Author ORCID Identifier
0000-0002-2982-8165
Date of Award
Spring 5-6-2024
Degree Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Biology
First Advisor
Dr. Kuk-Jeong Chin
Second Advisor
Dr. Zehava Eichenbaum
Third Advisor
Dr. Eric Gilbert
Fourth Advisor
Dr. Irene Weber
Abstract
The recalcitrant nature of petroleum hydrocarbons makes the bioremediation of oil-contaminated environments challenging. These contaminants accumulate in environments with limited oxygen, exacerbating remediation. Preliminary analysis of the genome of Geotalea daltonii strain FRC-32 revealed the presence of putative aromatic-degrading genes and homologs, indicating the potential for it to break down aromatic hydrocarbons. We aimed to investigate the metabolic capacity of G. daltonii to oxidize various aromatic hydrocarbons anaerobically as a sole carbon and electron source.
A metabolic pathway of anaerobic benzene oxidation by G. daltonii was proposed and the putative aromatic-degrading genes were examined. The hbs gene was upregulated during benzene oxidation while bss was upregulated during toluene oxidation. Expression of BbsEF and BbsG during benzene degradation indicated beta oxidation of benzylsuccinate. Addition of benzene to the whole cell lysate resulted in toluene formation, indicating methylation of benzene. Oxidation of p-cresol and phenol resulted in expression of bamY and bamR, indicating that p-cresol and phenol both utilized the benzoate degradation pathway.
In naphthalene-oxidizing G. daltonii cultures, the genes hbs, bbsF, bbsA, bamR, and bamA, were expressed. Accumulation of benzene in whole cell lysate from naphthalene-oxidizing cultures indicated utilization of the benzene degradation pathway. Functional site analysis of Bss and Hbs revealed active S-adenosylmethionine sites on the activating subunits of both enzymes. The presence of two cysteine residues proximal to a glycine residue suggested the ability of Hbs to catalyze methylation of aromatic compounds. Complementation of rpoN into σ54-deficient E. coli transformed with the bss operon restored the ability of E. coli to grow in the presence of toluene, revealing bss to be regulated by σ54. Binding sites for σ70 and the transition state regulator AbrB were identified in the promoter region of the σ54 encoding gene rpoN and protein-DNA binding was confirmed by electrophoretic mobility shift assay. Induced expression of abrB during benzene and toluene degradation resulted in G. daltonii cultures transitioning to death phase. These results suggested that G. daltonii can anaerobically oxidize benzene via the toluene degradation pathway that is regulated by AbrB, representing a uniquely efficient approach to anaerobic aromatic oxidation by G. daltonii.
DOI
https://doi.org/10.57709/36958795
Recommended Citation
Bullows, James, "Regulations and Mechanisms of Anaerobic Aromatic Hydrocarbon Oxidation in Geotalea daltonii strain FRC-32." Dissertation, Georgia State University, 2024.
doi: https://doi.org/10.57709/36958795
File Upload Confirmation
1