- Isolation and Characterization of 2,4-Dichlorophenoxyacetic Acid-degrading Bacteria from Paddy Soils
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Chung, Min Jae , Ka, Jong Ok
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J. Microbiol. 1998;36(4):256-261.
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Nine numerically dominant 2,4-dichlorophenocyacetic acid (2,4-D)-degrading bacteria were isolated from rice field soils. Most of the isolates were identified as Brukholderia of Sphingomonas species by fatty acid methyl ester (FAME) analysis, and they exhibited diverse chromosomal DNA patterns in polymerase chain reaction (PCR)amplification of repetitive extragenic palindromic (REP) sequences. The isolates utilized 2,4-D as the sole source of carbon and two Sphingomonas species were capable of mineralizing both 3-chlorobenzoate (3-CB) and 4-chlorobenzoate (4-CB), in addition to 2,4-D. Plasmid DNAs were detected from all of the isolates, and conjugation analysis revealed that 2,4-D degradative genes were located on transferable plasmids in most of the isolates.
PCR analysis with specific primers selected from tfd genes showed that 67% of the isolates had DNA sequences homologous to the five tfd genes of the 2,4-D degradative plasmid pJP4 of Alcaligenes eutrophus JMP134. Among the isolates, strain TFD7 appeared to be a new genotype in that it contained a transmissible 2,4-D degradative plasmid nonhomologous to the tfd genes. 2,4-D was persistent in natural paddy soils which contained no indigenous 2,4-D-degrading microorganisms, but the application of the 2,4-D-degrading isolates resulted in rapid decline of the soil 2,4-D residues.
- Identification and Characterization of Aniline-Degrading Bacteria Isolated from Soil and Wastewater
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Shin, Se Young , Chung, Min Jae , Yi, Haak Rho , Kim, Chi Kyung , Min, Kyung Hee , Ka, Jong Ok
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J. Microbiol. 1998;36(2):80-85.
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Twenty two aniline-degrading bacteria were isolated from agricultural soil and wastewater. The isolates were able to utilize aniline as the sole source of carbon and nitrogen. A total of 45% of the isolates were identified to the species level by fatty acid methyl ester (FAME) analysis; the isolates were found to be strains of the Burkholderia, Nocardia, Arthrobacter, and Rhodococcus species. Their chromosomal patterns, obtained by polymerase chain reaction (PCR) amplification of repetitive extrogenic palindromic(REP) sequences, were distinct from each other. Plasmid DNAs were detected from 45% of the isolates, but only one strain, ANL5, was shown to have a transmissible, aniline degradative plasmid. The isolates were very restricted in their substrate utilization abilities. Aniline degradative enzymes were inducible by the presence of aniline, and most of the isolates appeared to mineralize aniline through an ortho-cleavage path-way using catechol 1,2-dioxygenase.
- Characteristics of Catechol 2,3-dioxygenase Produced by 4-Chlorobenzoate-degrading Pseudomonas sp. S-47
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Kim, Ki Pil , Seo, Dong In , Min, Kyung Hee , Ka, Jong Ok , Park, Yong Keun , Kim, Chi Kyung
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J. Microbiol. 1997;35(4):295-299.
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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.
- Isolation and characterization of 4-chlorophenoxyacetic acid-degrading bacteria from agricultural soils
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Chung, Min Jae , Shin, Se Young , Park, Yong Keun , Min, Kyung Hee , Ka, Jong Ok
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J. Microbiol. 1997;35(2):117-122.
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Several dominant 4-CPA-degrading bacteria were isoalted from agricultural soils. Most of the isolates were identified as Burkholderia species by fatty acid methyl ester (FAME) analysis, but they were distinct in chromosomal patterns obtained by PCR amplification of repetitive extragenic palindromic (REP) sequences. These strains were generally restricted in their substrate utilization capabilities. The 4-CPA degradative enzymes were idnducible by 4-CPA and some isolates appeared to mineralize 4-CPA via formation of 4-chlorophenol and 4-chlorocatechol as intermediates during its biodegradation pathway. Plasmid DNAs were not detected from most of the isolates and their 4-CPA genes wer on the chromosomal DAN. The 4-CPA degradation patterns in axenic cultures and natural soils varied depending on the strains and soils. The inoculation of 4-CPA degraders much improved the removal of 4-CPA from the 4-CPA treated soils.
- Characterization of biphenyl biodegradation, and regulation of biphenyl catabolism in alcaligenes xylosoxydans
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Lee, Na Ri , On, Hwa Young , Jeong, Min Seon , Kim, Chi Kyung , Park, Young Keun , Ka, Jong Ok , Min, Kyung Hee
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J. Microbiol. 1997;35(2):141-148.
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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.
- Improvement of 4-chlorobiphenyl degradation bya recombinant strain, pseudomonas sp. DJ12-C
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Kim, Ji Young , Kim, Young Chang , Lim, Jai Yun , Lee, Ki Sung , Ka, Jong Ok , Min, Kyung Hee , Kim, Chi Kyung
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J. Microbiol. 1997;35(1):53-60.
