Development of a system for genetic manipulation of Bartonella bacilliformis

Lack of a system for site specific genetic manipulation has severely hinder ed studies on the molecular biology of all Bartonella species. We report th e first site-specific mutagenesis and complementation for a Bartonella spec ies. A highly transformable strain of B. bacilliformis, termed JB584, was i solated and found to exhibit a significant increase in transformation effic iency with the broad-host-range plasmid pBBR1MCS-2, relative to wild-type s trains. Restriction analyses of genomic preparations with the methylation-s ensitive restriction enzymes ClaI and StuI suggest that strain JB584 posses ses a dcm methylase mutation that contributes to its enhanced transformabil ity. A suicide plasmid, pUB1, which contains a polylinker, a pMB1 replicon, and a nptI kanamycin resistance cassette, aas constructed. An internal 508 -bp fragment of the B. bacilliformis flagellin gene (fla) was cloned into p UB1 to generate pUB508, a fla-targeting suicide vector. Introduction of pUB 508 into JB584 by electroporation generated eight Kan(r) clones of B. bacil liformis, Characterization of one of these strains, termed JB585, indicated that allelic exchange between pUB508 and fla had occurred. Analysis by sod ium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and electron microscopy showed that synthesis of flagellin encoded by fla and secretion/assembly of flagella were abolished. Complementation of fla in tr ans was accomplished with a pBBR1MCS recombinant containing the entire wild -type fla gene (pBBRFLAG), These data conclusively show that inactivation o f fla results in a bald, nonmotile phenotype and that pMB1 and REP replicon s make suitable B. bacilliformis suicide and shuttle vectors, respectively. When used in conjunction with the highly transformable strain JB584, this system for site-specific genetic manipulation and complementation provides a new venue for studying the molecular biology of B. bacilliformis.