Journal of Microbiology

Research Support, Non-U.S. Gov't

Isolation and Characterization of a Rhodococcus Species Strain Able to Grow on ortho- and para-Xylene: Jung Yeon Jang , Dockyu Kim , Hyun Won Bae , Ki Young Choi , Jong-Chan Chae , Gerben J. Zylstra , Young Min Kim , Eungbin Kim; J. Microbiol. 2005;43(4):325-330.; DOI: https://doi.org/2258 [pii]

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Abstract: Rhodococcus sp. strain YU6 was isolated from soil for the ability to grow on o-xylene as the sole carbon and energy source. Unlike most other o-xylene-degrading bacteria, YU6 is able to grow on p-xylene. Numerous growth substrate range experiments, in addition to the ring-cleavage enzyme assay data, suggest that YU6 initially metabolizes o- and p-xylene by direct aromatic ring oxidation. This leads to the formation of dimethylcatechols, which was further degraded largely through meta-cleavage pathway. The gene encoding meta-cleavage dioxygenase enzyme was PCR cloned from genomic YU6 DNA using previously known gene sequence data from the o-xylene-degrading Rhodococcus sp. strain DK17. Subsequent sequencing of the 918-bp PCR product revealed a 98% identity to the gene, encoding methylcatechol 2,3-dioxygenase from DK17. PFGE analysis followed by Southern hybridization with the catechol 2,3-dioxygenase gene demonstrated that the gene is located on an approximately 560-kb megaplasmid, designated pJYJ1 <br>

Characterization of biphenyl biodegradation, and regulation of biphenyl catabolism in alcaligenes xylosoxydans: Lee, Na Ri , On, Hwa Young , Jeong, Min Seon , Kim, Chi Kyung , Park, Young Keun , Ka, Jong Ok , Min, Kyung Hee; J. Microbiol. 1997;35(2):141-148.

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Abstract: Alcaligenes xylosoxydans strain SMN3 capable of utilizing biphenyl grew not only on phenol, and benzoate, but also on salicylate. Catabolisms of biphenyl and salicylate appear to be interrelated since benzoate is a common metabolic intermediate of these compounds. Enzyme levels in the excatechol 2,3-dioxygenas which is meta-cleavage enzyme of catechol, but did not induce catechol 1, 2-dioxygenase. All the oxidative enzymes of biphenyl and 2,3-dihydroxybiphenyl (23DHBP) were induced when the cells were grown on biphenyl and salicylate, respectively. Biphenyl and salicylate could be a good inducer in the oxidation of biphenyl and 2, 3-dihydroxybiphenyl. The two enzymes for the degradation of biphenyl and salicylate were induced after growth on either biphenyl or salicylate, suggesting the presence of a common regulatory element. However, benzoate could not induce the enzymes responsible for the oxidation of these compounds. Biphenyl and salicylate were good inducers for indigo formation due to the activity of biphenyl dioxygenase. These results suggested that indole oxidation is a property of bacterial dioxygenase that form cis-dihydrodiols from aromatic hydrocarbon including biphenyl.

Characteristics of Catechol 2,3-dioxygenase Produced by 4-Chlorobenzoate-degrading Pseudomonas sp. S-47: Kim, Ki Pil , Seo, Dong In , Min, Kyung Hee , Ka, Jong Ok , Park, Yong Keun , Kim, Chi Kyung; J. Microbiol. 1997;35(4):295-299.

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Abstract: Pseudomonas sp. S-47 is capable of transforming 4-chlorobenzoate to 4-chlorocatechol which is subsequently oxidized bty meta-cleavage dioxygenase to prodyce 5-chloro-2-hydroxymuconic semialdehyde. Catechol 2,3-dioxygenase (C23O) produced by Pseudomonas sp. S-47 was purified and characterized in this study. The C23O enzyme was maximally produced in the late logarithmic growth phase, and the temperature and pH for maximunm enzyme activity were 30~35℃ and 7.0, respectively. The enzyme was purified and concentrated 5 fold from the crude cell extracts through Q Sepharose chromatography and Sephadex G-100 gel filtration after acetone precipitation. The enzyme was identified as consisting of 35 kDa subunits when analyzed by SDS-PAGE. The C23O produced by Pseudomonas sp. S-47 was similar to Xy1E of Pseudomonas putida with respect to substrate specificity for several catecholic compounds.

