The rol (cld) gene encodes a protein involved in the expression of lipopolysaccharides in some members of the family Enterobacteriaceae. Rol interacts with one or more components of Rfc-dependent O-antigen biosynthetic complexes to regulate the chain length of lipopolysaccharide O antigens. The Rfc-Rol-dependent pathway for O-antigen synthesis is found in strains with heteropolysaccharide O antigens, and, consistent with this association, rol-homologous sequences were detected in chromosomal DNAs from 17 different serotypes with heteropolysaccharide O antigens. Homopolymer O antigens are synthesized by a pathway that does not involve either Rfc or Rol. It was therefore unexpected when a survey of Escherichia coli strains possessing mannose homopolymer O8 and O9 antigens showed that some strains contained rol. All 11 rol-positive strains coexpressed a group IB capsular K antigen with the O8 or O9 antigen. In contrast, 12 rol-negative strains all produced group IA K antigens in addition to the homopolymer O antigen. Previous research from this and other laboratories has shown that portions of the group I K antigens are attached to lipopolysaccharide lipid A-core, in a form that we have designated K LPS . By constructing a hybrid strain with a deep rough rfa defect, it was shown that the K40 (group IB) K LPS antigen exists primarily as long chains. However, a significant amount of K40 antigen was surface expressed in a lipid A-core-independent pathway. The typical chain length distribution of the K40 antigen was altered by introduction of multicopy rol, suggesting that the K40 group IB K antigen is equivalent to a Rol-dependent O antigen. The prototype K30 (group IA) K antigen is expressed as short oligosaccharides (primarily single repeat units) in K LPS , as well as a high-molecular-weight lipid A-core-independent form. Introduction of multicopy rol into the K30 strain generated a novel modal pattern of K LPS with longer polysaccharide chains. Collectively, these results suggested that group IA K LPS is also synthesized by a Rol-dependent pathway and that the typically short oligosaccharide K LPS results from the absence of Rol activity in these strains.

In Escherichia coil 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

Escherichia coli K antigens (capsular polysaccharides) are divided into two broad classes, designated groups I and II, on the basis of a number of chemical, physical, and genetic criteria. Group I K antigens can be further subdivided on the basis of the absence (group IA) or presence (group IB) of amino sugars in the repeating unit of the K antigen. One criterion proposed for inclusion in group I is covalent linkage of the capsular polysaccharide to the lipid A-core of lipopolysaccharide (LPS). E. coli O9:K30 is a strain with a representative group IA K antigen. This organism synthesizes an LPS-associated low-molecular-weight form of K30 antigen which is called K(LPS). To determine the involvement of LPS lipid A-core in expression of the K30 capsular polysaccharide, E. coli K30/K-12 hybrid strains were constructed with mutations in the E. coli K-12 rfa locus, responsible for the biosynthesis of the LPS core oligosaccharide. These strains lack K(LPS), indicating that a full-length core is required for K(LPS) expression. However, formation of a K30 capsule was unaffected by rfa defects, indicating that attachment to lipid A-core is not an obligatory step for either export of high-molecular-weight capsular polysaccharide or maintenance of the capsular structure on the cell surface. Silver-stained tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of lipopolysaccharides from other E. coli K serotypes showed that all strains with group IB K antigens expressed some K(LPS). In contrast, some strains with group IA K antigens appear to lack K(LPS). Consequently, although association of group 1 K antigens with lipid A-core is common, it is not a universal marker for inclusion in group I.

The rfbg,, gene cluster, which is responsible for the synthesis of the lipopolysaccharide 09 antigen, was cloned from Escherichia coi 09:130. The gnd gene, encoding 6-phosphogluconate dehydrogenase, was identified adjacent to the rJb09 cluster, and by DNA sequence analysis the gene order gnd-rJbM-rjbK was established. This order differs from that described for other members of the family Enterobacteriaceae.Nucleotide sequence analysis was used to identify the rftK and rJbM genes, encoding phosphomannomutase and GDP-mannose pyrophosphorylase, respectively. In members of the family Enterobacteriaceae, these enzymes act sequentially to form GDP-mannose, which serves as the activated sugar nucleotide precursor for mannose residues in cell surface polysaccharides. In the E. coli 09:K30 strain, a duplicated rJbM2-rJbK2 region was detected approximately 3 kbp downstream of rJbM'-dbK' and adjacent to the remaining genes of the rib09cluster. The rJbM isogenes differed in upstream flanking DNA but were otherwise highly conserved. In contrast, the rfbK isogenes differed in downstream flanking DNA and in 3'-terminal regions, resulting in slight differences in the sizes of the predicted RfbK proteins. RfbMog and RfbKog are most closely related to CpsB and CpsG, respectively. These are isozymes of GDP-mannose pyrophosphorylase and phosphomannomutase, respectively, which are thought to be involved in the biosynthesis of the slime polysaccharide colanic acid in E. coi K-12 and Salmonella enterica serovar Typhimurium. An E. col 0-:K30 mutant, strain CWG44, lacks rJbM2-rJb0K2 and has adjacent essential rib09 sequences deleted. The remaining chromosomal genes are therefore suflicieift for GDP-mannose formation and K30 capsular polysaccharide synthesis. A mutant of E. coli CWG44, strain CWG152, was found to lack GDP-anannose pyrophosphorylase and lost the ability to synthesize K30 capsular polysaccharide. Wild