Functional Analysis of Genes in the rfb Locus of Leptospira borgpetersenii Serovar Hardjo Subtype Hardjobovis

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Abstract
Lipopolysaccharide (LPS) is a key antigen in immunity to leptospirosis. Its biosynthesis requires enzymes for the biosynthesis and polymerization of nucleotide sugars and the transport through and attachment to the bacterial membrane. The genes encoding these functions are commonly clustered into loci; for Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis, this locus, named rfb , spans 36.7 kb and contains 31 open reading frames, of which 28 have been assigned putative functions on the basis of sequence similarity. Characterization of the function of these genes is hindered by the fact that it is not possible to construct isogenic mutant strains in Leptospira . We used two approaches to circumvent this problem. The first was to clone the entire locus into a heterologous host system and determine if a “recombinant” LPS or polysaccharide was synthesized in the new host. The second approach used putative functions to identify mutants in other bacterial species whose mutations might be complemented by genes on the leptospiral rfb locus. This approach was used to investigate the function of three genes in the leptospiral rfb locus and demonstrated function for orfH10 , which complemented a wbpM strain of Pseudomonas aeruginosa , and orfH13 , which complemented an rfbW strain of Vibrio cholerae . However, despite the similarity of 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 GalE was used as a background in which orfH8 produced detectable GalE enzyme activity.