- Cloning and Sequence Analysis of Two Catechol-degrading Gene Clusters from a Phenol-utilizing Bacterium Pseudomonas putida SM25
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Young-Hee Jung , Jong-Ok Ka , Choong-Ill Cheon , Myeong-Sok Lee , Eun-Sook Song , Soon-Young Choi , Daeho Cho , Sang-Ho Choi , Kwon-Soo Ha , Young Mok Park , Jong-Soon Choi , Kyung-Hee Min
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J. Microbiol. 2003;41(2):102-108.
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A 6.1 kb Sph I fragment from the genomic DNA of Pseudomonas putida SM 25 was cloned into the vector pUC19. The open reading frame of catB was found to consist of 1,122 nucleotides. The sequence alignment of the catB gene products from different kinds of bacteria revealed an overall identity ranging from 40 to 98%. The catC gene contained an open reading frame of 96 codons, from which a protein with a molecular mass of about 10.6 kDa was predicted. The amino acids in the proposed activesite region of CatC were found to be almost conserved, including the charged residues. Since the catBC genes in P. putida SM25 were tightly linked, they could be regulated under coordinate transcription, and transcribed from a single promoter located upstream of the catB gene, as in P. putida RB1.
- Assessing Survival and Detection of Catechol-bidegrading Strains in Waste Water microcosms
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Soo-Jin Choi , Min-Sup Song , Soo-youn Lee , Seong-Karp Hong , Kyung-Hee Min , Jong-Ok Ka , Chi-Kyung Kim , Young-Keun Park
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J. Microbiol. 1998;36(4):239-248.
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Catechol-degrading bacteria, Escherichia coli JM101 carrying pIB1343 (strain pIB1343) and Flavimonas orizihabitans KHi (strain KH1), were chosen as model bacteria to estimate the sur vival and catechol-degradability in waste water microcosms. Before their application to ecosystem, survival and catechol degradation of these bacteria were investigated in waste water microcosms. It was found that strain pIB1343 adapted much faster to waste water environments and degraded catechol in the case of Namdong samples, whereas the strain KH1 degraded catechol much faster in Sihwa samples. When catechol was added to the microcosms, indigenous microorganisms in Sihwa samples used catechol as a carbon and energy source much better than those in Namdong samples. A modified filter extraction technique was used to obtain the high-yield purified DNA from 50 ml of waste water samples and the extracted DNA (polymorphic DNA [20~23 kb]) was of sufficient quantity and quality for the amplification. PCR was performed with catA-specicic promers, C120U and C120L, which specifically detected the catA gene encoding catechol 1,2-dioxygenase in waste water microcosms, and then the 320 bp PCR products were amplified. PCR products were quantified by densitomenter. Using the standard curve of detection limit by catA-specific PCR products, the number of catA genes for their corresponding intensities of PCR products was obtained. the number of total catA genes PCR in waste water microcosms was correalated with catechol degradation.
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