Cloning and Analysis of Duplicated rJbM and rJbK Genes Involved in the Formation of GDP-Mannose in Eschenrchia coli

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 rib09 cluster. 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-type capsular polysaccharide could be restored in CWG152, by transformation with plasmids containing either fM' or JM2. Introduction of a complete rib09 gene cluster into CWG152 restored synthesis of both 09 and K30 polysaccharides. Consequently, rJbM is sufficient for the biosynthesis of GDP-mannose for both 0 antigen and capsular polysaccharide in E. coil 09:K30. Analysis of a collection of serotype 08 and 09 isolates by Southern hybridization and PCR amplification experiments demonstrated extensive polymorphism in the rJbM-rJbK region.