Presenter Information

Finley J. AndrewFollow

Presentation Type

Poster

Faculty Mentor’s Full Name

Michael Minnick

Faculty Mentor’s Department

Division of Biological Sciences

Abstract

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. bacilliformisgenome. One mutant, designated JB584-4B2, was identified as having its flgI gene disrupted by the Tn. The flgIgene 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.

Category

Life Sciences

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Development of a System for Transposon Mutagenesis of Bartonella bacilliformis

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. bacilliformisgenome. One mutant, designated JB584-4B2, was identified as having its flgI gene disrupted by the Tn. The flgIgene 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.