Date of Award

May 2013

Degree Type

Thesis

Degree Name

Master of Science

Department

Biological Sciences

First Advisor

Daad Saffarini

Committee Members

Mark McBride, Sonia Bardy

Abstract

Porphyromonas gingivalis is an oral Gram-negative anaerobic bacterium implicated in periodontal disease, a polymicrobial inflammatory disease that is correlated with cardiovascular disease, diabetes and preterm birth. Therefore understanding the physiology and metabolism of P.gingivalis through genetic manipulation is important in identifying mechanisms to eliminate this pathogen. Although numerous genetic tools have been developed for the manipulation of other bacterial species, they either do not function in P.gingivalis or they have limitations. We modified a Mariner transposon pHimarEM1 system that was developed for Flavobacterium johnsoniae for mutagenesis of P. gingivalisWe introduced the P. gingivalis fimA promoter upstream of the transposase gene to improve the efficiency of the transposition in P.gingivalis. Transposon mutants of P.gingivalis were screened for growth or pigmentation defects and analyzed. Using this technique, strains with mutations in genes encoding cation efflux system Proteins, Na+-translocating NADH-quinone reductase were identified thus demonstrating the effectiveness of this new transposon. Therefore pHimarEM1-PfimA can be used to generate new mutants in P. gingivalis that lead to explore the metabolic pathways of P. gingivalis

Shewanella oneidensis MR-1 is a facultative anaerobe and metal reducing bacterium that uses a large number of terminal electron acceptors for respiration. These include thiosulfate, polysulfide and sulfite. The dissimilatory sulfite reductase, Sir A is a c type cytochrome predicted to be a copper protein. It catalyses the six electron reduction of sulfite to sulfide and unlike other sulfite reductases, it appears to lack siroheme. Cu is essential for many respiratory enzymes such as cytochrome c oxidases and anaerobic nitrous oxide reductases. NosA is predicted to be a copper specific porin in the outer membrane that allows copper diffusion into the cell. In the second chapter of this thesis we hypothesized that NosA is expressed under aerobic and anaerobic conditions and is involved in biogenesis of the sulfite reductase (SirA). We also predict that additional porins may be needed for Cu entry into the cell.

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