• Journal of Microbiology
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• v.35 no.1
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• pp.53-60
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• 1997

Pseudomonas sp. P20 and Pseudomonas sp. DJ-12 isolated from the polluted environment are capable of degrading biphenyl and 4-chlorobiphenyl (4CB) to produce benzoic acid and 4-chlorobenzoic acid (4CBA) respectively, by pcbABCD-encoded enzymes. 4CBA can be further degraded by Pseudomonas sp. DJ-12, but not by Pseudomonas sp P20. However, the meta-cleavage activities of 2, 3-dihydroxybiphenyl (2, 3-DHBP) and 4-chloro-2, 3-DHBP dioxygenases (2, 3-DHBD) encoded by pcbC in Pseudomonas sp. P20 were stronger than Pseudomonas sp. DJ-12. In this study, the pcbC gene encoding 2, 3-DHBD was cloned from the genomic DNA of Pseudomonas sp. P20 by using pKT230. A hybrid plasmid pKK1 was constructed and E. coli KK1 transformant was selected by transforming the pKK1 hybrid plasmid carrying pcbC into E. coli XL1-Blue. By transferring the pKK1 plasmide of E. coli KK1 into Pseudomonas sp. DJ-12 by conjugation, a recombinant strain Pseudomonas sp. P20, Pseudomonas sp. DJ-12, and the recombinant cell assay methods. Pseudomonas sp. DJ12-C readily degraded 4CB and 2, 3-DHBP to produce 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoic acid (HOPDA), and the resulting 4CBA and benzoic acid were continuously catabolized. Pseudomonas sp. DJ12-C degraded 1 mM 4CB completely after incubation for 20 h, but Pseudomonas sp. P20 and Pseudomonas sp. DJ-12 showed only 90% and Pseudomonas sp. DJ-12 had, but its degradation activity to 2, 3-DHBP, 3-methylcatechol, and catechol was improved.

• Journal of Microbiology
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• v.35 no.4
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• pp.290-294
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• 1997

4-Chlorobiphenyl-degrading Pseudomonas sp. DJ-12 was able to degrade 4-chlorobenzoate(4CBA), 4-iodobenzoate, and 4-bromobenzoate completely under aerobic conditions. During. the degradation of 4CBA by Pseudomonas sp. DJ-12, chloride ions were released by dechlorination and 4-hydroxybenzoate was produced as an intermediate metabolite. The NotI-KNA fragments of pKC157 containing dechlorination genes hybridized with the gene encoding 4CBA:CoA dehalogenase of Pseudomonas sp. CBS3 which is responsible for the hydrolytic dechlorination of 4CBA. These results imply that Pseudomonas sp. DJ-12 degrades 4CBA to 40hydroxybenzoate via dechlorination as the initial step of its degradativ pathway. The genes responsible for dechlorination of 4CBA were found to be blcated on the chromosomal DNA of Pseudomonas sp. DJ-12.

• Korean Journal of Microbiology
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• v.40 no.2
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• pp.121-126
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• 2004

Comamonas sp. strain DJ-12 is a 4-chlobiphenyl(4CB)-degrading bacterium that was reidentified from Pseudomonas sp. DJ-12. The genomic DNA was isolated from the strain DJ-12 and amplified by PCR with primers for cloning pcbABCD genes responsible for degradation of 4CB. The amino acid sequences deduced from the nucleotide sequences of pcbA1, pcbA2, pcbA3, pcbA4, pcbB, pcbC2, and pcbD2 genes showed 91, 87, 99, 87, 97, 90 and 87％ homologies with those of Pseudomonas sp. KKS102, respectively. The pcbC1D1 genes that are involved in the degradation of (4-chloro)1,2-dihydroxybiphenyl produced from 4CB by pcbAB gene products were previously reported in the recombinant plasmid pCU1 from Pseudomonas sp. DJ-12. However, the pcbC2D2 genes in the plasmid pCT4 and pCT5 cloned from Comamonas sp. DJ-12 in this study showed 51 and 62％ homologies with those of pcbC1D1 in their nucleotide sequences. The pcbC1D1 and pcbC2D2 genes were found by Southern hybridization to be located at different loci on the chromosome of DJ-12 strain. These results indicate that Comamonas sp. strain DJ-12 has two different sets of pcbCD genes responsible for deg-radation of (4-chloro)1,2-dihydroxybiphenyl.

