- Cloning and Expression in E. coli of the Genes Responsible for Degradation of 4-Chlorobenzoate and 4-Chlorocatechol drom Pseudomonas sp. Strain S-47
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Kim, Ki Pil , Seo, Dong In , Lee, Dong Hun , Kim, Young Soo , Kim, Chi Kyung
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J. Microbiol. 1998;36(2):99-105.
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Pseudomonas sp. strain S-47 can grow on 4-chlorobenzoate (4CBA) and transform 4CBA to 4-chlorocatechol (4CC) under aerobic conditions, which is subsequently degraded to produce 2-hydroxypent-2, 4-dienoate (2H-2,4DA). The upper steps for conversion of 4CBA to 4CC are recognized to be conducted by the benzoate-1,2-dioxygenase (B12O) system encoded by benABC and benD. The ensving meta-cleabage reaction of 4CC is catalyzed by catechol 2,3-dioxygenase(C23O) encoded by the xylE gene. The benABCD and the xylE genes were cloned from the chromosome of Pseudomonas sP. S-47 into pCS1(48.7kb), pCS101(24.4kb), pCS201(17.7kb), and pCS202(6.7kb) recombinant plasmids, and were well ecpressed in E. coli XL1-Blue host cells. The PstI-insert (4.0kb) of pC202 was found to contain the benABCD and cylE genes and to have 2 EcoRV, 1 SphI, and 3 SacII restriction sites.
- Cloning and expression of pseudomonas cepacia catB gene in pseudomonas putida
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Song, Seung Yeon , Jung, Young Hee , Lee, Myeong Sok , Lee, Ki Sung , Kim, Young Soo , Kim, Chi Kyung , Choi, Sang Ho , Min, Kyung Hee
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J. Microbiol. 1996;34(4):334-340.
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The enzyme, cis,cis-muconate lactonizing enzyme has been proposed to play a key role in the β-ketoadipate pathway of benzoate degradation. A 3.2-kb EcoRI fragment termed as pRSU2, isolated from a Pseudomonas cepacia genomic library was able to complement the catB defective mutant. Several relevant restriction enzyme sites were determined within the cloned fragment. In Pseudomonas putida SUC2 carrying pRSU2, the enzyme activity was relatively higher than those of the induced or partially induced state of wild type P. putida PRS2000. It was probably due to higher expression of P. cepacia catB in P. putida PRS2000. It was probably due to higher expression of P. cepacia catB in P. putida. One possible interpretation of these results is that the catB promoter in P. cepacia is recognized within P. putida, resulting in the almost same expression level.
- Extracellular DNAs released form the genetically engineered E. coli CU103 during growth in different liquid media
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Kim, Chi Kyung , Park, Sang Ho , Lim, Jai Yun , Kim, Young Chang , Kim, Young Soo , Min Kyung Hee , Lee, Ki Sung
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J. Microbiol. 1996;34(2):144-150.
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During growth of the genetically engineered E. coli CU103 in different media, extracellular DNAs released from the cells were studied. The extracellular DNAs released in the medium were concentrated by an ethanol precipitation method and then quantified by a fluorescence method using Hoechst 33258. The released extracellular DNAs were also examined by gel electrophoresis and identified by Southern hybridization for the cloned pcbCD genes. The chromosomal DNAs and recombinant plasmid containing the cloned genes were observed to be released in an exponential growth phase. In Luria-Bertani (LB) broth and MM2-GLUCOSE, 210 and 69 ng/ml of DNAs were detected, respectively, after 3-4 days incubation at 30℃ and at pH 7.0. But the released DNAs were measured to be about 10-15 ng/ml in filtered river water (FW) and Tris-EDTA (TE). The at both 15℃ and 4℃, but the released DNAs were more easily degraded at the higher temperature. The extracellular DNAs were produced about 2 times more at pH 7.0 than at both pH 5.0 and pH 9.0 in MM2-glucose medium at 30℃. Therefore, the extracellular DNAs were found to be released actively from the cells during growth in liquid media.
- Effect of Temperature on Persistence of Recombinant Plasmid pCU103 in Different Waters
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Kwak, Myong Ja , Kim, Chi Kyung , Kim, Young Chang , Lim, Jae Yun , Kim, Young Soo , Lee, Ki Sung , Min, Kyung Hee
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J. Microbiol. 1995;33(3):178-183.
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The recombinant plasmid of pCU103 constructed by cloning pcbCD genes in pBluescript SK(+) was studied for the effect of temperature on its persistence in different waters by the methods of electrophoresis, Southern hybridization, quantification, and transformation. The plasmid was very rapidly degraded out in non-sterile FW water without regards to water temperature, probably due to the effect of biochemical factor such as nucleases. The pCU103 was most persistent at 4℃ in any water environments, moderately persistant at 15℃, but least stable at 30℃ such results could be explained by the facts that hydrogen bonds in double-stranded plasmid DNAs become unstable and that nucleases are activated by increasing temperature. The intact structure of pCU1-3 was generally observed by gel electrophoresis under the conditions which the plasmid should be 2.0 ng/㎕ or higher in concentration and that about 10² CFU/ml or more transformant cells should be recovered.
- Cloning of the Gene Responsible for Dechlorination of 4-Chlorobenzoate from Pseudomonas sp. DJ-12
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Chae, Jong Chan , Kim, Young Chang , Kim, Young Soo , Kim, Chi Kyung
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J. Microbiol. 1995;33(1):34-39.
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The gene responsible for dechlorination of 4-chlorobenzoate(4CBA) was cloned from chromosomal DNA of Pseudomonas sp. DJ-12 in Escherichia coli XL1-Blue by using pBluescript SK(+) phagemid vector. Two recombinant plasmids of pCJ1 and pCJ2 carrying dechlorinase gene were constructed. The inserted DNAs in the pCJ1 and pCJ2 were found to have the fragment of 9.5 kb carrying dechlorinase gene, but they were oriented in opposite direction. The inserted DNA of 3.4kb in the pCJ101 subclone carrying dechlorinase gene had two restriction sites for AccI and each one site for HincII, KpnI, PstI, and SacIi but the dechlorinase gene in pCJ101 was found to be placed over the HincII site. The dechlorinase fenes in the recombinant cells of E. coli CJ1 and CJ101 were well expressed to show the dechlorination activity of 4CBA. The proteins encoded by dechlorinase genes in E. coli CJ1 and CJ101 were about 49 kDa in molecular weight. But this protein was not produced by E. coli CJ102, CJ103 and E. coli XL1-Blue. Therefore, the proteins produced by Pseudomonas sp. DJ-12 and the cloned cells of E. coli CJ1 and CJ101 were thought to be the dechlorinase enzyme which was the product of dechlorinase gene.
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