Adaptation Of Pseudomonas Putida F1 To Growth On Styrene
Pseudomonas putida F1 is unable to grow on styrene, a common industrial pollutant, despite degrading it through the toluene degradation (tod) pathway. This dissertation investigates the biochemical and genetic aspects of styrene degradation by P. putida F1 and identifies substrate-level catechol-2,3-dioxygenase (C23O) inactivation as the factor which prevents growth on styrene. Novel adaptations to growth on styrene were investigated and found to mitigate C23O inactivation through management of 3-vinylcatechol production and consumption. Previous studies of P. putida F1 identified TodF, a hydrolase responsible for the degradation of styrene meta-fission product (MFP), as putatively responsible for F1's inability to grow on styrene. Through kinetic and growth analyses, we demonstrated that TodF is capable of styrene MFP degradation and thus not responsible for prohibiting growth on styrene. Preliminary genetic analysis of styrene adapted mutants strengthened this conclusion. It was found that cultures of F1 exposed to styrene accumulated 3vinylcatechol in the media, suggestive of inhibited C23O activity. Specifically, micromolar concentrations of 3-vinylcatechol were found to inactivate TodE during catalysis. Analysis of cells growing on styrene suggested that inactivation of TodE and the subsequent accumulation of 3-vinylcatechol resulted in toxicity and cell death. Over-expression of TodE or an inactivation resistant C23O on a plasmid was able to prevent catechol accumulation and confer the ability to grow on styrene. We characterized a spontaneous F1 mutant, designated SF1, which was capable of growth on styrene and did not accumulate 3-vinylcatechol. Resting cell assays demonstrated that the activity of toluene dioxygenase (TDO), the multicomponent enzyme responsible for the production of 3-vinylcatechol from styrene, was reduced in SF1. DNA sequence analysis of the tod operon revealed a single base pair mutation in todA (C479T), a gene encoding the reductase component of TDO. Replacement of the wild-type todA allele in F1 with todAC479T from SF1 reduced TDO activity, prevented 3-vinylcatechol accumulation, and conferred the ability to grow on styrene. Collectively, our data reveals a unique adaptation where slowing down the rate of vinylcatechol production via decreased TDO activity leads to reduced C23O inactivation and permits growth on styrene.
Biodegradation; Pseudomonas putida; catechol-2; 3-dioxygenase
Hay, Anthony G.
Madsen, Eugene Lewis; Wilson, David B
Ph. D., Environmental Toxicology
Doctor of Philosophy
dissertation or thesis