• Korean Journal of Microbiology
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• v.32 no.2
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• pp.126-131
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• 1994

The pchABCD genes in Pseudomonas sp. DJ-12 speciyin degradation o 4-chlorobiphenyl(4CB) were cloned in Eschericia coli. The cloned cells of E. coli CU1 and CU101 showed to produce 2,3-dihydroxybiphenyl (2,3-DHBP) from 4-chlorobiphenyl by dechlorination, as Pseudomonas so. DJ-12 produced 2,3-DHBP from both biphenyl and 4CB. In particular, E. coli CU101 transformed with the recombinant plasmid of pCU101 revealed dechlorination activity to produce 2,3-DHBP from 4CB without production of 4-chlorobenzoic acid. Therefore, the pcbAB genes (2.2 kb in size) cloned from the chromosome of Pseudomonas sp. DJ-12 were found to have dechlorination activity on 4CB to produce 2,3-DHNP.

• Journal of Microbiology and Biotechnology
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• v.12 no.3
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• pp.417-422
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• 2002

Gene fusions were constructed by the transcriptional fusion of the dnaK promoter of pseudomonas sp. DJ-12 or E. coli to the lux or luc marker gene. The dnaKp-DJ::luxCDABE bioluminescent fusion in the biosensor using the Pseudomonas sp. DJ-12 dnaK promoter exhibited about 5-fold more extensive response to ethanol than that of dnaKp-EC::luxCDABE. The bioluminescent response of the dnaK-DJ::luc fusion to ethanol was much weaker than those of the other fusions. The biosensor harboring the dnaKp-DJ::luCDABE fusion was examined for its bioluminescence production based on exposure to aromatic compounds, such as biphenyl, 4-chlorobiphenyl (4CB), 4-hydroxybenzoate (4HBA), and catechol. In particular, the bioluminescence produced by the dnaKp-DJ::luxCDABE fusion was most sensitive to 1 mM biphenyl and 4CB when exposed for 80 min, and the responses were also very strong to other aromatics. Therefore, the biosensor bearing the dnaKp-DJ::luxCDABE fusion would appear to be the most useful for the detection of aromatics and other pollutants.

• Journal of Microbiology and Biotechnology
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• v.8 no.6
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• pp.692-695
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• 1998

Pseudomonas sp. DJ-12 was able to degrade 4-chlorobenzoate by hydrolytic dechlorination to produce 4-hydroxybenzoate and chloride ion. The fcbABC genes responsible for the hydrolytic dechlorination were cloned from the chromosomal DNA of the organism. The genes were found to be organized in the order fcbB-fcbA-fcbC, but there was an intergenic space between the fcbA and fcbC genes.

• Journal of Microbiology
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• v.37 no.3
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• pp.123-127
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• 1999

A Pseudomonas sp. strain DJ-12 isolated by 4-cholrobiphenyl enrichment culture technique is capable of utilizing 4-hydroxybenzoic acid as a sole source of carbon and energy. The bacterium catabolized 4-hydroxybenzoic acid through the intermediate formation of protocatechuic acid, which was further metabolized. The cell free extracts of pseudomonas sp. DJ-12, grown on 4-hydroxybenzoic acid showed higher activities of 4-hydroxyenzoate 3-hydroxylase and protocatechuate 4,5-dioxygenase, but the activity of catechnol 2,3-dioxygenase was lower. The results suggest that 4-hydroxybenzoic acid is catabolized via protocatechuic acid rather than catechol or gentisic acid in this bacterium and that the protocatechuic acid formed was metabolized through a metacleavage pathway by protocatechuate 4,5-dioxygenase.

