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.     

Control of Colanic Acid Synthesis

The nucleotide pools of certain mucoid, colanic acid-synthesizing strains of Escherichia coli, Salmonella typhimurium, and Aerobacter cloacae were examined, and in all cases the nucleotide sugars uridine-5'-diphosphate glucose (UDPG), uridine-5'-diphosphate galactose (UDPGal), guanosine-5'-diphosphate fucose (GDPF), and uridine-5'-diphosphate glucuronic acid (UDPGA) were detected. It is postulated that these nucleotide sugars are precursors in the synthesis of colanic acid. The levels of these nucleotide sugars and of the enzymes involved in their synthesis were examined in a number of mucoid strains and compared with the levels found in certain strains which were repressed in the synthesis of colanic acid, only becoming mucoid when grown in the presence of p-fluorophenylalanine (PFA). The levels of UDPG and UDPGal and the enzymes involved in their synthesis were substantially the same in both mucoid and repressed types, but the levels of UDPGA and GDPF and of some of the enzymes involved in their synthesis were much higher in mucoid strains. When repressed strains were grown in the presence of PFA, the levels of UDPGA and GDPF approached those found in mucoid strains. The existence of an operon, containing genes coding for certain key enzymes involved in colanic acid synthesis has been suggested.     

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.     

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.     

A method for constructing single-copy lac fusions in Salmonella typhimurium and its application to the hemA-prfA operon.

This report describes a set of Escherichia coli and Salmonella typhimurium strains that permits the reversible transfer of lac fusions between a plasmid and either bacterial chromosome. The system relies on homologous recombination in an E. coli recD host for transfer from plasmid to chromosome. This E. coli strain carries the S. typhimurium put operon inserted into trp, and the resulting fusions are of the form trp::put::[Kanr-X-lac], where X is the promoter or gene fragment under study. The put homology flanks the lac fusion segment, so that fusions can be transduced into S. typhimurium, replacing the resident put operon. Subsequent transduction into an S. typhimurium strain with a large chromosomal deletion covering put allows selection for recombinants that inherit the fusion on a plasmid. A transposable version of the put operon was constructed and used to direct lac fusions to novel locations, including the F plasmid and the ara locus. Transductional crosses between strains with fusions bearing different segments of the hemA-prfA operon were used to determine the contribution of the hemA promoter region to expression of the prfA gene and other genes downstream of hemA in S. typhimurium.     

Isolation and characterization of Escherichia coli strains containing new gene fusions (soi::lacZ) inducible by superoxide radicals.

Gene fusions in Escherichia coli that showed increased beta-galactosidase expression in response to treatment with a superoxide radical (O2-) generator, methyl viologen (MV), were obtained. These fusions were constructed by using a Mud(Ap lac) phage to insert the lactose structural genes randomly into the E. coli chromosome. Ampicillin-resistant colonies were screened for increased expression of beta-galactosidase on X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) plates containing MV at 1.25 micrograms/ml. Other O2- generators, menadione and plumbagin, also induced beta-galactosidase activity in these fusion strains. The induction by these drugs occurred only under aerobic conditions. Hyperoxygenation also elicited an induction of the fusions. On the other hand, no significant induction was observed with hydrogen peroxide and cumene hydroperoxide. The induction of these fusions by MV was not dependent on the peroxide stress control mediated by the oxyR gene or on the recA-dependent SOS system. These fusions were named soi (superoxide inducible)::lacZ. The induction of beta-galactosidase was significantly reduced by introducing a soxS::Tn10 locus into the fusion strains, indicating that the soi genes are members of the soxRS regulon. Five of the fusions were located in 6 to 26 min of the E. coli genetic map, while three fusions were located in 26 to 36 min, indicating that these fusions are not related to genes already known to be inducible by O2- under the control of soxRS. At least five mutants containing the soi::lacZ fusion were more sensitive to MV and menadione than the wild-type strain, suggesting that the products of these soi genes play an important role in protection against oxidative stress.     

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.     

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.     

Maximizing gene expression on a plasmid using recombination in vitro.

Recombination in vitro has been used to place one or more copies of a strong promoter, the lac promoter, at varying distances from the cl (repressor) gene of bacteriophage lambda on the E. coli plasmid pMB9. In all constructions, lambda repressor synthesis is driven wholly or predominantly by the inserted lac promoter. One of our fusions directs the synthesis of very high levels of lambda repressor. In this case, the fused DNA encodes a ribosome binding site which is a "hybrid" of lambda and lac sequences. In principle, this method of construction should elicit high levels of expression in E. coli of any gene, whatever its source. We also described strains with different sequence arrangements that, for reasons not completely understood, produce less repressor.     

Effect of rfaH (sfrB) and temperature on expression of rfa genes of Escherichia coli K-12.

In order to study the regulation of a large block of contiguous genes at the rfa locus of Escherichia coli K-12 which are involved in synthesis and modification of the lipopolysaccharide core, the transposon TnlacZ was used to generate in-frame lacZ fusions to the coding regions of five genes (rfaQ, -G, -P, -B and -J) within this block. The beta-galactosidase activity of strains in which these fusions had been crossed into the chromosomal rfa locus was significantly decreased when the rfaH11 (sfrB11) allele was introduced and was restored to wild-type levels when these strains were lysogenized with a lambda phage carrying wild-type rfaH. This indicates that the positive regulatory function encoded by rfaH is required throughout this block of genes. In addition, expression of the lacZ fusion to rfaJ was reduced by growth at 42 degrees C, and this correlated with a temperature-induced change in the electrophoretic profile of the core lipopolysaccharide.     

New locus for exopolysaccharide overproduction in Escherichia coli K-12.

A new locus for exopolysaccharide overproduction in Escherichia coli K-12 was mapped by insertion mutagenesis. A 66% linkage to serA, which is located at 62 min on the E. coli K-12 linkage map, was shown by P1 transduction. The polysaccharide produced by the mutant was isolated and was shown to be similar to colanic acid.