Regulation of capsule synthesis and cell motility in Salmonella enterica by the essential gene igaA

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.     

Interaction of the regulator proteins RcsA and RcsB with the promoter of the operon for amylovoran biosynthesis in Erwinia amylovora

The RcsA and RcsB proteins of Erwinia amylovora and Escherichia coli were expressed in E. coli and purified. Their DNA-binding activity was examined using a 1-kb DNA region containing the putative promoter of the ams operon of Ew. amylovora, which is responsible for the biosynthesis of the exopolysaccharide amylovoran. Mobility shift assays indicated specific binding of RcsA and RcsB to a region of 78 bp spanning nucleotide positions −578 to −501 relative to the translational start of the first open reading frame of the operon. This region includes stretches of homology to E. coliσ ⁷⁰ promoter consensus sequences and to the E. coli cps promoter region. Binding of the Rcs proteins was not found at a JUMPstart consensus, typical for various promoters of polysaccharide gene clusters. DNA-binding activity was not detected for RcsA alone and only high concentrations of RcsB were able to interact with the ams promoter in our assay. The two proteins bind cooperatively at the indicated region of the ams promoter and further evidence is provided showing that the DNA-protein complex formed involves a heterodimer of RcsA and RcsB. The specific activity of RcsA, but not of RcsB, was enhanced when the protein was expressed in E. coli at 28° C, relative to expression at 37° C. In addition, DNA-protein complex formation is affected by temperature. The E. coli RcsA/RcsB proteins bind to the same region of the ams promoter and are able to interact with the Rcs proteins from Ew. amylovora. Received: 26 February 1997 / Accepted: 23 May 1997     

RcsF is an outer membrane lipoprotein involved in the RcsCDB phosphorelay signaling pathway in Escherichia coli

The RcsCDB signal transduction system is an atypical His-Asp phosphorelay conserved in gamma-proteobacteria. Besides the three proteins directly involved in the phosphorelay, two proteins modulate the activity of the system. One is RcsA, which can stimulate the activity of the response regulator RcsB independently of the phosphorelay to regulate a subset of RcsB targets. The other is RcsF, a putative outer membrane lipoprotein mediating the signaling to the sensor RcsC. How RcsF transduces the signal to RcsC is unknown. Although the molecular and physiological signals remain to be identified, the common feature among the reported Rcs-activating conditions is perturbation of the envelope. As an initial step to explore the RcsF-RcsC functional relationship, we demonstrate that RcsF is an outer membrane lipoprotein oriented towards the periplasm. We also report that a null mutation in surA, a gene required for correct folding of periplasmic proteins, activates the Rcs pathway through RcsF. In contrast, activation of this pathway by overproduction of the membrane chaperone-like protein DjlA does not require RcsF. Conversely, activation of the pathway by RcsF overproduction does not require DjlA either, indicating the existence of two independent signaling pathways toward RcsC.     

Interaction of the regulator proteins RcsA and RcsB with the promoter of the operon for amylovoran biosynthesis in Erwinia amylovora

The RcsA and RcsB proteins of Erwinia amylovora and Escherichia coli were expressed in E. coli and purified. Their DNA-binding activity was examined using a 1-kb DNA region containing the putative promoter of the ams operon of Ew. amylovora, which is responsible for the biosynthesis of the exopolysaccharide amylovoran. Mobility shift assays indicated specific binding of RcsA and RcsB to a region of 78?bp spanning nucleotide positions ?578 to ?501 relative to the translational start of the first open reading frame of the operon. This region includes stretches of homology to E. coliσ ⁷⁰ promoter consensus sequences and to the E. coli cps promoter region. Binding of the Rcs proteins was not found at a JUMPstart consensus, typical for various promoters of polysaccharide gene clusters. DNA-binding activity was not detected for RcsA alone and only high concentrations of RcsB were able to interact with the ams promoter in our assay. The two proteins bind cooperatively at the indicated region of the ams promoter and further evidence is provided showing that the DNA-protein complex formed involves a heterodimer of RcsA and RcsB. The specific activity of RcsA, but not of RcsB, was enhanced when the protein was expressed in E. coli at 28°?C, relative to expression at 37°?C. In addition, DNA-protein complex formation is affected by temperature. The E. coli RcsA/RcsB proteins bind to the same region of the ams promoter and are able to interact with the Rcs proteins from Ew. amylovora.     

