Date of Award

Summer 8-12-2014

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Kuk-Jeong Chin

Second Advisor

Adam Wilson

Third Advisor

Frank Cruz

Fourth Advisor

Eric Gilbert

Abstract

Petroleum spills causes contamination of drinking water with carcinogenic aromatic compounds including benzene and cresol. Current knowledge of anaerobic benzene and cresol degradation is extremely limited and it makes bioremediation challenging. Geobacter daltonii strain FRC-32 is a metal-reducing bacterium isolated from radionuclides and hydrocarbon-contaminated subsurface sediments. It is notable for its anaerobic oxidation of benzene and its unique ability to metabolize p-, m-, or o-cresol as a sole carbon source. Location of genes involved in aromatic compound degradation and genes unique to G. daltonii were elucidated by genomic analysis using BLAST.

Genes predicted to play a role in aromatic degradation cluster into an aromatic island near the start of the genome. Of particular note, G. daltonii has two copies of the bss genes, which are responsible for the first step in anaerobic toluene oxidation. This bacterium is unique among the family Geobacteraceae and other toluene degraders in this aspect. The a subunits have 74% identity to one another. The remaining genes in each operon are not identical. BssA was upregulated when G. daltonii was grown on benzene and toluene while the grlA was upregulated during growth on m-cresol. Toluene was accumulated during degradation of benzene by cell lysate. Cells grown with benzene and toluene exhibited a similar protein profile compared to cells grown with benzoate. These results indicate that benzene is converted to toluene and further degraded via the toluene pathway.

Both the bss and grl operons were predicted to have sigma54-dependent promoters. This was confirmed using 5' RACE and sequence analysis. E. coli transformed with the bss operon were able to grow in the presence of toluene but lost this capability when sigma 54 was knocked out. Growth was restored with complementation of sigma 54. The sigma 54-dependent signaling system bamVW was upregulated in the presence of all aromatic compounds tested. These results suggest that the bss operon is regulated via sigma 54-dependent mechanisms. This study significantly contributes to anaerobic aromatic gene regulation which is crucial in effective oil spill bioremediation.

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