Isolation from Klebsiella and characterization of two rcs genes that activate colanic acid capsular biosynthesis in Escherichia coli

Two genes, designated rcsA (regulation of capsule synthesis) and rcsB, that had been cloned from the chromosome of Klebsiella aerogenes (K. pneumoniae) capsular serotype K21 were capable of activating expression of colanic acid capsular polysaccharide in Escherichia coli K12. The Klebsiella rcsA gene encoded a polypeptide of 23 kDa that was required for the induction of a mucoid phenotype at less than or equal to 30 degrees C but not at greater than or equal to 37 C. The Klebsiella rcsB locus encoded no apparent polypeptides and was not capable by itself of causing the overproduction of colanic acid. However, when present in the same cell with rcsA, either in cis or in trans, rcsB caused expression of mucoidy in E. coli at all growth temperatures. These findings are best explained if the Klebsiella rcsA gene product acts as a positive regulator of colanic acid biosynthesis in E. coli and that activity of this protein is in turn subject to regulation by Lon protease. The Klebsiella rcsB locus may exert its effect by preferentially binding a negative regulator of capsular biosynthesis, possibly Lon itself. DNA sequences homologous to the Klebsiella K21b rcsA and rcsB genes were found in the genomes of all other capsular serotypes of klebsiellae examined, including K2, K12, K36 and K43. However, there was no homology between such genes and the chromosome of E. coli. The ability of these rcs genes to induce a mucoid phenotype explains the apparent conjugative transfer from klebsiellae to E. coli of the ability to produce K21 or other Klebsiella capsular polysaccharides that are structurally and antigenically related to colanic acid.     

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.     

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.     

An extraintestinal, pathogenic isolate of Escherichia coli (O4/K54/H5) can produce a group 1 capsule which is divergently regulated from its constitutively produced group 2, K54 capsular polysaccharide.

We are studying an O4/K54/H5 Escherichia coli bacteremic isolate (CP9) as a model pathogen for extraintestinal infection. Its group 2, K54 capsular polysaccharide is an important virulence determinant and confers serum resistance. In this study the effect of the group 1 capsule regulators, RcsA, RcsB, and Lon protease, on the regulation of CP9's capsular polysaccharides was assessed. It was established that in the presence of multicopy rcsA or with disruption of lon, CP9 can be induced to produce a group 1 capsule. RcsA, RcsB, and Lon are present in this K54 background and regulate group 1 capsule expression in a fashion similar to that described for K-12 strains. Two independent group 2 capsule gene protein fusions (cl1.29::TnphoA and cl1.137::TnphoA) were used to evaluate the effects of these regulators on group 2 K54 capsule production. Disruption of lon resulted in 1.9-fold (TR293 [cl1.29::TnphoA lon-146]) and 3.4-fold (TR1373 [cl1.137::TnphoA lon-146]) decreases in fusion activity at 28 degrees C, relative to the baseline level. However, decreases in fusion activity at 42 degrees C were only 1.2- and 1.4-fold, respectively. Inactivation of both lon and rcsA or lon and rcsB restored fusion activity to baseline levels at 28 degrees C, but only a partial restoration of activity was seen at higher temperatures. To assess whether these differences in fusion activity reflected a functional change in capsule production, the effects of 80% normal human serum (NHS) were tested against CP9 and TR93 (lon-146). Since the group 2 K54 capsule protects against the bactericidal activity of 80% NHS, a decrease in its production results in an increase in serum sensitivity. Viable counts of CP9 increased 10-fold in 80% NHS over 3 h at 28 degrees C, as expected. In contrast to CP9, TR93 (lon-146) incurred a 10-fold loss in viability under the same conditions. The levels of RcsA are increased in TR93 (lon 146) as consequence of lon disruption; therefore, these results in conjunction with the cl1::TnphoA protein fusion data establish RcsA as a negative regulator of the group 2 K54 capsular polysaccharide. Furthermore, these results also suggest existence of another Lon-sensitive negative regulator of group 2 K54 capsule production, which is active higher temperatures.     

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.     

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.     

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.     

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.