Functional analysis of genes in the rfb locus of Leptospira borgpeterseniiserovar Hardjo subtype Hardjobovis

Lipopolysaccharide (LPS) is a key antigen in immunity to leptospirosis. Its biosynthesis requires enzymes For the biosynthesis and polymerization of n ucleotide sugars and the transport through and attachment to the bacterial membrane. The genes encoding these functions are commonly clustered into lo ci; for Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis, this locus, named rfb, spans 36.7 kb and contains 31 open reading frames, of whi ch 28 have been assigned putative functions on the basis of sequence simila rity. Characterization of the function of these genes is hindered by the fa ct that it is not possible to construct isogenic mutant strains in Leptospi ra. We used two approaches to circumvent this problem. The first was to clo ne the entire locus into a heterologous host system and determine if a "rec ombinant" LPS or polysaccharide was synthesized in the new host, The second approach used putative functions to identify mutants in other bacterial sp ecies whose mutations might be complemented by genes on the leptospiral rfb locus. This approach was used to investigate the function of three genes i n the leptospiral rfb locus and demonstrated function for orfH10, which com plemented a wbpM strain of Pseudomonas auruginosa, and orfH13, which comple mented an rfbW strain of Vibrio cholerae. However, despite the similarity o f OrfH11 to WecC, a wecC strain of E. coli was not complemented by orfH11. The predicted protein encoded by orfH8 is similar to GalE from a number of organisms. A Salmonella enterica serovar Typhimurium strain producing no Ga lE was used as a background in which orfH8 produced detectable Calf enzyme activity.