pKT230 벡터를 이용한 Pseudomonas sp. P20 2,3-Dihydroxybiphenyl Dixygenase 유전자의 클로닝

김지영,김치경,가종억,민경희,박용근 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.6

Biphenyl과 4-chlorobiphenyl을 분해하는 자연계 분리 균주인 Pseudomonas sp. P20의 chromosomal DNA로부터 pBluescript SK(+)를 이용하여 pcbABCD 유전자를 클로닝하여 재조합플라스미드 pCK1을 제조하였고, 또 pcbCD 유전자를 포함하여 pCK102을 제조하였다. 방향족 탄화수소 화합물의 생분해는 벤젠고리의 개환 과정이 중요하기 때문에, 2,3-dihydroxybiphenyl(2,3-DHBP)의 벤젠고리의 개환에 관여하는 2,3-DHBP dioxygenase(2,3-DHBD) 유전자를 pKT230 벡터를 이용하여 pCK102로부터 클로닝하였다. EcoRI으로 절단한 pCK102와 pKT230 벡터를 ligation시켜 13.8 kb의 hybrid plasmid pKK1을 제조하였다. 2,3-DHBD 유전자를 포함하는 pKK1을 E. coli XL1-Blue에 형질전환시켜 E. coli KK1 재조합 균주를 얻은 후, 2,3-DHBD의 활성을 측정하였다. E. coli KK1의 2,3-DHBD의 효소활성은 pBluescript SK(+)를 이용하여 제조한 재조합 균주인 E. coli CK102의 효소활성과 유사하였으나, Pseudomonas sp. DJ-12와 Pseudomonas sp. P20과 같은 자연계 분리균주보다 훨씬 높았다. Pseudomonas sp. P20 isolated from the polluted environment is capable of degrading biphenyl and 4-chlorobiphenyl. The pcbABCD genes responsible for degradation of biphenyl and 4-chlorobiphenyl were cloned using pBluescript SK(+) from the chromosomal DNA of Pseudomonas sp. P20 to construct pCK1 and pCK102, harbouring pcbABCD and pcbCD, respectively. The 2,3-DHBP dioxygenase gene, pcbC, was cloned again from pCK102 by using pKT230 which is known as a shuttle vector and pKK1 hybrid plasmid was constructed. The E. coli KK1 transformant obtained by transforming the pKK1 into E. coli XL1-Blue showed 2,3-DHBP dioxygenase activity. The specific 2,3-DHBP dioxygenase activity of E. coli KK1 was similar to that of the E. coli CK102, but much higher than those of the natural isolates, Pseudomonas sp. DJ-12 and Pseudomonas sp. P20

Microbial Ecology : Use of Gene Probes for Tracking 2,4 - Dichlorophenoxyacetic Acid - Degrading Microbial Populations in Soil

Jong Ok Ka,W . E . Holben,J . M . Tiedje 한국미생물생명공학회(구 한국산업미생물학회) 1995 FUNCTIONAL BIOMATERIALS Vol.- No.-

DNA probes 에 의한 토양의 이사디 ( 2,4 - D ) 분해세균의 검출

가종억 ( Jong Ok Ka ) 한국응용생명화학회 1996 Applied Biological Chemistry (Appl Biol Chem) Vol.39 No.5

Total bacterial community DNA, which was extracted from microcosm soil and field soil after 2.4D amendments, was analyzed on Southern blots, using the tfdA gene probe derived from plasmid pJP4 and the Spa probe from Sphingomonas paucimobilis. Southern blot analyses with total bacterial DNA extracted from soils inoculated with Pseudomonas cepacia/pJP4 revealed that DNA probe method could detect the 2,4-D degrading bacteria down to 10^6 cells/g dry soil. In the microcosm experiment, there was a good correlation between 2,4-D degradation and banding patterns in hybridization analyses performed after each 2,4-D treatment using the two probes. When bacterial DNA extracted from microcosm soil was hybridized with the tfdA probe. a change in the position of hybrid bands was observed over time in a Southern blot, suggesting that population change or possibly genetic rearrangement in 2,4-D degrading microbial populations occurred in this soil. With the Spa probe, one hybrid DNA band was persistently observed throughout the five 2,4-D additions. When bacterial DNA isolated from the field soil was probed with the tfdA and Spa, strong hybridization signal was observed in the 100 ppm-treated subplot, weak signal in the 10 ppm-treated subplot, and no significant signal in the 1 ppm-treated and control subplots. The data show that DNA probe analyses were capable of detecting and discriminating the indigenous 2,4-D degrading microbial populations in soil amended with 2,4-D under laboratory and field conditions.

