Identification of a second RcsA protein, a positive regulator of colanic acid capsular polysaccharide genes, in Escherichia coli.

A second form of RcsA, a positive activator of the capsular polysaccharide genes (cps), has been identified in Escherichia coli. Ferguson plot analysis suggests that the two RcsA proteins differ by size rather than by charge. Both RcsA proteins are expressed from a single rcsA gene. Detection of both RcsA proteins in delta lon cells is RcsB dependent.     

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.     

Identification, Genomic Organization, and Analysis of the Group III Capsular Polysaccharide Genes kpsD, kpsM, kpsT, and kpsE from an Extraintestinal Isolate of Escherichia coli (CP9, O4/K54/H5)

Group III capsular polysaccharides (e.g., K54) of extraintestinal isolates of Escherichia coli, similar to group II capsules (e.g., K1), are important virulence traits that confer resistance to selected host defense components in vitro and potentiate systemic infection in vivo. The genomic organization of group II capsule gene clusters has been established as a serotype-specific region 2 flanked by regions 1 and 3, which contain transport genes that are highly homologous between serotypes. In contrast, the organization of group III capsule gene clusters is not well understood. However, they are defined in part by an absence of genes with significant nucleotide homology to group II capsule transport genes in regions 1 and 3. Evaluation of isogenic, TnphoA-generated, group III capsule-minus derivatives of a clinical blood isolate (CP9, O4/K54/H5) has led to the identification of homologs of the group II capsule transport genes kpsDMTE. These genes and their surrounding regions were sequenced and analyzed. The genomic organization of these genes is distinctly different from that of their group II counterparts. Although kps_K54DMTE are significantly divergent from their group II homologs at both the DNA and protein levels phoA fusions and computer-assisted analyses suggest that their structures and functions are similar. The putative proteins Kps_K54M and Kps_K54T appear to be the integral membrane component and the peripheral ATP-binding component of the ABC-2 transporter family, respectively. The putative Kps_K54E possesses features similar to those of the membrane fusion protein family that facilitates the passage of large molecules across the periplasm. At one boundary of the capsule gene cluster, a truncated kpsM (kpsM_truncated) and its 5′ noncoding regulatory sequence were identified. In contrast to the complete kps_K54M, this region was highly homologous to the group II kpsM. Fifty-three base pairs 3′ from the end of kpsM_truncated was a sequence 75% homologous to the 39-bp inverted repeat in the IS110 insertion element from Streptomyces coelicolor. Southern analysis established that two copies of this element are present in CP9. These findings are consistent with the hypothesis that CP9 previously possessed group II capsule genes and acquired group III capsule genes via IS110-mediated horizontal transfer.     

Generation of isogenic K54 capsule-deficient Escherichia coli strains through TnphoA-mediated gene disruption

Transposon muta genesis, using IS50^L::phoA(Tn-phoA), was performed in a K54/O4/H5 blood isolate of Escherichia coli (CP9), to generate a library of random mutants. Five hundred and twenty-six independent CP9 TnphoA mutants were isolated with active gene fusions to alkaline phosphatase. From this mutant library, eight capsule-deficient strains were detected and were found to have a single copy of TnphoA. Sixteen additional capsule deficient mutants with TnphoA inserts were subsequently obtained that did not possess active PhoA fusions. In conjunction with the initial eight capsule-deficient isolates we have defined genes on three different XbaI fragments as being involved in capsule production. Generalized transduction with the bacteriophage T4 established that these insertions were responsible for the loss of capsule and that they are linked. These capsule-deficient strains can be used to assess the pathogenic role of the K54 capsular polysaccharide.     

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.     

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.     

Expression of the tolQRA genes of Escherichia coli K-12 is controlled by the RcsC sensor protein involved in capsule synthesis

The tolQRABpal cluster of Escherichia coli K-12 encodes proteins involved in the maintenance of cell-envelope integrity. In addition, toi/pal mutations result in a mucoid colony phenotype at low temperature. The synthesis of capsular polysaccharides by the cps genes is controlled by the positive regulator RcsA and the two-component RcsC/RcsB system. It was shown that the mucoid phenotype of the tol/pal mutants was due to an rcsCB-dependent activation of the cps genes. Furthermore, we have identified a mutation in the rcsC gene that decreased the activity of a tolA-lac operon fusion independently of RcsA and partially independently of RcsB activators. The corresponding rcsC338 mutation resulted in a Glu to Lys substitution at residue 338 of RcsC. This mutation induced mucoidy even at high temperature. We propose that RcsC modulates the phosphorylated forms of RcsB and an uncharacterized regulatory protein involved in the control of the tolQRA genes in an opposite manner. Moreover, our findings strengthen the previous suggestion that RcsC senses some alterations in the cell surface such as those induced by tol, pal or rfa mutations, and activates capsule synthesis to protect the cell against deleterious agents.     

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.     

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     

Regulation of breakdown of canavanyl proteins in Escherichia coli by growth conditions in Ion+ and Ion− cells

In vivo rates of proteolysis of canavanyl proteins were compared in lon+ and lon- Escherichia coli strains following growth in a variety of media. Both lon+ and lon- cells grown rapidly in complex media possessed higher levels of constitutive degradative activity than when cultured in minimal media. Pre-growth of lon+ cells in the presence of canavanine induced proteolytic activity following growth in minimal media as did stress agents such as heat, alcohol and puromycin: the lon mutant did not show the increased activity following canavanine treatment. The results suggest the presence of a proteolytic activity which selectively degrades aberrant proteins which does not involve protease La, the product of the lon gene, and which furthermore is regulated in part by growth conditions independently of the stress response.