Cloning, Complementation, and Characterization of a rfaD Homolog from Bradyrhizobium japonicum

초록

We have previously cloned and characterized the gene region involved in lipopolysaccharide (LPS) biosynthesis in Bradyrhizobium japonicum. The LPS, a major component of the outer membrane of gram-negative bacteria, is composed of three domains: lipid A; the core, which is an oligosaccharide consisting of an inner and an outer region; and a distal repeating unit known as the O-antigenic chain. Heptose is a structural unit of the inner core. The rfaD gene encodes ADP-L-glycero-D-manno-heptose-6-epimerase, which converts ADP-D-glycero-D-manno-heptose -6-epimerase to ADP-L-glycero-D-manno-heptose-6-epimerase. The rfaD mutants produce a deep rough LPS and are highly susceptible to hydrophobic antibiotics such as novobiocin; however they become resistance to novobiocin if transformed with a plasmid carrying the rfaD gene. In this study, we describe cloning and knockout mutation of the rfaD gene in B. japonicum. To study the role of the rfaD gene in B. japonicum LPS biosynthesis, the rfaD gene was inactivated in chromosome by insertion of kanamycin-resistance gene cassette where knockout fragment replaced the rfaD gene in B. japonicumby double cross-over homologous recombination.