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Development of a System for Transposon Mutagenesis of Bartonella Bacilliformis

Finley J. Andrew, Linda D. Hicks, and Dr. Michael F. Minnick, Division of Biological Sciences, The University of Montana, 32 Campus Drive HS 104 Missoula, MT 59812-1002

Bartonella bacilliformis is a tropical bacterial pathogen responsible for Carrión’s disease in humans. The lack of a system for random mutagenesis has greatly hindered our ability to efficiently study the agent’s molecular biology. Here, we report the first transposon (Tn) mutagenesis of B. bacilliformis, generation of a mutant library, and confirmation of five mutant strains by arbitrarily-primed PCR coupled with nucleotide sequencing. To accomplish this, B. bacilliformis strain JB584 was transformed by electroporation with the plasmid pSAM-R1; a vector initially intended for use in Rhizobium species. pSAM-R1 contains a transposase and a Tn encoding a kanamycin-resistance gene, allowing for selection and maintenance of the integrated Tn in the B. bacilliformis genome. One mutant, designated JB584-4B2, was identified as having its flgI gene disrupted by the Tn. The flgI gene encodes the FlgI protein, an essential component in the P-ring used in the flagellar motor of bacteria. Thus, the motility phenotype of JB584-4B2 was subsequently examined on a novel motility medium. Results conclusively demonstrated that interruption of flgI gene created a non-motile mutant of B. bacilliformis. Taken as a whole, our results show that: 1) pSAM-R1 is a viable transposon vector for B. bacilliformis, 2) the plasmid can be employed to create a Tn library, and 3) arbitrarily-primed PCR is a suitable method for identifying and locating mutations generated by this procedure. When used in conjunction with B. bacilliformis strain JB584, this system of transposon mutagenesis and mutation identification allows for a new and expanded way to investigate the molecular biology of this emerging human pathogen.




Additional Abstract Information

Presenter: Finley Andrew

Institution: University of Montana Missoula

Type: Poster

Subject: Microbiology

Status: Approved


Time and Location

Session: Poster 8
Date/Time: Tue 5:00pm-6:00pm
Session Number: 5629