Regulation and mode of action of the second small RNA activator of RpoS translation,RprA

Translation of the stationary phase sigma factor RpoS is stimulated by at least two small RNAs, DsrA and RprA. DsrA disrupts an inhibitory secondary structure in the rpoS leader mRNA by pairing with the upstream RNA. Mutations in rprA and compensating mutations in the rpoS leader demonstrate that RprA interacts with the same region of the RpoS leader as DsrA. This is the first example of two different small RNAs regulating a common target. Regulation of these RNAs differs. DsrA synthesis is increased at low temperature. We find that RprA synthesis is regulated by the RcsC/RcsB phosphorelay system, previously found to regulate capsule synthesis and promoters of ftsZ and osmC. An rcsB null mutation abolishes the basal level, whereas mutations in rcsC that activate capsule synthesis also activate expression of the rprA promoter. An essential site with similarity to other RcsB-regulated promoters was defined in the rprA promoter. Activation of the RcsC/RcsB system leads to increased RpoS synthesis, in an RprA-dependent fashion. This work suggests a new signal for RpoS translation and extends the global regulation effected by the RcsC/RcsB system to coregulation of RpoS with capsule and FtsZ.     

The Effects of Regulatory Proteins RcsA and RcsB on the Expression of the Vibrio fischeri lux Operon in Escherichia coli

A study was made of the effect of RcsA and RcsB on the Vibrio fischeri lux expression in Escherichia coli. RcsA suppressed the LuxR activity and thereby inhibited expression of the lux genes coding for luciferase and reductase. In osmotic shock, RcsA–RcsB activated lux expression and, consequently, the bioluminescence of E. coli cells in the early log phase.     

Acetyl phosphate-sensitive regulation of flagellar biogenesis and capsular biosynthesis depends on the Rcs phosphorelay

As part of our attempt to map the impact of acetyl phosphate (acetyl approximately P) on the entire network of two-component signal transduction pathways in Escherichia coli, we asked whether the influence of acetyl approximately P on capsular biosynthesis and flagellar biogenesis depends on the Rcs phosphorelay. To do so, we performed a series of epistasis experiments: mutations in the components of the pathway that controls acetyl approximately P levels were combined with mutations in components of the Rcs phosphorelay. Cells that did not synthesize acetyl approximately P produced no capsule under normally permissive conditions, while those that accumulated acetyl approximately P synthesized capsule under conditions previously considered to be non-permissive. Acetyl approximately P-dependent capsular biosynthesis required both RcsB and RcsA, while the lack of RcsC restored capsular biosynthesis to acetyl approximately P-deficient cells. Similarly, acetyl approximately P-sensitive repression of flagellar biogenesis was suppressed by the loss of RcsB (but not of RcsA), while it was enhanced by the lack of RcsC. Taken together, these results show that both acetyl approximately P-sensitive activation of capsular biosynthesis and acetyl approximately P-sensitive repression of flagellar biogenesis require the Rcs phosphorelay. Moreover, they provide strong genetic support for the hypothesis that RcsC can function as either a kinase or a phosphatase dependent on environmental conditions. Finally, we learned that RcsB and RcsC inversely regulated the timing of flagellar biogenesis: rcsB mutants elaborated flagella prematurely, while rcsC mutants delayed their display of flagella. Temporal control of flagella biogenesis implicates the Rcs phosphorelay (and, by extension, acetyl approximately P) in the transition of motile, planktonic individuals into sessile biofilm communities.     

Polysialic and colanic acids metabolism in Escherichia coli K92 is regulated by RcsA and RcsB

We have shown previously that Escherichia coli K92 produces two different capsular polymers known as CA (colanic acid) and PA (polysialic acid) in a thermoregulated manner. The complex Rcs phosphorelay is largely related to the regulation of CA synthesis. Through deletion of rscA and rscB genes, we show that the Rcs system is involved in the regulation of both CA and PA synthesis in E. coli K92. Deletion of either rcsA or rcsB genes resulted in decreased expression of cps (CA biosynthesis cluster) at 19°C and 37°C, but only CA production was reduced at 19°C. Concerning PA, both deletions enhanced its synthesis at 37°C, which does not correlate with the reduced kps (PA biosynthesis cluster) expression observed in the rcsB mutant. Under this condition, expression of the nan operon responsible for PA catabolism was greatly reduced. Although RcsA and RcsB acted as negative regulators of PA synthesis at 37°C, their absence did not reestablish PA expression at low temperatures, despite the deletion of rcsB resulting in enhanced kps expression. Finally, our results revealed that RcsB controlled the expression of several genes (dsrA, rfaH, h-ns and slyA) involved in the thermoregulation of CA and PA synthesis, indicating that RcsB is part of a complex regulatory mechanism governing the surface appearance in E. coli.     

