Characterization of the rfaD gene region of Bradyrhizobium japonicum 61A101C

In our previous studies, we have cloned and characterized a gene region from Bradyrhizobium japonicum ,which is involved in the synthesis of lipopolysaccharide (LPS). In this study, we have expanded the sequence analysis of the region and found an additional open reading frame (orf), which appeared to be divergently transcribed from the rfaF gene. Sequence alignment of the orf revealed a significant similarity with rfaD genes of Salmonella typhimurium , Escherichia coli, and Neisseria gonorrhoeae. These genes encode a heptose-6-epimerase, which catalyzes the interconversion of ADP -D -glycerol-D-manno-heptose to ADP-L-glycero-D-manno-heptose. This divergent organization of the rfaF and rfaD genes is different from that of other Gram-negative bacteria where two genes form an operon. A rfaD- mutant of E. coli was successfully transformed with plasmid constructs containing the rfaD gene of B. japonicum. Novobiocin sensitivity test showed that the rfaD gene from B. japonicum could complement the rfaD mutation in E. coli, which confirms the functionality of the cloned B. japonicum gene.