Genetic and Phenotypic Diversity of (R/S)-Mecoprop [2-(2-Methyl-4-Chlorophenoxy)Propionic Acid]-Degrading Bacteria Isolated fromSoils

Jong-Sung Lim,Mee-Kum Jung,Mi-Soon Kim,Jae-Hyung Ahn,Jong-Ok Ka 한국미생물학회 2004 The journal of microbiology Vol.42 No.2

Twelve mecoprop-degrading bacteria were isolated from soil samples, and their genetic and phenotypic characteristics were investigated. Analysis of 16S rDNA sequences indicated that the isolates were related to members of the genus Sphingomonas. Ten different chromosomal DNA patterns were obtained by polymerase-chain-reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences from the 12 isolates. The isolates were found to be able to utilize the chiral herbicide mecoprop as a sole source of carbon and energy. While seven of the isolates were able to degrade both (R)- and (S)-mecoprop, four isolates exhibited enantioselective degradation of the (S)-type and one isolate could degrade only the (R)-enantiomer. All of the isolates were observed to possess plasmid DNAs. When certain plasmids were removed from isolates MP11, MP15, and MP23, those strains could no longer degrade mecoprop. This compelling result suggests that plasmid DNAs, in this case, conferred the ability to degrade the herbicide. The isolates MP13, MP15, and MP24 were identified as the same strain; however, they exhibited different plasmid profiles. This indicates that these isolates acquired different mecoprop-degradative plasmids in different soils through natural gene transfer.

Genetic and Phenotypic Diversity of (R/S)-Mecoprop [2-(2-Methyl-4- Chlorophenoxy)Propionic Acid]-Degrading Bacteria Isolated from Soils

Lim, Jong-Sung,Jung, Mee-Kum,Kim, Mi-Soon,Ahn, Jae-Hyung,Ka, Jong-Ok The Microbiological Society of Korea 2004 The journal of microbiology Vol.42 No.2

Twelve mecoprop-degrading bacteria were isolated from soil samples, and their genetic and phenotypic characteristics were investigated. Analysis of 16S rDNA sequences indicated that the isolates were related to members of the genus Sphingomonas. Ten different chromosomal DNA patterns were obtained by polymerase-chain-reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences from the 12 isolates. The isolates were found to be able to utilize the chiral herbicide meco-prop as a sole source of carbon and energy. While seven of the isolates were able to degrade both (R)-and (S)-mecoprop, four isolates exhibited enantioselective degradation of the (S)-type and one isolate could degrade only the (R)-enantiomer. All of the isolates were observed to possess plasmid DNAs. When certain plasmids were removed from isolates MPll, MP15, and MP23, those strains could no longer degrade mecoprop. This compelling result suggests that plasmid DNAs, in this case, conferred the ability to degrade the herbicide. The isolates MP13, MP15, and MP24 were identified as the same strain; however, they exhibited different plasmid profiles. This indicates that these isolates acquired dif-ferent mecoprop-degradative plasmids in different soils through natural gene transfer.

Revegetation of a Lakeside Barren Area by the Application of Plant Growth-promoting Rhizobacteria

안태석,Jong-Ok Ka,이건형,송홍규 한국미생물학회 2007 The journal of microbiology Vol.45 No.2

The growth stimulation of wild plants by several bacterial species showing plant growth-promoting capabilities was examined in a barren lakeside area at Lake Paro, Korea. Microbial numbers and activities in the field soil were monitored for 73 days after inoculation of the bacteria. The acridine orange direct counts for the total soil bacterial populations ranged between 2.0-2.3×109 cells/g soil and 1.4-1.8×109 cells/g soil in the inoculated and uninoculated soils, respectively. The numbers of Pseudomonas spp., which is known as a typical plant growth-promoting rhizobacteria, and the total microbial activity were higher in the inoculated soil compared to those in the uninoculated soil. The average shoot and root lengths of the wild plants grown in the inoculated soil were 17.3 cm and 12.4 cm, respectively, and longer than those of 11.4 cm and 8.5 cm in the uninoculated soil. The total dry weight of the harvested wild plants was also higher in the inoculated soil (42.0 g) compared to the uninoculated soil (35.1 g). The plant growth-promoting capabilities of the inoculated bacteria may be used for the rapid revegetation of barren or disturbed land, and as biofertilizer in agriculture. The growth stimulation of wild plants by several bacterial species showing plant growth-promoting capabilities was examined in a barren lakeside area at Lake Paro, Korea. Microbial numbers and activities in the field soil were monitored for 73 days after inoculation of the bacteria. The acridine orange direct counts for the total soil bacterial populations ranged between 2.0-2.3×109 cells/g soil and 1.4-1.8×109 cells/g soil in the inoculated and uninoculated soils, respectively. The numbers of Pseudomonas spp., which is known as a typical plant growth-promoting rhizobacteria, and the total microbial activity were higher in the inoculated soil compared to those in the uninoculated soil. The average shoot and root lengths of the wild plants grown in the inoculated soil were 17.3 cm and 12.4 cm, respectively, and longer than those of 11.4 cm and 8.5 cm in the uninoculated soil. The total dry weight of the harvested wild plants was also higher in the inoculated soil (42.0 g) compared to the uninoculated soil (35.1 g). The plant growth-promoting capabilities of the inoculated bacteria may be used for the rapid revegetation of barren or disturbed land, and as biofertilizer in agriculture.

