This study cloned and characterized the rfb region from Yersinia pseudotuberculosis serogroup IIA, which encodes genes for producing the 3,6-dideoxyhexose sugar abequose in the bacterial outer membrane. The researchers created a gene library from Y. pseudotuberculosis strain M85 and screened it using antibodies against abequose. A 39 kb cosmid clone (pPR981) was identified that conferred O antigen expression in E. coli K12 strains. Using transposon mutagenesis, they narrowed the functional rfb region to a 19.3 kb segment, which was further subcloned into plasmid pPR1197. Both clones produced O antigen detectable by SDS-PAGE and silver staining. Southern hybridization experiments showed significant DNA sequence similarity between the Y. pseudotuberculosis rfb region and Salmonella enterica genes rfbF and rfbG, which encode early steps in abequose biosynthesis. However, no homology was detected to the abequose synthase gene rfbJ from S. enterica or the paratose synthase gene from S. enterica Ty2, suggesting that different genes within the abequose pathway have distinct evolutionary origins in these two bacterial species.

Key findings

• The entire 39 kb Y. pseudotuberculosis serogroup IIA rfb region was successfully cloned and shown to produce functional O antigen in E. coli K12 strains
• Transposon mutagenesis identified a minimal 19.3 kb functional rfb region required for O antigen expression
• Southern hybridization revealed homology between Y. pseudotuberculosis rfb genes and S. enterica rfbF and rfbG, indicating shared evolutionary origin for early abequose biosynthesis steps
• The abequose synthase gene (rfbJ) showed no sequence similarity between Y. pseudotuberculosis and S. enterica, suggesting independent evolutionary origins