppositely to those required for motility and invasion. Previous studies suggest that osmoregulation of motility, invasion and capsule expression is mediated through the RcsC/RcsD/RcsB phosphorelay system. Here we performed gene expression profiling and functional studies to determine the role of TviA, an auxiliary protein of the RcsB response regulator, in controlling virulence gene expression in S. Typhi. TviA repressed expression of genes encoding flagella and the invasion-associated type III secretion system (T3SS-1) through repression of the flagellar regulators flhDC and fliZ, resulting in reduced invasion, reduced motility and reduced expression of FliC. Both RcsB and TviA repressed expression of flhDC, but only TviA altered flhDC expression in response to osmolarity. Introduction of tviA into S. enterica serotype Typhimurium rendered flhDC transcription sensitive to changes in osmolarity. These data suggest that the auxiliary TviA protein integrates a new regulatory input into the RcsB regulon of S. Typhi, thereby altering expression of genes encoding flagella, the Vi antigen and T3SS-1 in response to osmolarity.