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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.
- Fate of genetically engineered 2,4-D-Degrading microorganisms in natural soils and waters
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Hong, Seok Myeong , Lee, Yin Won , Kim, Chi Kyung , Ka, Jong Ok
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J. Microbiol. 1996;34(4):320-326.
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To analyze the effects of host versus plasmid on survival of 2,4-degrading bacteria in environmental samples, strains Pseudomonas cepacia/pJP4, Alcaligenes JMP228/pJP4, P. cepacia/p712, and Alcaligenes JMP228/p712 were separately inoculated into samples of field soil, paddy soil, lake water, and river water, and then the changes of their populations were measured. The strains used contained a 2,4-D degradative plasmid, either pJP4 conferring fast-growing property to the host or p712 conferring slow-growing property, and were resistant to antibiotics such that the inoculated strains could be enumerated against the indigenous microbial populations. In sterile environmental samples, these strains were stably maintained at the levels used for inoculation, except in sterile paddy soil where Alcaligenes JMP228 strains died drapidly. In natural soil samples for four strains declined steadily with time, but in natural water samples their populations fell rapidly at the early phase and then remained almost constant. When the environmental samples were treated with 2, 4-D, P. cepacia/pJP4 and P. cepacia/p712 maintained significant numbers, while Alcaligenes JMP228/pJP4 and Alcaligenes JMP228/p712 declined significantly in most of the samples. The results indicated that the survivability of genetically modified microorganisms could vary depending on the environments and that their abundance in the environments under 2,4-D selection was markedly influenced by the nature of the 2,4-D degradative plasmid as well as type of the host strain.
- Construction of genetically engineered miroorganisms for overexpression of xylE gene encoding catechol 2, 3-dioxygenase and the functional stability of the recombinant plasmid pSW3a containing xylE in aquatic environment
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Han, Hyo Yung , Kim, Chi Kyung , Park, yong Keun , Ka, Jong Ok , Lee, Byeong Jae , Min, Kyung Hee
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J. Microbiol. 1996;34(4):341-348.
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The regulation of xylE gene expression was examined by using vector promoter and construction of genetically engineered microorganisms (GEMs) for application in microcosm. When the xylE gene wsa subcloned into pBluscript SK(+) under the control of lac promoter (pTY1) in E. coli, and the expression was induced by IPTG, the enzyme activity of catechol 2, 3-dioxygenase was increased 4.7 times more than that of the crude extracts from transformants harboring pTY1. We suggest that the xylE gene has its own promoter at the upstream portion, because it was able to be expressed even in the absence of IPTG. A recombinant plasmid, pSW3a harboring the xylE gene under the T7 promotor, showed the activity of 14.5 units/mg protein, higher than that of parental strain, E. coli PYT1. The xylE gene in recombinant plasmid pSW3a was used as reporter gene for the application in microcosm ecosystem, since it was used for detection of xylE-positive clones by catechol spray on the agar plates. The pSW3a in E. coli was introduced into Pseudomonas putida to construct GEM strain, and examined for the expression and functional stability in microcosms.
- Effects of Genetically Different 2.4-D-degradative Plasmids on Degradation Phenotype and Competitiveness of Soil Microorganisms
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Hong, Seok Myeong , Ahn, Young Joon , Park, Yong Keun , Min, Kyung Hee , Kim, Chi Kyung , Ka, Jong Ok
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J. Microbiol. 1995;33(3):208-214.
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The effects of various 2, 4-D-degradative plasmids on the axenic growth patterns, the degradation phenotypes, and the competitiveness of different host bacteria were evaluated in liquid cultures; the organisms and plasmids used were Alcaligenes eutrophus JMP134/pJP4, Alcaligenes paradoxus/p2811, Pseudomonas pickettii/p712, pJP4, and p712 or p 2811 exhibited very different restriction fragment profiles in restriction endonuclease digests. These plasmids were transferred to the recipients (P. cepacia and Alcaligenes JMP228) at relatively high frequencies ranging from 8.9 × 10^-3 to 1.6 × 10^-5 per donor cell. In the axenic liquid cultures the fast-growing strains, such as P. pseudomallei/p745 and P. cepacia/pJP4, exhibited short lag periods, high specific growth rates, and high relative fitness coefficients, while the slow-growing strains, such as P. pickettii/p712 and A. paradoxus/p2811, had long lag periods, low specific growth rates, and low relative fitness coefficients. Depending on the type of plasmid containing the genes for the 2, 4-D pathway, some transconjugants exhibited intermediate growth patterns between the fast-growing strains and the slow-growing strains. The plasmid and plasmid-host interactions determined specific growth rate and lag time, respectively, which were shown to be principal determinants of competitiveness among the strains, but relative fitness coefficient derived from the axenic culture was not always predictive for the mixed culture condition.
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