Research Of Regulation Mechanism Of Phenol Degradation In Corynebacterium Glutamicum

A large number of bacteria are able to degrade aromatic carbon sources employing different strategies. All these pathways are objects of regulatory control at the level of gene expression. Corynebacterium glutamicum is a fast growing, aerobic, and non-pathogenic Gram-positive soil bacterium. C. glutamicum is able to utilize a large variety of aromatic compounds as the sole carbon source. Under aerobic conditions, phenol is normally converted into catechol, which depends on the gene ncgl2588(phe) operon encoding phenol hydroxylase. Then catechol is further degraded via an ortho-cleavage pathway to central metabolism intermediates of the Krebs cycle in C. glutamicum. An additional gene, ncgl2587(pheR), was found upstream of phe gene. The pheR gene encodes an AraC/XylS-type regulator protein with 377 amino acid residues and is transcribed in the same direction to phe gene. Disruption of the gene pheR by homologous recombination resulted in the loss of phenol degradation in C. glutamicum. We used several methods to demonstrate the pheR active the expression of phe encoding phenol hydroxylase. C. glutamicum thus possesses a more elaborate mechanism for regulating the phe operon expression than has been found in other bacteria. More extensive studies are needed to fully understand mechanisms involved in the regulation of catabolic genes and to further improve the ability of Corynebacterium glutamicum to degrade aromatic environmental pollutants. Efficient aerobic biodegradation of aromatic compounds has become an attractive alternative to physical and chemical methods to remove these toxic pollutants from the environment.1. Recombinant plasmid pk18 mobsac B- pheR through overlapping PCR was constructed successfully to knockout the pheR gene in Corynebacterium glutamicum RES167, and the deletion mutant was constructed by homologous recombination. The mutant pheR and wild type were cultured in MS media containing 3 mM phenol, the mutant lost the ability to utilize phenol as the sole carbon source, and complementation of the mutant strain recovered the normal ability to grow on 3 mM phenol as fast as wild type. Results from gene disruption analyses and genetic complementation test demonstrate that pheR is required for the growth of C. glutamicum on phenol. We conducted pK18mobsacB-Pphe:: lacZ transcription fusion vector, then observed the promoter activity for Pphe was the same results as above. The result of quantitative real-time RT-PCR was demonstrated that pheR is involved in the activation of the phenol catabolic pathway.2. The pheR gene was cloned into pET21 a vector containing an N-terminal hexahistidine tag followed by a TEV cleavable maltose binding protein(MBP). At this stage, now the PheR protein is soluble, well behaved biochemically, and can be purified and obtained the full PheR protein following several simple purification steps by simple SDS gel electrophoresis. We performed electrophoretic mobility shift assays to investigate the manner in which PheR regulates the transcription of the phe operons.3. The DNA binding sites of phe operon were identified by DNase I footprinting assay. The binding site was T-G-T-T-C-T-C-A-G-G-C-G-G-C-A-T-C-A-T-C-T-T-G-C-C-T-A-A-CA-C-A-G-A-CA-A. When we mutated PheR binding site at Pphe, PheR could not bind to the promoter region of the phe operon any more. But the β-galactosidase assays demonstrated that the phe operon could express a little.4. According to multiple alignment of proteins belonging to the AraC/XylS family relating to PheR protein, we analyze the conserved region of these protein and the HTH regions of PheR. In the AraC regulator, the first HTH motif was predicted to be located as positions 247 to 273 in PheR and the second HTH motif was located between amino acid residues 288 and 328 in PheR.