Genetic and functional characterization of biphenyl, diphenylmethane, and diphenylether degradation

Diphenylmethane, biphenyl, and diphenylether are common contaminants in a former industrial site along the Passaic River with similar structures that are of interest due to their toxicity. This work identifies strains involved in their degradation, and the genes involved in the pathways for their degradation. Based on 16s rRNA analysis three phylogenetically diverse Pseudomonas species were isolated from the contaminated site based on their ability to grow on diphenylmethane. They were named Pseudomonas sp. AJR09, Pseudomonas stutzeri AJR13, and Pseudomonas sp. AJR20. The strains were later tested on biphenyl and were found to be able to metabolize it using the same pathway as for diphenylmethane degradation. All three strains were found to contain identical dioxygenase gene sequences. The presence of the identical genes in three diverse species led us to the conclusion that the genes must be horizontally transferred in the environment. Based on the phenotype conferred by the element and the fact that one strain had previously lost the ability to grow on the substrates tested, we hypothesized that the element was an Integrative and Conjugative Element. Integrative and Conjugative Elements (ICE) are a family of mobile genetic elements that can be transferred between different cells/organisms, and once in the recipient they integrate into the host’s chromosome using specific recombination sites. One of the three strains, the P. stutzeri AJR13, was successfully mated with the well characterized P. putida KT2440 which subsequently gained the ability to grow on biphenyl, diphenylmethane, and salicylate. This demonstrated the ability of the genes to self-mobilize leading us to believe that the genes for degradation of the three compounds must be horizontally transferred in the environment. The whole genomes of the three Passaic River strains and the KT2440 recipient were sequenced and assembled to reveal that the degradative genes are indeed present on an ICE. The ICE is about 128 kb in length and inserts at a short sequence at the end of a tRNA Gly (CCC). It contains an integrase and other genes involved in the transfer of the ICE, and genes for diphenylmethane/biphenyl and salicylate degradation. It also contains a number of repeated sequences. We noticed that mutations in the ICE were necessary in order to demonstrate growth similar to the wildtype. Our work demonstrates that integrative and conjugative elements play a large role in the spread of biodegradative genes in the environment. Another aim was to characterize a dioxygenase gene involved in the degradation of diphenylether. Only a few bacteria have been isolated for growth on diphenylether (DPE) as the sole carbon and energy source. Sphingobium sp. strain SS3 is perhaps the best studied diphenylether degrading strain with characterization of the catabolic pathway in the 1990s by investigators at the University of Hamburg. The DPE catabolic pathway is initiated by a dioxygenase attack resulting in the formation of catechol and phenol. We have sequenced the SS3 genome and identified 13 genes encoding Rieske type oxygenases. Analysis of the genome environments and comparison to related enzymes in the database identified promising candidate genes that could possibly be involved in the pathway for DPE degradation. We carried out an RT-PCR experiment to identify which of the dioxygenase genes are expressed when SS3 is growing in the presence of DPE. A gene encoding a putative benzoate dioxygenase was upregulated most likely because the gene was situated in an operon encoding the catechol branch of the beta-ketoadipate pathway. A gene encoding a putative DPE dioxygenase was also upregulated. Gene knockout experiments and heterologous expression of the angular dioxygenase confirmed its catalytic activity. This adds to our knowledge of angular dioxygenase and helps us genetically characterize only the second diphenylether degrading strain.Ph.D.Includes bibliographical reference