- Catabolism of 4-Hydroxybenzoic Acid by Pseudomonas sp. DJ-12
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Karegoudar, Timmanagouda B. , Chae, Jong Chan , Kim, Chi Kyung
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J. Microbiol. 1999;37(3):123-127.
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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 catechol 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.
- Degradation of Anthracene by a Pseudomonas strain, NGK1
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Manohar, Shinde , Kim, Chi Kyung , Karegoudar, Timmanagouda B.
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J. Microbiol. 1999;37(2):73-79.
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Pseudomonas sp. NGK1, isolated by naphthalene enrichment culture technique, is capable of degrading anthracene as a sole source of carbon and energy. The organism degraded anthracene through the intermediate formation of 1,2-dihydroxyanthracene, 2-hydroxy-3-naphthoic acid, salicylate, and catechol. The intermediates were isolated and characterized by TLC, spectrophotometry, and HPLC analysis. The cell free extract of anthracene-grown cells showed activities of anthracene dioxygenase, 2-hydroxy-3-naphthylaldehyde dehydrogenae, 2-hydroxy-3-naphthoate hydroxylase, salicylate hydroxylase and catechol 2,3-dioxygenase. The formed catechol as a metabolite is degraded through meta-cleavage with the formation of α-hydroxymuconic semi-aldehyde.
- Production of Stress-shock Proteins in Pseudomonas sp. DJ-12 Treated with 4-Hydroxybenzoate
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Park, Sang Ho , Oh, Kye Heon , Lee, Kil Jae , Kim, Chi Kyung
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J. Microbiol. 1998;36(4):273-279.
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Pseudomonas sp. DJ-12 can grow on 4-hydroxybenzoate (4HBA) at concentration of 5 mM or lower by degrading 4HBA for carbon and energy sources. The organisms were found to produce DnaK stress-shock protein when treated with several aromatic hydrocarbons including 4HBA. Those cells treated with 5 mM 4HBA exhibited increased tolerance to 10 mM concentration. In this study, the production of other stress-shock kproteins besides KnaK was examined in Pseudomonas sp. DJ-12 exposed to various concentrations of 4HBA, compraing the production of the proteins with their survival and degradation of 4HBA. The organisms could degrade 4HBA at 0.5 to 5 mM concentrations after 60 to 90 minutes of incubation. The survival rate of the organism decreased when treated with 4HBA at 10 mM or higher concentrations. The stress-shock proteins of DnaK, GroEL, and GroES were produced in the cells which were treated with 4HBA at 0.5 mM or higher concentrations for 10 minutes. Fifteen additional stress-shock proteins were produced in the cells which were treated with 5 mM 4HBA for 40 minutes. The DnaK and GroEL proteins in the cells gradually decreased upto 6 hours after the stress was removed from the culture.
- 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.
- Cellular Response of Pseudomonas sp. DJ-12 to the Stresses of Several Aromatic Pollutants
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Park, Sang Ho , Ko, Yeon Ja , Oh, Kye Heon , Kim, Chi Kyung
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J. Microbiol. 1998;36(2):93-98.
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Pseudomonas sp. DJ-12 is capable of degrading biphenyl, 4-chlorobiphenyl (4CB), and 4-hydroxybenzoate (4HBA) at relatively low concentrations. The degradability and cellular responses of the organism to the aromatic pollutants at higher concentrations have not been studied. In this study, the survival of the cells, production of stress proteins, and induction of tolerance were examined by exposing cells to various concentrations of the aromatics. The survival rates of the organism were reversely proportional to the concentration of each aromatic during incubation for 6 hs. Stress protein specifically reacting with anti-DnaK monoclonal antibody was commonly produced when cells were treated with each aromatic. Those cells pretreated with each aromatic at lower concentrations exhibited a certain degree of tolerance to higher concentrations of the aromatics. Such cellular responses of the organism to water-soluble 4HBA were more clearly distinguished than those to insoluble 3CB and biphenyl. Therefor, induction of DnaK stress protein in the cells by exposure to the aromatiocs could be used to develop an indicator for pollution by the aromatics.
