Expression of the O9 polysaccharide of Escherichia coli: sequencing of the E. coli O9 rfb gene cluster, characterization of mannosyl transferases, and evidence for an ATP-binding cassette transport system

Abstract

The rfb gene cluster of Escherichia coli O9 directs the synthesis of the O9-specific polysaccharide which has the structure 32-␣-Man- (132) (18,55), and the O3-and O5-specific polysaccharides of Klebsiella strains are identical to the O8 and O9 polysaccharides of E. coli (8,18,29). Mannans of algal origin were found to exert antitumor activity (37). Such an activity could later be attributed also to the mannan-containing LPS of E. coli and Klebsiella strains (13, 37).The genetics of LPS biosynthesis in enteric bacteria is well documented in recent reviews (33,43,48,55). Two mechanisms, block and monomeric, have been described for O-polysaccharide synthesis (49). In the block mechanism, observed for Salmonella typhimurium and related Salmonella serotypes, the oligosaccharide repeating units are assembled on undecaprenol phosphate (antigen carrier lipid [ACL]) under the direction of rfb genes. The first sugar transferred was found to be galactose-1-phosphate, and the corresponding transferase gene was termed rfbP (48). The repeating units are polymerized under the direction of the rfc gene, which may be located outside of or within the rfb gene cluster (41). The chain length is controlled by the rol gene, located between gnd and his (3, 4). The monomeric mechanism, experimentally proven only for E. coli O8 and O9 (18, 55), consists of the direct and sequential transfer of the monosaccharide residues from their nucleotideactivated precursors to the nonreducing end of the growing polysaccharide chain.The synthesis of some O polysaccharides requires the rfe gene. According to this requirement, LPS biosynthesis can also be divided into rfe-dependent and rfe-independent pathways. The rfe gene, first described by Mäkelä et al. (31), was found to be essential for the synthesis of the O polysaccharide in Salmonella strains of O groups C1 and L, and E. coli O8 and O9 (18, 33) and more recently in E. coli O4, O7, O18, O75, and O111 (1, 23a). It was reported to determine the tunicamycinsensitive transfer of N-acetylglucosamine (GlcNAc)-1-phosphate from UDP-GlcNAc to undecaprenol monophosphate

Polymorphism, duplication, and IS1-mediated rearrangement in the chromosomal his-rfb-gnd region of Escherichia coli strains with group IA and capsular K antigens

“…1) (13). This eliminates wzm and wzt, which encode an ABC-2-type transporter required for O9 antigen export, and wbdA, whose product has not been functionally characterized (22). Since the IS1 element and inverted repeat sequence down- stream of ugd are intact, the deletion in CWG44 is not simply explained by loss of an IS1 element and flanking DNA.…”

“…Although the K30 cps cluster has not been cloned or mapped in detail, available data point to a location at approximately 45 min on the E. coli chromosome, near his (the operon responsible for histidine biosynthesis) and rfb (the cluster responsible for O9 antigen biosynthesis) (24,37,48). The rfb cluster, responsible for the synthesis and expression of the O9 antigen, has been cloned and partially sequenced from strain E69 (21), and a complete sequence is available for the rfb cluster from a different O9 strain (22). These data place the rfb cluster between his and gnd (encoding gluconate-6-phosphate dehydrogenase).…”

Nonreducing Terminal Modifications Determine the Chain Length of Polymannose O Antigens of Escherichia coli and Couple Chain Termination to Polymer Export via an ATP-binding Cassette Transporter

“…Structural and biosynthetic data for the E. coli and K. pneumoniae analogs are interchangeable. The mechanistic details of assembling the E. coli O8 and O9a O-PSs on the lipid carrier have not been fully elucidated, but a working biosynthetic model has been proposed based on the available structural and genetic evidence (13,16). O-PS synthesis begins with the conserved formation of a "primer" by transfer of a GlcNAc-1-phosphate residue to und-P.…”

“…WecA activity is not confined to O-PS biosynthesis, and the structural gene is located outside of the O-PS biosynthesis gene cluster (18). Using the primed lipid intermediate as an acceptor, an adaptor disaccharide is assembled by the mannosyltransferases WbdC and WbdB (16). Adaptor synthesis commits the lipid intermediate to the O-PS biosynthetic pathway.…”

Substrate specificity of bacterial oligosaccharyltransferase suggests a common transfer mechanism for the bacterial and eukaryotic systems

“…The O9a polymannan is linked to the lipid A core, whereas the K30 antigen forms the capsular polysaccharide. The O9a antigen is synthesized by the ABC transporter-dependent mechanism (18)(19)(20). The K30 capsular polysaccharide is the established prototype for group 1 capsules (21).…”