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.     

Characterization of the RcsC→YojN→RcsB Phosphorelay Signaling Pathway Involved in Capsular Synthesis in Escherichia coli

Escherichia coli and other enteric microorganisms produce an extracellular polysaccharide capsule, called colanic acid, under certain environmental conditions. This capsular synthesis is regulated by the RcsC (sensor kinase)→YojN (phosphotransfer intermediate)→RcsB (response regulator) phosphorelay signal transduction under certain growth conditions. Nonetheless, little is known about signals that exaggerate the Rcs-system. To gain insight into signals that activate the Rcs-system, here we searched for genes that activate the Rcs-system, provided that those on a multicopy plasmid were introduced into E. coli. We identified several such genes, namely, rcsB, rcsA, djlA, lolA, and ompG. The DjlA, LolA, and OmpG proteins are particularly interesting in that they are all located on the cell surface, where the primary sensor RcsC histidine-kinase is localized. Implications of these findings are discussed with special reference to the mechanism by which RcsC perceives external signals.     

Characterization of the RcsC-->YojN-->RcsB phosphorelay signaling pathway involved in capsular synthesis in Escherichia coli.

Escherichia coli and other enteric microorganisms produce an extracellular polysaccharide capsule, called colanic acid, under certain environmental conditions. This capsular synthesis is regulated by the RcsC (sensor kinase)-->YojN (phosphotransfer intermediate)-->RcsB (response regulator) phosphorelay signal transduction under certain growth conditions. Nonetheless, little is known about signals that exaggerate the Rcs-system. To gain insight into signals that activate the Rcs-system, here we searched for genes that activate the Rcs-system, provided that those on a multicopy plasmid were introduced into E. coli. We identified several such genes, namely, rcsB, rcsA, djlA, lolA, and ompG. The DjlA, LolA, and OmpG proteins are particularly interesting in that they are all located on the cell surface, where the primary sensor RcsC histidine-kinase is localized. Implications of these findings are discussed with special reference to the mechanism by which RcsC perceives external signals.     

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.     

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.     

Solution structure of the Escherichia coli YojN histidine-phosphotransferase domain and its interaction with cognate phosphoryl receiver domains

The Rcs signaling system in Escherichia coli controls a variety of physiological functions, including capsule synthesis, cell division and motility. The activity of the central regulator RcsB is modulated by phosphorylation through the sensor kinases YojN and RcsC, with the YojN histidine phosphotransferase (HPt) domain representing the catalytic unit that coordinates the potentially reversible phosphotransfer reaction between the receiver domains of the RcsB and RcsC proteins. Heteronuclear high-resolution NMR spectroscopy was employed to determine the solution structure of the YojN-HPt domain and to map the interaction with its two cognate receiver domains. The solution structure of YojN-HPt exhibits a well-ordered and rigid protein core consisting of the five helices alphaI to alphaV. The helices alphaII to alphaV form a four-helix bundle signature motif common to proteins of similar function, and helix alphaI forms a cap on top of the bundle. The helix alphaII is separated by a proline induced kink into two parts with different orientations and dynamic behavior that is potentially important for complex formation with other proteins. The N-terminal part of YojN-HPt spanning the first 26 amino acid residues seems to contain neither a regular secondary structure nor a stable tertiary structure and is disordered in solution. The identified YojN-HPt recognition sites for the regulator RcsB and for the isolated receiver domain of the RcsC kinase largely overlap in defined regions of the helices alphaII and alphaIII, but show significant differences. Using the residues with the largest chemical shift changes obtained from titration experiments, we observed a dissociation constant of approximately 200microM for YojN-HPt/RcsC-PR and of 40microM for YojN-HPt/RcsB complexes. Our data indicate the presence of a recognition area in close vicinity to the active-site histidine residue of HPt domains as a determinant of specificity in signal-transduction pathways.     

Dam Methylation Participates in the Regulation of PmrA/PmrB and RcsC/RcsD/RcsB Two Component Regulatory Systems in Salmonella enterica Serovar Enteritidis

The absence of Dam in Salmonella enterica serovar Enteritidis causes a defect in lipopolysaccharide (LPS) pattern associated to a reduced expression of wzz gene. Wzz is the chain length regulator of the LPS O-antigen. Here we investigated whether Dam regulates wzz gene expression through its two known regulators, PmrA and RcsB. Thus, the expression of rcsB and pmrA was monitored by quantitative real-time RT-PCR and Western blotting using fusions with 3×FLAG tag in wild type (wt) and dam strains of S. Enteritidis. Dam regulated the expression of both rcsB and pmrA genes; nevertheless, the defect in LPS pattern was only related to a diminished expression of RcsB. Interestingly, regulation of wzz in serovar Enteritidis differed from that reported earlier for serovar Typhimurium; RcsB induces wzz expression in both serovars, whereas PmrA induces wzz in S. Typhimurium but represses it in serovar Enteritidis. Moreover, we found that in S. Enteritidis there is an interaction between both wzz regulators: RcsB stimulates the expression of pmrA and PmrA represses the expression of rcsB. Our results would be an example of differential regulation of orthologous genes expression, providing differences in phenotypic traits between closely related bacterial serovars.     

Novel persistence genes in Pseudomonas aeruginosa identified by high-throughput screening

Salmonella enterica polymyxin B (PM) resistance is modulated mainly by substitutions of the acyl chains and the phosphate groups on the lipid A moiety of lipopolysaccharide. These modifications are mediated by genes under the control of the PmrA/PmrB and PhoP/PhoQ two-component regulatory systems. In this study, a deletion in the gene encoding the alternative σ⁵⁴ factor, rpoN , was shown to increase PM resistance without affecting protamine sensitivity. The results presented here showed that the increased polymyxin resistance observed in the Δ rpoN mutant occurs through a PmrA/PhoP-independent pathway. Downregulation of one or more genes belonging to the RpoN regulon may provide an additional mechanism of defence against membrane-permeabilizing antimicrobial peptides that helps the pathogen to survive in different environments.