| My research project focuses on the genetic and biochemical studies of PCP degradation by S. chlorophenolica ATCC 39723. In this study, two new prospective genes, pcpM and pcpE, were identified. The functions of pcpE and pcpM as well as other two prospective pcp genes, pcpD and pcpR, were studied by genetic and biochemical analyses. All the 4 pcp genes were inactivated by homologous recombination. Gene pcpE and pcpD were cloned and expressed in E. coli, and the over-expressed proteins were purified. The product of gene pcpE was characterized as a maleylacetate reductase that converted 2-chloromaleylacetate to 3-oxoadipate by consuming 2 NADH. Thus, the catabolic pathway that converted substrate PCP to a central intermediate 3-oxoadipate is completely elucidated in strain ATCC 39723. The product of gene pcpD was characterized as PcpB reductase. It was not required for PCP degradation in strain ATCC 39723 but it enhanced the rate of PCP degradation. The analysis with the purified PcpD in vitro indicated that PcpD enhanced the reduction and catalysis of PcpB. Both pcpR and pcpM are hypothetical LysR type transcriptional regulators. By genetic studies, gene pcpR was characterized as the transcriptional activator for the operons of pcpE, pcpA, and pcpE. It was required for the expression of these PCP degradative genes, but not required for the expression of pcpC. PCP or other chlorophenols were able to serve as the inducer to activate the transcriptions of these PCP degradative genes. Gene pcpM was not required for PCP degradation. The function of gene pcpM is unclear. The overall organization of PCP-degrading genes was investigated, showing all the characterized PCP degradative genes, i.e. pcpB, pcpC, pcpA and pcpE were located in different operons and were separated from each other by at least a few unrelated open reading frames. Thus, the genes encoding PCP catabolic pathway were not organized as a single operon in strain ATCC 39723. |
