Molecular characterization of a gene region involved in lipopolysaccharidebiosynthesis in Bradyrhizobium japonicum: cloning, sequencing and expression of rfaf gene

A 3.0-kb legion involved in lipopolysaccharide biosynthesis in Bradyrhizobi um japonicum was sequenced. One complete open reading frame was identified which encodes a polypeptide of 354 amino acid residues with a predicted mol ecular mass of 38 209 Da. Expression of the protein using a T7 gene express ion system revealed a band of similar molecular mass after sodium dodecyl s ulfate-polyacrylamide gel electrophoresis. A database search against known gene sequences revealed a significant sequence similarity to the rfaF gene cloned from several Gram-negative bacteria. The rfaF gene is known to encod e heptosyltransferase II that transfers a second heptose to the inner core of lipopolysaccharide. The cloned B. japonicum open reading frame was able to functionally complement a rfaF mutant of Salmonella typhimurium SL3789. Transformation of this mutant with the B. japonicum gene restored productio n of an intact lipopolysaccharide and resistance to the hydrophobic antibio tic, novobiocin. An additional open reading frame having a significant sequ ence similarity to the rfaD gene was found to be divergently oriented to th e rfaF gene. (C) 2000 Published by Elsevier Science B.V. on behalf of the F ederation of European Microbiological Societies.