Regulation by a novel protein of the bimodal distribution of lipopolysaccharide in the outer membrane of Escherichia coli

Abstract

We report on the cloning and characterization of the rJb gene cluster of the 075 lipopolysaccharide from a urinary tract isolate of Escherichia coli. Deletion cloning defined the minimum region of DNA that expressed the 075 antigen in E. coli host strains to be on a 12.4-kb insert. However, the E. coli strain expressing this region did not produce a polymerized 0 chain as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. A slightly larger DNA clone of 13.4 kb produced a polymerized 0 chain in E. coli S0874 but was found to be abnormal in its distribution over the surface membrane. Normal wild-type E. coli, as with Salmonella spp., has a bimodal distribution of the lipopolysaccharide on the surface which is seen as an abundance of long and short 0 chains attached to the lipid A-core structure. We found in a region adjacent to the cloned rib region, and on the opposite side from where the putative polymerase (rfc) is encoded, a novel protein of 35.5 kDa expressed from a 1.75-kb DNA fragment. This protein was shown to complement in trans the E. coli strains carrying plasmids that expressed abnormal, unregulated lipopolysaccharides. The expression of these complemented strains was bimodal in distribution. Mutation of the gene encoding this protein destroyed its ability to regulate 0-chain distribution. We propose to call this regulator gene rol, for regulator of 0 length.The lipopolysaccharide (LPS) antigen of Escherichia coli and Salmonella spp. consists of three different molecular species (lipid A, core oligosaccharide, and 0 polysaccharide) that are synthesized independently of each other and are ligated together in or on the inner membrane (17). The O-polysaccharide subunit typically consists of one to five sugar molecules linked together to form a macromolecular polymer of repeated units that vary from 1 to more than 40 in number (11). After assembly, the entire LPS molecule is translocated to the outer membrane by an unknown mechanism (11, 15). The lipid A and core units are embedded in the outer membrane, while the 0 antigen extends outward from the cell. The 0 antigen has been shown to be important in mediating the effects of serum resistance, infection by certain bacteriophages, and interaction with the microenvironment (4,20,22,25). The presence of 0 antigen on the surface of these bacteria confers a smooth phenotype and has been correlated to increased virulence (18).On sodium dodecyl sulfate (SDS)-polyacrylamide gels, silver-stained or radiolabeled LPS has a ladderlike appearance, with each rung or step of the ladder corresponding to a complete lipid A-core unit plus an 0 chain of a specific length. Several groups of investigators have noted a bimodal distribution of LPS from wild-type strains in stained SDSpolyacrylamide gels (3, 10, 21). High-molecular-weight 0 antigens with chain lengths of 19 to 34 make up to 77% of the total antigen, with low-molecular-weight 0 antigen (1 to 5 chain lengths) forming the bulk of the remainder. Intermediate sizes of 0 antigen ...

Engineering N-linked protein glycosylation with diverse O antigen lipopolysaccharide structures in Escherichia coli

“…LPS is subsequently transported to the outer leaflet of the outer membrane. O antigen exhibits a preferred and strain-specific size distribution pattern, which is mediated by Wzz protein (20).…”

Biochemical and Structural Analysis of Bacterial O-antigen Chain Length Regulator Proteins Reveals a Conserved Quaternary Structure

“…Homologs of the PCP-1a protein, Wzz, regulate the polymerization of LPS O-antigens to generate the characteristic strain-specific modality evident in silver-stained SDS-PAGE profiles. Batchelor et al (22,23) first identified a gene that was originally called rol (regulator of O chain length) or cld (chain length determinant) (16) in E. coli. Homologs of rol/cld have now been identified in many Gram-negative bacteria, and the genes have been renamed wzz (24).…”

Identification of O-antigen polymerase transcription and translation start signals and visualization of the protein in Salmonella enterica serovar Typhimurium

“…The wzy genes of some smooth strains of Escherichia coli have been identified either by detectable homology to S. typhimurium wzy (Yao & Valvano, 1994) or by the complementation of mutations in rfb regions (Batchelor et al, 1991 ;Liu & Reeves, 1994 ;Lukomski et al, 1996). Complementation analysis has also indicated that the wzy genes of Shigella dysenteriae (Klena & Schnaitman, 1993), Shigella flexneri (Morona et al, 1994), Yersinia enterocolitica (Zhang et al, 1996) and Pseudomonas aeruginosa (Coyne & Goldberg, 1995 ;De Kievit et al, 1995) are located in their respective rfb gene clusters.…”