Subcloning, transposon insertion, and deletion analysis revealed that the Escherichia coli O9 rfb region is about 12 kb in size. The region encodes at least seven polypeptides of 89, 74, 55, 50, 44, 41 and 39.5 kDa. Southern hybridization analysis of rfb regions of E. coli O8 and O9, and Klebsiella O3 and O5 serotypes (all of these O polysaccharides are mannose homopolymers and the structures of the repeating unit of E. coli O9 and Klebsiella O3 are identical) showed that a central region specific for E. coli O9 and Klebsiella O3 is flanked by two regions common to all four. Complementation experiments using strains with known defects and specific tests for the enzymic activity showed that the 50 and 55 kDa polypeptides, encoded by the common region, are phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GMP), respectively. Nucleotide sequencing of the region revealed the presence of two genes, rfbK and rfbM, analogous to the corresponding genes of Salmonella typhimurium. In E. coli O9, rfbK and rfbM encode proteins of 460 amino acids (50,809 Da) and 471 amino acids (52,789 Da). The amino acid sequence of GMP was conserved in RfbMs of E. coli O7 and Salmonella groups B, C1 and C2, CpsB of S. typhimurium, AlgA of Pseudomonas aeruginosa, and XanB of Xanthomonas campestris. The phylogenetic trees of PMM and GMP were different in topology and in the evolutionary distances from ancestors.