Author ORCID Identifier

0009-0009-3878-5708

Date of Award

5-5-2025

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Dr. Kuk-Jeong Chin

Second Advisor

Dr. Eric Gilbert

Third Advisor

Dr. Irene Weber

Abstract

Accumulation of crude oil-derived contaminants in anoxic environments necessitates investigations into anaerobic microorganisms that can utilize these contaminants as carbon sources. Geotalea daltonii strain FRC-32 anaerobically can oxidize various crude oil derivatives, including aromatic compounds such as benzoate or plastics such as cellulose acetate butyrate (CAB) as a sole carbon and electron source. We aimed to investigate genetic and metabolic mechanisms of anaerobic degradation of aromatic compounds and plastics in G. daltonii.

The metabolic pathways employed by G. daltonii were elucidated in anaerobic benzoate-oxidizing cultures in the presence of acetate, a key intermediate in anaerobic organic matter degradation, to predict carbon source transport and utilization strategies. Benzoate accumulation in G. daltonii whole cell lysates indicated that intracellular benzoate transport occurred during benzoate oxidation in the presence of acetate. Expression analyses of putative benzoate transporter BenK and protein-ligand binding affinity prediction suggested BenK’s specificity for transporting benzoate. Relative expression levels for the gene benK, encoding BenK, and the genes bamNOPQ, involved in the benzoyl-CoA pathway, were significantly higher in cultures grown on both benzoate and acetate than in cultures grown on acetate as sole carbon source, indicating that intracellular benzoate accumulation facilitated the regulation of bamNOPQ. These results demonstrated that G. daltonii can perform differential benzoate oxidation in the presence of acetate, by either simultaneous or sequential carbon source oxidation, indicating the metabolic plasticity of G. daltonii in response to varying carbon source availability.

Modulation of benzoate-CoA ligase gene bamY, specifically, the role of BamVW and σ54 on transcription of bamY in G. daltonii, was elucidated to understand the regulation of the benzoyl-CoA pathway during anaerobic aromatic degradation. The two-component-system (TCS) genes bamV, encoding a histidine kinase, bamW, encoding a σ54-dependent response-regulator, the xylR-like gene, encoding a σ54-binding enhancer-binding protein, and the σ54-dependent pY promoter upstream of bamY, encoding benzoate-CoA ligase, were identified via genomic analysis. Arrangement of bamVW and a xylR-like gene in an operon suggested that BamVW functions as σ54-dependent kinase-regulator pair. Expression levels for bamVW, bamY, and rpoN, encoding σ54, were significantly higher in benzoate-oxidizing cultures compared to those in benzene- and toluene-oxidizing cultures, suggesting that these genes are essential in anaerobic benzoate oxidation by G. daltonii. The transcription-start-site of bamY was identified, and binding of σ54 to the pY promoter was confirmed. These results suggested that regulation of bamY is σ54-dependent and that BamVW and σ54 are essential in initiating transcription of bamY in anaerobic aromatic oxidation by G. daltonii. The results of in silico analysis of BgeR suggested that the benzoyl-CoA pathway was globally regulated by transcription-repressor BgeR in response to benzoyl-CoA, the central intermediate of anaerobic aromatic degradation in G. daltonii.

Lastly, the role of selective trace element availability in anaerobic degradation of benzoate and CAB plastics by G. daltonii was investigated. Increased availability of trace elements cobalt, molybdenum, selenite, and tungsten stimulated anaerobic growth of G. daltonii cultures on benzoate and CAB plastic and enhanced the efficacy of anaerobic benzoate and CAB plastic oxidation in G. daltonii. In summary, our findings unraveled regulatory mechanisms that allow G. daltonii to adapt their metabolic strategies and capacity in order to overcome energetic and thermodynamic challenges during anaerobic degradation of aromatic compounds and plastics. Furthermore, the findings of our study indicated the efficiency of anaerobic aromatic and plastic biodegradation and contributed to build the foundation of knowledge that is necessary to translate laboratory-based findings into sustainable bioremediation efforts.

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