- 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.
- Dechlorination of 4-Chlorobenzoate by Pseudomonas sp. DJ-12
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Chae, Jong Chan , Kim, Chi Kyung
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J. Microbiol. 1997;35(4):290-294.
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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.
- 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 of pseudomonas sp. S-47 and its degradation of 4-chlorobenzoic acid
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Seo, Dong In , Lim, Jai Yun , Kim, Young Chang , Min, Kyung Hee , Kim, Chi Kyung
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J. Microbiol. 1997;35(3):188-192.
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The strain of S-47 degrading 4-chlorobenzoic acid (4CBA) was isolated from Ulsan chemical industrial complex by enrichment cultivation with 1 mM 4CBA. The strain was Gram-negative rod and grew optimally at 30℃ and pH 7 under aerobic condition, so that the organism was identified as a species of Pseudomonas. Pseudomonas sp. S-47 degraded 4-chlorobenzoic acid to produce a yellow-colored meta-cleavage product, which was confirmed to be 5-chloro-2-hydroxymuconic semialdehyde (5C-2HMS) by UV-visible spectrophotometry. 5C-3HMS was proved trometry. This means that Pseudomonas sp. S-47 degraded 4CBA via 4-chlorocatechol to 5C-2HMS by meta-cleavage reaction and then to 5C-2HMA by 5C-2HMS dehydrogenase.
- 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.
- 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.
- 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.
- Cloning and expression in E. coli of the HOPDA hydrolase gene from pseudomonas sp. P 20
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Lim, Jong Chul , Chae, Jong Chan , Kim, Hyong Bai , Kim, Chi Kyung
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J. Microbiol. 1996;34(4):349-354.
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Pseudomonas sp. P20 is a natural isolate which is capable of degrading biphenyl and 4-chlorobiphenyl. From a clone of pCK1022 harboring pcbCD genes of Pseudomonas sp P20, a pcbD gene encoding 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoic acid (HOPDA) hydrolase was subcloned in Escherichia coli XL-1-Blue by using pBluescript SK(+) vector. The 2.8-kb HindII fragment harboring the pcbD gene cloned in pCK 1024 had a single site for each of XhoI, SalI, BstXI, and XbaI restriction enzymes. Escherichia coli CK1024 had a single site for each of XhoI, SalI, BstXI, and XbaI restriction enzymes. Escherichia coli CK1024 carrying pCK0124 degraded HOPDA to benzoate and 2-hydroxypenta-2, 4-dienoate by HOPDA hydrolase encoded by pcbD gene as effectively as E coli CK 1022 HARBORING pcbCD genes.
- Structural and functional stability of the genetic recombinant plasmid pCU103 in different water environments
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Kim, Chi Kyung , Kwak, Myoung Ja , Lee, Sung Gie
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J. Microbiol. 1996;34(3):241-247.
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The stbility of the genetically engineered microorganisms and their recombinant plasmids released in natural environments has been regarded as one of the molecular ecological topics. In this study, the recombinant plasmids pCU103 in which the pcbCD genes involved in biodegradation of biphenyl and 4-chlorobiphenyl were cloned in pBluescript SK(+) vector, were examined for their structural and functional stability in different waters at 15℃ by the methods of electrophoresis, Southern hybridization, quantification with fluorescent dye, and transformation. The recombinant plamids maintained their stabilities for about 30 days in sterilized distilled water (SDW), 15 days in autoclaved creek water (AW), 25 days in filtered and autoclaved non-sterible creek water (FAW), 4 days in Luria-Bertani (LB) broth, and less than one day in filtered non-sterile creek water (FW). The covalently closed circular (CCC) form of the plasmid was decreased and open circular (OC) form was increased as a function of incubation time, and then linear (L) form was produced to be ultimately degraded out. The degradation rates of the plasmid were proportionally correlated to trophic level of the water, and the biological factor such as DNases was found to be one of the most critical factors affecting structural and functional stability of the plasmid in non-sterile natural water.
- 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.
- 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.
- 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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