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theses

Dioxins and dioxin-like compounds are highly toxic, ubiquitous polycyclic aromatic hydrocarbons with important environmental and human health impacts. Dioxins are listed among the most relevant environmental organic pollutants due to their high persistence and extreme lipophilicity. In recent years, a number of bacterial strains have been isolated and identified for their diverse metabolic capabilities to grow on and metabolize a broad range of environmentally recalcitrant compounds including, dibenzo-p-dioxin and dibenzofuran. Among these strains Sphingomonas wittichii RW1 has been one of the most effective dioxin degraders studied so far. RW1 is of great interest for its diverse metabolic activities and unusual genome structure. The complete genome sequence of RW1 reveals that this strain contains one chromosome and two circular megaplasmids referred to as pSWIT01 and pSWIT02. Many of the important catabolic genes that are involved in dibenzofuran and dibenzo-p-dioxin degradation are located on the small megaplasmid pSWIT02; particularly genes encoding the initial dioxygenase system DxnA1A2, a ferredoxin Fdx3, and a reductase RedA2. This unusual dioxygenase system initiates the oxygenolytic attack of dibenzofuran and dibenzo-p-dioxin in an angular fashion. Here we report the first physiological identification of the important proteins that together function as the initial dibenzofuran and dibenzo-p-dioxin dioxygenase enzyme in RW1. Knock out mutagenesis showed that two reductases RedA1 and RedA2 and two ferredoxins Fdx1 and Fdx3 are interchangeable where either RedA1/RedA2 in combination with Fdx1/Fdx3 can function as an electron donor to the terminal oxygenase. The knockout mutants were also screened on substrates other than dibenzofuran and dibenzo-p-dioxin such as salicylate and benzoate. We discovered that the reductase RedA2 is involved in supplying electrons to the salicylate oxygenase since deletion of the redA2 gene blocked the growth of RW1 on salicylate. Interestingly, single knockout mutants of redA1 and redA2 had no effect on RW1's ability to grow on benzoate while a double knockout mutant resulted in the loss of the ability to grow on benzoate. In addition, we constructed stable and unstable cloning vectors for sphingomonads based on the Sphingobium yanoikuyae B1 pKG2 plasmid and the Sphingomonas wittichii RW1 pSWIT02 plasmid. Stable vectors included the rep and par plasmid regions for replication and partitioning while unstable vectors included only the rep region. The stable vector pSEZ_RW1RP was used to cure RW1 of pSWIT02 by plasmid incompatibility. The cured strain RW1c was no longer able to grow on dibenzo-p-dioxin or dibenzofuran. A complementation test with the broad host range plasmid pRK415 carrying the initial dioxygenase system allows RW1c to grow on both dibenzo-p-dioxin and dibenzofuran. These results demonstrate that the only major role that pSWIT02 plays in the degradation of dibenzo-p-dioxin and dibenzofuran is to supply the initial dioxygenase responsible for the first step in the catabolic pathway.

ETD_10464

Ph.D.

Includes bibliographical references

doi:10.7282/t3-438v-5j54

ETD doctoral

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