• Microbiology and Biotechnology Letters
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• v.21 no.2
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• pp.150-156
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• 1993

The 2,3-dihydroxybiphenyl(2,3-DHBP) dioxygenase, the product of pcbC gene, was purified from the biphenyl and 4-chlorobiphenyl degrading Pseudomonas sp. DJ-12 by the methods of acetone precipitation, DEAE-Sephadex A-50 ion exchange chromatography, and Sephadex G-150 gel filtration chromatography. The enzyme was estimated to be about 260 kilodaltons in molecular weight and to be consisted of eight subunits. The Km value of the enzyme was 61 nM to 2,3-DHBP and the highest activity of the enzyme was observed at pH 8 and 30C.

• Korean Journal of Microbiology
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• v.35 no.2
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• pp.139-145
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• 1999

Aromatic hydrocarbons which are not easily degraded by microorganisms can be accumulated in the conlaminated environment for a long lime, producing toxic effects on wild lives and humans. However, the sublethal concentrations of the chemicals induce the synthesis of stress-shock proteins in the cells and increase the adaptability of the organisms to the environmental stresses. In this study, therefore, the cells of Psezido~nonus sp. DJ- 12 treated with catechol at various concentrations were inveshgated for their survival, biodegtadability of catechol, production of stress-shock proteins, and cytological changes. The organisms were capable of degrading catechol at the range of 0.5 to 1.0 mM concentration wilhin 6 hours incubation, but they were killed by $10^2$-10$^3$ celllinl at 3 mM or higel- concentration without any catechol degradation. These cells treated with catechol begm lo produce DnaK and GroEL at 1 mM and 0.5 mM. respectively. Pseudumonas sp. DJ-12 treated with 10 mM catechol for I hour exhihiled some punctuated pores on the cell wall and contortion of the rod shape. The cells treated with he sublethal concentration of catechol showed the increased tolerance for suvival when exposed to the lethal concentration, and such tolerant effects were functioned crossly among benzoate, 4-chlorobenzoate, 'and catechol.

• Journal of Microbiology and Biotechnology
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• v.9 no.4
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• pp.422-428
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• 1999

Aromatic hydrocarbons can be utilized as carbon and energy sources by some microorganisms at lower concentrations. However, they can also act as stresses to these organisms at higher concentrations. Pseudomonas sp. DJ-12 is capable of degrading 0.5 mM concentration of benzoate and 4-chlorobenzoate (4CBA). In this study, the exposure of Pseudomonas sp. DJ-12 to the pollutant stresses of benzoate and 4CBA at various concentrations was comparatively studied for its cellular responses, including survival tolerance, degradability of the aromatics, and morphological changes. Pseudomonas sp. DJ-12 utilized 0.5 to 1.0mM benzoate and 4CBA as carbon and energy sources for growth. However, the organism could not degrade the aromatics at concentrations of 3 mM or higher, resulting in reduced cell viability due to the destruction of the cell envelopes. Pseudomonas sp. DJ-12 cells produced stress-shock proteins such as DnaK and GroEL when treated with benzoate and 4CBA at concentrations of 0.5mM, or higher as sublethal dosage; Yet, there were differing responses between the cells treated with either benzoate or 4CBA. 4CBA affected the degradability of the cells more critically than benzoate. The DnaK and GroEL stress-shock proteins were produced either by 1mM benzoate with 5 min treatment or by higher concentrations after 10min. The proteins were also induced by 0.5mM 4CBA, however, it needed at least 20min treatment or longer. These results indicate that the chlorination of benzoate increased the recalcitrance of the pollutant aromatics and changed the conditions to lower concentrations and longer treatment times for the production of stress-shock proteins. of stress-shock proteins produced by the aromatics at sublethal concentrations functioned interactively between the aromatics for survival tolerance to lethal concentrations.