CLONING AND ANALYSIS OF DUPLICATED RFBM AND RFBK GENES INVOLVED IN THE FORMATION OF GDP-MANNOSE IN ESCHERICHIA-COLI O9 K30 AND PARTICIPATION OF RFB GENES IN THE SYNTHESIS OF THE GROUP-I K30 CAPSULAR POLYSACCHARIDE/

The rfb(O9) gene cluster, which is responsible for the synthesis of th e lipopolysaccharide O9 antigen, was cloned from Escherichia coli O9:K 30. The gnd gene, encoding 6-phosphogluconate dehydrogenase, was ident ified adjacent to the rfb(O9) cluster, and by DNA sequence analysis th e gene order gnd-rfbM-rfbK was established. This order differs from th at described for other members of the family Enterobacteriaceae. Nucle otide sequence analysis was used to identify the rfbK and rfbM genes, encoding phosphomannomutase and GDP-mannose pyrophosphorylase, respect ively. 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 polysacchari des. In the E. coli O9:K30 strain, a duplicated rfbM(2)-rfbK(2) region was detected approximately 3 kbp downstream of rfbM(1)-rfbK(1) and ad jacent to the remaining genes of the rfb(O9) cluster. The rfbM isogene s differed in upstream flanking DNA but were otherwise highly conserve d. In contrast, the rfbK isogenes differed in downstream flanking DNA and in 3'-terminal regions, resulting in slight differences in the siz es of the predicted RfbK proteins. RfbM(O9) and RfbK(O9) are most clos ely 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 polysaccha ride colanic acid in E. coli K-12 and Salmonella enterica serovar Typh imurium. An E. coli O-:K30 mutant, strain CWG44, lacks rfbM(2)-rbK(2) and has adjacent essential rfb(O9) sequences deleted. The remaining ch romosomal genes are therefore sufficient for GDP-mannose formation and K30 capsular polysaccharide synthesis. A mutant of E. coli CWG44, str ain CWG152, was found to lack GDP-mannose pyrophosphorylase and lost t he ability to synthesize K30 capsular polysaccharide. Wild-type capsul ar polysaccharide could be restored in CWG152, by transformation with plasmids containing either rfbM(1) or rfbM(2). Introduction of a compl ete rfb(O9) gene cluster into CWG152 restored synthesis of both O9 and K30 polysaccharides. Consequently, rfbM is sufficient for the biosynt hesis of GDP-mannose for both O antigen and capsular polysaccharide in E. coli O9:K30. Analysis of a collection of serotype O8 and O9 isolat es by Southern hybridization and PCR amplification experiments demonst rated extensive polymorphism in the rfbM-rfbK region.