The pathogenicity island encoded PvrSR/RcsCB regulatory network controls biofilm formation and dispersal in Pseudomonas aeruginosa PA14

Pseudomonas aeruginosa biofilm formation is linked to persistent infections in humans. Biofilm formation is facilitated by extracellular appendages, some of which are assembled by the Chaperone Usher Pathway (Cup). The cupD gene cluster is located on the PAPI‐1 pathogenicity island of strain PA14 and has probably been acquired together with four genes encoding two‐component signal transduction proteins. We have previously showed that the RcsB response regulator activates expression of the cupD genes, which leads to the production of CupD fimbriae and increased attachment. Here we show that RcsB activity is tightly modulated by two sensors, RcsC and PvrS. While PvrS acts as a kinase that enhances RcsB activity, RcsC has a dual function, first as a phosphorelay, and second as a phosphatase. We found that, under certain growth conditions, overexpression of RcsB readily induces biofilm dispersal. Microarray analysis shows that RcsB positively controls expression of pvrR that encodes the phosphodiesterase required for this dispersal process. Finally, in addition to the PAPI‐1 encoded cupD genes, RcsB controls several genes on the core genome, some of which encode orphan response regulators. We thus discovered that RcsB is central to a large regulatory network that fine‐tunes the switch between biofilm formation and dispersal.     

Identification, cloning, and characterization of rcsF, a new regulator gene for exopolysaccharide synthesis that suppresses the division mutation ftsZ84 in Escherichia coli K-12.

A new gene, designated rcsF, was located adjacent to drpA at the 5.2-min position of the genetic map of Escherichia coli. The deduced amino acid sequence encoded by the rcsF gene indicates a small protein of 133 amino acid residues with a calculated pI of 10.8 that is rich in proline, serine, alanine, and cysteine residues. When overexpressed as a result of its presence on a multicopy plasmid, rcsF confers a mucoid phenotype and restores colony formation to ftsZ84 mutant cells on L agar medium containing no added NaCl. These two phenotypes are not observed in rcsB mutant cells. Ion mutant cells harboring an rcsF mutation accumulate considerably lower levels of exopolysaccharides, whereas the presence of a multicopy rcsF plasmid not only increases capsule synthesis but also confers a mucoid phenotype at 37 degrees C, a temperature at which ion mutant cells are known not to form mucoid colonies. RcsF does not stimulate the expression of rcsB, indicating that it exerts its action through the RcsB protein, possibly by phosphorylation. It is also shown that RcsF stimulation of capsule synthesis is RcsA-dependent, whereas colony formation of ftsZ84 mutant cells can be restored by RcsF in the absence of RcsA.     

A novel feature of the multistep phosphorelay in Escherichia coli: a revised model of the RcsC --> YojN --> RcsB signalling pathway implicated in capsular synthesis and swarming behaviour

In this study, we re-investigated the previously characterized RcsC (sensor His-kinase) --> RcsB (response regulator) phosphorelay system that is involved in the regulation of capsular polysaccharide synthesis in Escherichia coli. The previously proposed model hypothesized the occurrence of a direct phosphotransfer from RcsC to RcsB in response to an unknown external stimulus. As judged from the current general view as to the His --> Asp phosphorelay, this RcsC --> RcsB framework is somewhat puzzling, because RcsC appears to contain both a His-kinase domain and a receiver domain, but not a histidine (His)-containing phosphotransmitter domain (e.g. HPt domain). We thus suspected that an as yet unknown mechanism might be underlying in this particular His --> Asp phosphorelay system. Here, we provide several lines of in vivo and in vitro evidence that a novel and unique His-containing phosphotransmitter (named YojN) is essential for this signalling system. A revised model is proposed in which the multistep RcsC --> YojN --> RcsB phosphorelay is implicated. It was also demonstrated that this complex signalling system is somehow involved in the modulation of a characteristic behaviour of E. coli cells during colony formation on the surface of agar plates, namely swarming.     

RcsB and RcsC: a two-component regulator of capsule synthesis in Escherichia coli.

Colanic acid capsule synthesis in Escherichia coli K-12 is regulated by RcsB and RcsC. The amino acid sequences of these two proteins, deduced from the nucleotide sequence reported here, demonstrate their homology to environmentally responsive two-component regulators that have been reported in both gram-positive and gram-negative bacteria. In our model, RcsC acts as the sensor and RcsB acts as the receiver or effector to stimulate capsule synthesis from cps genes. In addition, RcsC shows limited homology to the other effectors in its C terminus. Fusions of rcsC to phoA that resulted in PhoA+ strains demonstrated that RcsC is a transmembrane protein with a periplasmic N-terminal domain and cytoplasmic C-terminal domain. Additional control of this regulatory network is provided by the dependence on the alternate sigma factor, RpoN, for the synthesis of RcsB. The rcsB and rcsC genes, which are oriented convergently with their stop codons 196 base pairs apart, are separated by a long direct repeat including two repetitive extragenic palindromic sequences.     

The RcsCDB signaling system and swarming motility in Salmonella enterica serovar typhimurium: dual regulation of flagellar and SPI-2 virulence genes

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.