Fine-structure mapping and identification of two regulators of capsule synthesis in Escherichia coli K-12

Abstract

Positive and negative regulatory elements involved in the synthesis of colanic acid, the capsular polysaccharide ofEscherichia coli K-12, have been identified previously. RcsB, a positive regulator for transcription of the structural genes of colanic acid synthesis (cps), is a protein of about 26 kilodaltons which probably acts as a multimer. rcsC, which maps close to rcsB at 48 min on the E. coli chromosome, exerts a negative effect on expression of the structural genes and codes for a protein of about 100 kilodaltons. The two genes appear to be transcribed in opposite directions, with the C-terminal ends of the genes being less than 0.3 kilobases apart. Multicopy expression of rcsB is lethal in rcsC mutants which carry cps-lac fusions, probably owing to accumulation of intermediates in the capsule synthesis pathway in these cells. Examination of double mutants and cells carrying multicopy rcsB+ plasmids reveal an rcsA-independent pathway for capsule synthesis. We hypothesize that RcsC may act as an environmental sensor, transmitting information to the RcsB positive regulator.The capsular polysaccharide colanic acid is synthesized by a variety of enteric bacteria. Although a function for colanic acid has not been clearly demonstrated, similar capsules in other gram-negative organisms serve to protect cells from dehydration, from bacteriophage infection, and from cellular immunity systems (3, 10, 17). In plant pathogens such as Erwinia stewartii, the capsule acts as an important virulence factor (4, 9). Capsular polysaccharides have also been implicated in the plant-bacterium interactions of Rhizobium species (21).We have been investigating the regulation of colanic acid synthesis in Escherichia coli K-12 by monitoring the expression of lac operon fusions to genes necessary for colanic acid synthesis (cpsA to cpsF) (13,39,41). Using these fusions, we have identified four loci which have major effects on cps-lac expression. Two negative regulators, lon and rcsC, and two positive regulators, rcsA and rcsB, have been identified (13,41).Lon is a major ATP-dependent protease in E. coli (6,8,11). Among the pleiotropic effects of lon are filamentation after DNA-damaging treatments such as UV irradiation and methyl methanesulfonate treatment, failure to degrade abnormal and some normal proteins, and the mucoidy associated with overproduction of colanic acid (12,15,16,25). We have hypothesized that the stabilization of regulatory proteins in lon mutants is responsible for the phenotypes of lon cells (14). SulA, an inhibitor of cell septation induced after DNA damage, seems to be responsible for the filamentation phenotype; it has a half-life of 1. The other two regulators of capsule synthesis, rcsB and rcsC, map near to each other at 48 min and have opposite effects on the synthesis of capsular polysaccharide. Mutations in rcsB, like those in rcsA, reduce synthesis in lon cells, whereas rcsC mutations increase expression in lon+ hosts. Preliminary complementation studies with these mutations, carried out with a cosmid v...

Elevated levels of σ S inhibit biofilm formation in Escherichia coli: a role for the Rcs phosphorelay

“…The increase in RcsB-P levels in the rcsC mutant background would be expected to be quite modest since there is no significant induction in the production of colanic acid (our unpublished observations; Brill et al, 1988). Nonetheless, if the increased level of RcsB-P is directly responsible for the observed defect in biofilm formation, we would expect that full activation of the Rcs phosphorelay would have the same phenotype.…”

Exploring the relationship between lipoprotein mislocalization and activation of the Rcs signal transduction system in Escherichia coli

“…When pHR725 was introduced into the rcsC137 mutant SG20803, in which the Rcs system is constitutively activated (Brill et al, 1988), suppression of the Rcs activation was not observed on LB agar containing X-Gal and 0.5 mM IPTG: the blue colony colour did not differ from that of the mutant carrying the vector plasmid pHR718 (data not shown). This suggests that lgt overexpression does not affect the Rcs signal transduction pathway downstream of RcsC.…”

Virulence attenuation in Salmonella enterica rcsC mutants with constitutive activation of the Rcs system

“…Two sets of genes whose regulation by the Rcs system is well known, both in E. coli and in S. enterica serovar Typhimurium, are colanic acid genes (Brill et al, 1988;Cano et al, 2002), and genes involved in flagella synthesis and in chemotaxis (Cano et al, 2002;Francez-Charlot et al, 2003). Control of flagella and chemotaxis is exercised through the master operon flhDC (Macnab, 1996).…”

“…The Rcs phosphorelay system was initially characterized in Escherichia coli as a two-component positive regulator of colanic acid capsule synthesis encoded by rcsC and rcsB (Brill et al, 1988;Gottesman et al, 1985;Stout & Gottesman, 1990). The sensor protein RcsC, a hybrid histidine kinase, and the transcriptional activator RcsB are the main components of the system, which also includes a second transcriptional activator, RcsA (Stout et al, 1991) and an intermediate phosphotransmitter, YojN (Takeda et al, 2001).…”