Graduation Year

2020

Graduation Month

May

Document Type

Thesis - Campus Access Only

Degree Name

Bachelor of Science

School or Department

Biological Sciences, Division of

Major

Microbiology

Faculty Mentor Department

Biological Sciences, Division of

Faculty Mentor

Dr. Michael F. Minnick

Keywords

Bartonella, bacilliformis, transposon, arbitrarily-primed PCR, mutagenesis

Subject Categories

Bacteria | Bacterial Infections and Mycoses | Bacteriology | Biology | Diseases | Hemic and Lymphatic Diseases | Immunology and Infectious Disease | Laboratory and Basic Science Research | Life Sciences | Microbiology | Molecular Genetics | Pathogenic Microbiology | Research Methods in Life 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-Rl; a vector initially intended for use in Rhizobium species. pSAM-Rl 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.

Honors College Research Project

Yes

GLI Capstone Project

no

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© Copyright 2020 Finley J. Andrew, Michael F. Minnick Ph.D., and Linda D. Hicks