Assessing Survival and Detection of Catechol-bidegrading Strains in Waste Water microcosms: Soo-Jin Choi , Min-Sup Song , Soo-youn Lee , Seong-Karp Hong , Kyung-Hee Min , Jong-Ok Ka , Chi-Kyung Kim , Young-Keun Park; J. Microbiol. 1998;36(4):239-248.

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Abstract: 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.

Cloning and Expression in E. coli of the Genes Responsible for Degradation of 4-Chlorobenzoate and 4-Chlorocatechol drom Pseudomonas sp. Strain S-47: Kim, Ki Pil , Seo, Dong In , Lee, Dong Hun , Kim, Young Soo , Kim, Chi Kyung; J. Microbiol. 1998;36(2):99-105.

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Abstract: 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.

Construction of a Bioluminescent Reporter Using the luc Gene and meta-Cleavage Dioxygenase Promoter for Detection of Catecholic Compounds: Sang-Ho Park , Dong-Hun Lee , Kye-Heon Oh , Chi-Kyung Kim; J. Microbiol. 2000;38(3):183-186.

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Abstract: Several types of bioluminescent reporter strains have been developed for the detection and monitoring of pollutant aromatics contaminating the environment. In this study, a bioluminescent reporter strain, E. coli SHP3, was constructed by fusing the luc gene of firefly luciferase with the promoter of pcbC responsible for the meta-cleavage of aromatic hydrocarbons. The bioluminescence expressed by the luc gene in the reporter was well triggered by the promoter when it was exposed to 2,3-dihydroxybiphenyl (2,3-DHBP) at 0.5 to 1 mM concentrations. The bioluminescent response was more extensive when the reporter strain was exposed to 5 mM catechol and 2 mM 4-chlorocatechol. These different types of bioluminescent responses by E. coli SHP3 appeared to be characterized by the nature of the aromatics to stress. Since E. coli SHP3 responded to 2,3-DHBP quite sensitively, this reporter strain could be applied for detecting some catecholic pollutants.

Toxic Effects of Catechol and 4-Chlorobenzoate Stresses on Bacterial Cells: Sang-Ho Park , Yeon-Ja Ko , Chi-Kyung Kim; J. Microbiol. 2001;39(3):206-212.

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Abstract: Catechol and 4-chlorobenzoate (4CBA) which are produced from the biodegradation of a variety of aromatic and chloroaromatics have been recognized as toxic to living organisms. In this study, the toxic effects of catechol and 4-chlorobenzoate on gram-positive and -negative bacteria were examined in terms of survival, morphology, change in fatty acids and membrane protein composition. The survival rate of the organisms during treatment for 6 h was decreased, as the concentration of each aromatic was increased. Escherichia coli and Pseudomonas cells treated with catechol and 4CBA at concentrations causing a significant decrease in their viability, showed destructive openings in their cell envelopes. Bacillus subtilis treated with the aromatics were reduced in cell size and Staphylococcus aureus cells displayed irregular rod shapes with wrinkled surfaces. The bacterial cells treated with 20 mM catechol showed increases in unsaturated fatty acids, but several saturated fatty acids were decreased. In the E. coli cells treated with 20 mM catechol, inner membrane proteins of 150 kDa and 105 kDa were decreased. But several kinds of the inner and outer membrane proteins were increased. In B. subtilis treated with 20 mM catechol, several kinds of proteins were increased or decreased in membrane proteins.

Cloning and Sequence Analysis of Two Catechol-degrading Gene Clusters from a Phenol-utilizing Bacterium Pseudomonas putida SM25: 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; J. Microbiol. 2003;41(2):102-108.

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Abstract: 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.

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