The rfbO9 gene cluster, which is responsible for the synthesis of the lipopolysaccharide O9 antigen, was cloned from Escherichia coli O9:K30. The gnd gene, encoding 6-phosphogluconate dehydrogenase, was identified adjacent to the rfbO9 cluster, and by DNA sequence analysis the gene order gnd-rfbM-rfbK was established. This order differs from that described for other members of the family Enterobacteriaceae. Nucleotide sequence analysis was used to identify the rfbK and rfbM 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 O9:K30 strain, a duplicated rfbM2-rfbK2 region was detected approximately 3 kbp downstream of rfbM1-rfbK1 and adjacent to the remaining genes of the rfbO9 cluster. The rfbM 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. RfbMO9 and RfbKO9 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. coli K-12 and Salmonella enterica serovar Typhimurium. An E. coli O-:K30 mutant, strain CWG44, lacks rfbM2-rfbK2 and has adjacent essential rfbO9 sequences deleted. The remaining chromosomal genes are therefore sufficient for GDP-mannose formation and K30 capsular polysaccharide synthesis. A mutant of E. coli CWG44, strain CWG152, was found to lack GDP-mannose pyrophosphorylase and lost the ability to synthesize K30 capsular polysaccharide. Wild-type capsular polysaccharide could be restored in CWG152, by transformation with plasmids containing either rfbM1 or rfbM2. Introduction of a complete rfbO9 gene cluster into CWG152 restored synthesis of both O9 and K30 polysaccharides. Consequently, rfbM is sufficient for the biosynthesis of GDP-mannose for both O antigen and capsular polysaccharide E. coli O9:K30. Analysis of a collection of serotype O8 and O9 isolates by Southern hybridization and PCR amplification experiments demonstrated extensive polymorphism in the rfbM-rfbK region.