Members of the phylum<i>Acidobacteria</i>are dominant and metabolically active in rhizosphere soil

Lee, Sang-Hoon,Ka, Jong-Ok,Cho, Jae-Chang Oxford University Press 2008 FEMS microbiology letters Vol.285 No.2

<P>A culture-independent survey was performed to search for 16S rRNA gene sequences representing dominant and metabolically active bacteria in rhizosphere soil. PCR- and reverse transcription-PCR-derived clone libraries were constructed from DNA and RNA directly extracted from the soil sample. Acidobacteria-related sequences occupied an unusually large proportion (>50%) of both rDNA- and rRNA-derived clone libraries. This study suggested that the bacteria belonging to the phylum Acidobacteria might be numerically dominant as well as metabolically active in the soil sample, implying that the phylum Acidobacteria might be highly involved in the biogeochemical cycles of the rhizosphere soil.</P>

Genetic and Phenotypic Diversity of Dichlorprop-Degrading Bacteria Isolated from Soils

Park, Hae-Dong,Ka, Jong-Ok The Microbiological Society of Korea 2003 The journal of microbiology Vol.41 No.1

Nine dichlorprop-degrading bacteria and three pairs of bacteria showing syntrophic metabolism of the herbicide were isolated from soils, and their genetic and phenotypic characteristics were investigated. Analysis of 16S rDNA sequences indicated that the isolates were related to members of the genera, Sphingomonas, Herbaspirillum, and Bradyrhizobium. Twelve different chromosomal DNA patterns were obtained by polymerase-chain-reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences from the 15 isolates. The isolates were able to utilize the herbicide dichlorprop as a sole source of carbon and energy and their dichlorprop derogative pathways were induced by the presence of dichlorprop. Most of the isolates and syntrophic pairs were able to degrade both (R)- and (S)-dichlorprop, but strain DP522 exhibited enantioselective degradation of (S)-dichlorprop. The isolates degraded 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid , and mecoprop, in addition to dichlorprop. Oxygen uptake experiments indicated that most of the isolates degraded dichlorprop through 2,4-dichlorophenol.

Revegetation of a Lakeside Barren Area by the Application of Plant Growth-promoting Rhizobacteria

Ahn, Tae-Seok,Ka, Jong-Ok,Lee, Geon-Hyoung,Song, Hong-Gyu The Microbiological Society of Korea 2007 The journal of microbiology Vol.45 No.2

The growth stimulation of wild plants by several bacterial species showing plant growth-promoting capabilities was examined in a barren lakeside area at Lake Paro, Korea. Microbial numbers and activities in the field soil were monitored for 73 days after inoculation of the bacteria. The acridine orange direct counts for the total soil bacterial populations ranged between $2.0-2.3{\times}10^{9}\;cells/g$ soil and $1.4-1.8{\times}10^{9}\;cells/g$ soil in the inoculated and uninoculated soils, respectively. The numbers of Pseudomonas spp., which is known as a typical plant growth-promoting rhizobacteria, and the total microbial activity were higher in the inoculated soil compared to those in the uninoculated soil. The average shoot and root lengths of the wild plants grown in the inoculated soil were 17.3 cm and 12.4 cm, respectively, and longer than those of 11.4 cm and 8.5 cm in the uninoculated soil. The total dry weight of the harvested wild plants was also higher in the inoculated soil (42.0 g) compared to the uninoculated soil (35.1 g). The plant growth-promoting capabilities of the inoculated bacteria may be used for the rapid revegetation of barren or disturbed land, and as biofertilizer in agriculture.

Isolation and Characterization of 4-(2,4-Dichlorophenoxy)Butyric Acid-Degrading Bacteria from Agricultural Soils

( In Hyun Park ),( Jong Ok Ka ) 한국미생물생명공학회 2003 Journal of microbiology and biotechnology Vol.13 No.2