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theses

Dibenzofuran and dibenzo-p-dioxin are ubiquitous environmental pollutants in soil and sediment. Sphingomonas wittichii RW1 is one of a few strains known for the ability to grow on the related compounds dibenzofuran (DBF) and dibenzo-p-dioxin (DXN) as the sole source of carbon. The genes for the initial steps in the DBF catabolic pathway (ring hydroxylating dioxygenase, ring cleavage dioxygenase, and a hydrolase) which result in the formation of salicylate and a five-carbon fragment have been localized to a mega-plasmid designated pSWIT02 in RW1. Plasmids highly similar to pSWIT02 have been found in other DBF degrading Sphingomonas strains. However, despite having the pSWIT02-encoded DBF degradation pathway these other bacteria are not capable of growth on DXN. This thesis describes involvement of chromosomally encoded genes in dibenzo-p-dioxin degradation by RW1. RW1 lacking the pSWIT02 dbfB gene grows extremely slowly on DBF and accumulates the ring cleavage substrate 2,2',3-trihydroxybiphenyl. The mutant grows normally on DXN as the sole source of carbon indicating that dbfB is not necessary for the DXN catabolic pathway and suggesting involvement of other ring cleavage dioxygenases in DXN pathway. Knockout of gene SWIT3046 resulted in a strain that grows normally on DBF but that does not grow on DXN. The double knockout does not grow on either DBF or DXN. These results prove that separate ring cleavage enzymes are necessary for DBF and DXN degradation.
We then examined the third enzymatic step in RW1, the hydrolase. RW1 lacking the pSWIT02 encoded gene dxnB1 or the chromosome encoded gene dxnB2 grow normally on both DBF and DXN. A double knockout of both genes does not grow on DBF but still grows on DXN. We then examined previously published transcriptomic data that showed that the SWIT0910 encoded hydrolase is up regulated during growth on DBF and DXN. A knockout of SWIT0910 grows normally on DBF but does not grow on DXN. Our results demonstrate that a chromosomally encoded hydrolase, SWIT0910, is absolutely required for growth on DXN and that two different hydrolases (chromosomally and plasmid encoded) contribute equally to growth on DBF.
Genes for three biphenyl ring cleavage dioxygenases from Burkholderia xenovorans LB400, Sphingomonas yanoikuyae B1, and Pseudomonas putida F1 were moved into a mutant lacking the RW1 DBF and DXN ring cleavage genes. All three ring cleavage dioxygenases allowed the mutant RW1 to grow on DBF at different rates. Interestingly, only bphC from Burkholderia xenovorans LB400 allowed RW1 mutant to grow on DXN.

ETD_10109

Ph.D.

Includes bibliographical references

doi:10.7282/t3-vc8x-cs80

ETD doctoral

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