Activation of the Rcs signal transduction system is responsible for the thermosensitive growth defect of an Escherichia coli mutant lacking phosphatidylglycerol and cardiolipin

The lethal effect of an Escherichia coli pgsA null mutation, which causes a complete lack of the major acidic phospholipids, phosphatidylglycerol and cardiolipin, is alleviated by a lack of the major outer membrane lipoprotein encoded by the lpp gene, but an lpp pgsA strain shows a thermosensitive growth defect. Using transposon mutagenesis, we found that this thermosensitivity was suppressed by disruption of the rcsC, rcsF, and yojN genes, which code for a sensor kinase, accessory positive factor, and phosphotransmitter, respectively, of the Rcs phosphorelay signal transduction system initially identified as regulating the capsular polysaccharide synthesis (cps) genes. Disruption of the rcsB gene coding for the response regulator of the system also suppressed the thermosensitivity, whereas disruption of cpsE did not. By monitoring the expression of a cpsB'-lac fusion, we showed that the Rcs system is activated in the pgsA mutant and is reverted to a wild-type level by the rcs mutations. These results indicate that envelope stress due to an acidic phospholipid deficiency activates the Rcs phosphorelay system and thereby causes the thermosensitive growth defect independent of the activation of capsule synthesis.     

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.     

Regulation of capsular polysaccharide synthesis in Escherichia coli K-12: characterization of three regulatory genes.

The synthesis of the Escherichia coli capsular polysaccharide varies with growth medium, temperature of growth, and genetic background. lac fusions to genes necessary for capsule synthesis (cps) demonstrated that these genes are regulated negatively in vivo by the lon gene product. We have now isolated, characterized, and mapped mutations in three new regulatory genes (rcs, for regulator of capsule synthesis) that control expression of these same fusions. rcsA and rcsB are positive regulators of capsule synthesis. rcsA is located at min 43 on the E. coli map, whereas rcsB lies at 47 min. rcsC, a negative regulator of capsule synthesis, is located at min 47, close to rcsB. All three regulatory mutations are unlinked to either the structural genes cpsA-F or lon. Mutations in all three rcs genes are recessive to the wild type. We postulate that lon may regulate capsule synthesis indirectly, by regulating the availability of one of the positive regulators.     

Constitutive expression of Pasteurella multocida toxin

Abstract The introduction of a plasmid containing skc (streptokinase-coding gene) fused with ompA signal sequence into Escherichia coli K-12 strains, rendered the bacteria mucoid. Measurement of the synthesis of β-galactosidase from a cps-lacZ fusion ( lacZ fusion to a gene necessary for capsule synthesis) showed that the mucoid phenotype was due to induction of the capsular polysaccharide colanic acid synthesis. The introduction of a plasmid carrying skc fused with malE (gene encoding maltose-binding protein) also induced cps-lacZ expression, but intracellular expression of streptokinase in E. coli did not. The cps expression by secretion of streptokinase was diminished to the basal level in a cps-lacZ strain carrying a rcsC mutation. These results show that the secretion of streptokinase in E. coli induces colanic acid synthesis through the RcsC-dependent pathway.     

Characterization of the rcsB gene from Erwinia amylovora and its influence on exoploysaccharide synthesis and virulence of the fire blight pathogen.

RcsB belongs to a family of positive regulators of exopolysaccharide synthesis in various enterobacteria. The rcsB gene of the fire blight pathogen Erwinia amylovora was cloned by PCR amplification with consensus primers, and its role in exopolysaccharide (EPS) synthesis was investigated. Its overexpression from high-copy-number plasmids stimulated the synthesis of the acidic EPS amylovoran and suppressed expression of the levan-forming enzyme levansucrase. Inactivation of rcsB by site-directed mutagenesis created mutants that were deficient in amylovoran synthesis and avirulent on host plants. In addition, a cosmid which complemented rcsB mutants was selected from a genomic library. The spontaneous E. amylovora mutant E8 has a similar phenotype and was complemented by the cloned rcsB gene. The rcsB region of strain E8 was also amplified by PCR, and the mutation was characterized as a nine-nucleotide deletion at the start of the rcsB gene. Nucleotide sequence analysis of the E. amylovora rcsB region and the predicted amino acid sequence of RcsB revealed extensive homology to rcsB and the encoded protein of other bacteria such as Escherichia coli and Erwinia stewartii. In all three organisms, rcsB is localized adjacent to the rcsC gene, which is transcribed in the opposite direction of rcsB. The E. amylovora rcsB gene has now been shown to strongly affect the formation of disease symptoms of a plant pathogen.     

Coexpression of colanic acid and serotype-specific capsular polysaccharides in Escherichia coli strains with group II K antigens.

In Escherichia coli K-12, the rcsA and rcsB gene products are positive regulators in expression of the slime polysaccharide colanic acid. We have previously demonstrated the presence of rcsA sequences in E. coli K1 and K5, strains with group II capsular K antigens, and shown that introduction of multicopy rcsA into these strains results in the expression of colanic acid. We report here the presence of rcsB sequences in E. coli K1 and K5 and demonstrate that RcsB also plays a role in the biosynthesis of colanic acid in strains with group II K antigens. In E. coli K1 and K5 grown at 37 degrees C, multicopy rcsB and the resulting induction of colanic acid synthesis had no significant effect on synthesis of the group II K antigens. K-antigen-specific sugar transferase activities were not significantly different in the presence or absence of multicopy rcsB, and introduction of a cps mutation to eliminate colanic acid biosynthesis in a K1-derivative strain did not influence the activity of the polysialyltransferase enzyme responsible for synthesis of the K1 polymer. Furthermore, immunoelectron microscopy showed no detectable difference in the size or distribution of the group II K-antigen capsular layer in cells which produced colanic acid. Colanic acid expression therefore does not appear to significantly affect synthesis of the group II K-antigen capsule and, unlike for group I K antigens, expression of group II K antigens is not positively regulated by the rcs system.     

Reconstruction of Escherichia coli mrcA (PBP 1a) mutants lacking multiple combinations of penicillin binding proteins

Previously, we constructed a set of mutants from which eight penicillin binding protein (PBP) genes were deleted in 192 combinations from Escherichia coli (S. A. Denome, P. K. Elf, T. A. Henderson, D. E. Nelson, and K. D. Young, J. Bacteriol. 181:3981-3993, 1999). Although these mutants were constructed correctly as determined by restriction mapping and the absence of relevant protein products, we recently discovered by PCR mapping that strains from which mrcA (PBP 1a) was deleted were also missing two neighboring genes of unknown function (yrfE and yrfF). We created a new deletion mutation in mrcA and reconstructed 63 strains lacking PBP 1a and other PBP mutant combinations. The new mrcA mutants do not exhibit mucoidy, phage resistance, temperature sensitivity, growth rate defects, or antibiotic resistance, suggesting that these phenotypes require the loss of either yrfE or yrfF alone or in combination with the absence of multiple PBPs.     

Characterization of rcsB and rcsC from Escherichia coli O9:K30:H12 and examination of the role of the rcs regulatory system in expression of group I capsular polysaccharides.

In Escherichia coli K-12, RcsC and RcsB are thought to act as the sensor and effector components, respectively, of a two-component regulatory system which regulates expression of the slime polysaccharide colanic acid (V. Stout and S. Gottesman, J. Bacteriol. 172:659-669, 1990). Here, we report the cloning and DNA sequence of a 4.3-kb region containing rcsC and rcsB from E. coli O9:K30:H12. This strain does not produce colanic acid but does synthesize a K30 (group I) capsular polysaccharide. The rcsB gene from E. coli K30 (rcsBK30) is identical to the rcsB gene from E. coli K-12 (rcsBK-12). rcsCK30 has 16 nucleotide changes, resulting in six amino acid changes in the predicted protein. To examine the function of the rcs regulatory system in expression of the K30 capsular polysaccharide, chromosomal insertion mutations were constructed in E. coli O9:K30:H12 to independently inactivate rcsBK30 and the auxiliary positive regulator rcsAK30. Strains with these mutations maintained wild-type levels of K30 capsular polysaccharide expression and still produced a K30 capsule, indicating that the rcs system is not essential for expression of low levels of the group I capsular polysaccharide in lon+ E. coli K30. However, K30 synthesis is increased by introduction of a multicopy plasmid carrying rcsBK30. K30 polysaccharide expression is also markedly elevated in an rcsBK30-dependent fashion by a mutation in rcsCK30, suggesting that the rcs system is involved in high levels of synthesis. To determine whether the involvement of the rcs system in E. coli K30 expression is typical of group I (K antigen) capsules, multicopy rcsBK30 was introduced into 22 additional strains with structurally different group I capsules. All showed an increase in mucoid phenotype, and the polysaccharides produced in the presence and absence of multicopy rcsBK30 were examined. It is has been suggested that E. coli strains with group I capsules can be subdivided based on K antigen structure. For the first time, we show that strains with group I capsules can also be subdivided by the ability to produce colanic acid. Group IA contains capsular polysaccharides (including K30) with repeating-unit structures lacking amino sugars, and expression of group IA capsular polysaccharides is increased by multicopy rcsBK30. Group IB capsular polysaccharides all contain amino sugars. In group IB strains, multicopy rcsBK30 activates synthesis of colanic acid.     

Repression of the RcsC-YojN-RcsB phosphorelay by the IgaA protein is a requisite for Salmonella virulence

Bacterial pathogenesis relies on regulators that activate virulence genes. Some of them act, in addition, as repressors of specific genes. Intracellular-growth-attenuator-A (IgaA) is a Salmonella enterica membrane protein that prevents overactivation of the RcsC-YojN-RcsB regulatory system. This negative control is critical for growth because disruption of the igaA gene is only possible in rcsC, yojN or rcsB strains. In this work, we examined the contribution of this regulatory circuit to virulence. Viable igaA point mutant alleles were isolated and characterized. These alleles encode IgaA variants leading to different levels of activation of the RcsC-YojN-RcsB system. IgaA-mediated repression of the RcsB-YojN-RcsC system occurred at the post-translational level, as shown by chromosomal epitope tagging of the rcsC, yojN and rcsB genes. The activity of the RcsC-YojN-RcsB system, monitored with the product of a tagged gmd-3xFLAG gene (positively regulated by RcsC-YojN-RcsB), was totally abolished by wild-type bacteria in mouse target organs. Such tight repression occurred only in vivo and was mediated by IgaA. Shutdown of the RcsC-YojN-RcsB system is a requisite for Salmonella virulence since all igaA point mutant strains were highly attenuated. The degree of attenuation correlated to that of the activation status of RcsC-YojN-RcsB. In some cases, the attenuation recorded was unprecedented, with competitive index (CI) values as low as 10(-6). Strikingly, IgaA is a protein absolutely dispensable for virulence in mutant strains having a non-functional RcsC-YojN-RcsB system. To our knowledge, IgaA exemplifies the first protein that contributes to virulence by exclusively acting as a negative regulator upon host colonization.     

Regulation of Escherichia coli cell division genes ftsA and ftsZ by the two-component system rcsC-rcsB

Genes rcsC and rcsB form a two-component system in which rcsC encodes the sensor element and rcsB the regulator. In Escherichia coli, the system positively regulates the expression of the capsule operon, cps, and of the cell division gene ftsZ. We report the identification of the promoter and of the sequences required for rcsB-dependent stimulation of ftsZ expression. The promoter, ftsA1p, located in the ftsQ coding sequence, co-regulates ftsA and ftsZ. The sequences required for rcsB activity are immediately adjacent to this promoter.     

Inactivation of mdoH leads to increased expression of colanic acid capsular polysaccharide in Escherichia coli.

Capsule gene (cps) expression, which normally occurs at low levels in Escherichia coli lon+ cells, increased 38-fold in lon+ cells carrying a Tn10::delta kan insertion mapping to 24 min on the E. coli chromosome. Null mutations in rcsA, rcsB, or rcsC abolished the effect of the Tn10::delta kan insertion. Sequencing of both sides of the Tn10::delta kan insertion localized the insertion to the previously reported mdoH gene, which encodes a protein involved in biosynthesis of membrane-derived oligosaccharides (MDOs). A model suggesting that the periplasmic levels of MDOs act to signal RcsC to activate cps expression is proposed.     

外环境压力对福氏志贺菌荚膜异多糖酸合成调节子转录的影响

荚膜异多糖酸合成调节子(regulator of colanic acid capsule synthesis,Rcs)对大肠埃希菌适应外环境压力具有重要调控功能,但其在志贺菌中的功能尚未见报道。为探索外环境压力对福氏志贺菌Rcs编码基因rcs转录水平的影响,本研究采用核苷酸序列比对及蛋白结构域预测等生物信息学方法分析福氏志贺菌的Rcs编码基因簇rcsBDC,利用实时定量聚合酶链反应(quantitative real-time polymerase chain reaction,qRT-PCR),对该菌不同生长时期的rcsB、rcsD、rcsC基因转录水平进行分析,并检测在不同pH值培养基、渗透压条件下的基因转录水平。结果显示,福氏志贺菌rcsBDC在培养5～6 h(对数中期)时转录水平较高,8～10 h(稳定期)时转录水平较低(P<0.001);10 h时,rcsB和rcsD在酸性、渗透压条件下的转录水平均显著高于正常条件下的转录水平(P<0.05)。结果提示,外环境刺激可提高福氏志贺菌在稳定期的rcsB和rcsD转录水平,为志贺菌适应胃肠道酸性、渗透压环境的机制研究提供了一定理论基础。     

Expression of Vi antigen in Escherichia coli K-12: characterization of ViaB from Citrobacter freundii and identity of ViaA with RcsB.

The Vi antigen in Salmonella typhi is stably expressed and may act to protect the strain against the defensive system of the host. Citrobacter freundii, not usually a common human pathogen, also expresses the Vi antigen but expresses it unstably, exhibiting a reversible transition between the Vi+ and Vi- states. Two widely separated chromosomal regions, ViaA and ViaB, are needed for Vi synthesis. Escherichia coli K-12 harboring a functional ViaB plasmid can also express Vi antigen, but the cloned ViaB sequence can only be stably maintained and expressed in recA hosts. Vi- derivatives arise either through IS1-like insertional events occurring in ViaB sequences or by chromosomal mutations at the ViaA region. P1vir mapping indicates that the ViaA mutations are located at min 47.75 on the E. coli chromosome. All the spontaneous viaA mutants isolated from E. coli and S. typhi were identified as rcsB mutants by complementation tests using plasmid pJB100. Introduction of rcsA::Tn10 into E. coli harboring functional ViaB sequences eliminates the expression of Vi antigen. These results indicate that Vi antigen synthesis is regulated by the same regulatory proteins involved in colanic acid synthesis in E. coli.