다환방향족 탄화수소에 오염된 토양에서 분리한 Pseudomonas sp. KK1의 생리학적 특성

윤병준,강형일,오덕철 제주대학교 생명과학연구소 2001 제주생명과학연구 Vol.4 No.-

Strain KK1 was isolated from soil contaminated with polycyclic aromatic hydrocarbons and able to mineralize anthracene, naphthalene, and phenanthrene. In this study, strain KK1 was tested to evaluate carbazole-degrading potential using radiorespirometry. When KK1 was pre-grown on phenanthrene the cells were able to mineralize carbazole much more rapidly, suggesting a possible close linkage between the pathways for catabolism of carbazole and phenanthrene. Such an increase in carbazole degradation was not appreciable when KK1 was pre-grown on naphthalene. Strain KK1 was identified as the genus Pseudomonas with over 90 % confidence based on BIOLOG system and FAME analysis. Analysis of PLFAs extracted from kk1 cells on carbazole medium revealed that lipids 10:0 3OH, 17:0 cyclo, and 18:0 were representatives produced or significantly increased in response to carbazole. Tests for antibiotics provided the data that strain KK1 was resistant to antibiotic ampicillin, but susceptibile to chloramphenicol, gentamycin, kanamycin, streptomycin, and tetracycline. Strain KK1 demonstrated strong resistance to most heavy metals such as Ba, Cd, Fe, Hg, Pb used in this study.

광합성 세균 Rhodospirillum rubrum S1이 생성한 Catalase-Peroxidase의 부분 정제 및 특성 규명

김영미,이동헌,강형일,오덕철 濟州大學校 基礎科學硏究所 2001 基礎科學硏究 Vol.14 No.2

호기적으로 배양한 광합성 세균 Rhodospirillum rubrum SI은 5 가지의 catalase를 생성함을 알 수 있었다. 그 중 peroxidase의 기능도 동시에 갖는 catalase-peroxidase(Cat-3)를 부분 정제하고 그 특성을 조사하였다. 부분정제된 catalase-peroxidase의 수율은 catalase가 1.6% 그리고 peroxidasse가 5.1% 였으며, 정제 배수가 4.6배와 14배로 증가한 효소를 얻을 수 있었는데, catalase보다 peroxidase의 정제배수가 더 높았다. pH에 대한 영향은 catalase활성은 pH6에서, peroxidase활성은 pH5에서 가장 높게 나타났다. 온도에 대한 영향은 catalase와 peroxidase 활성이 모두 30℃에서 최고의 활성을 보이는 것으로 나타났으며, 온도에 대한 효소의 안정성은 50℃에서 catalase가 peroxidase보다 더 안정함을 알 수 있었다. 부분정제된 catalase-peroxidase에 유기용매와 10mM 3-amino-1,2,4-triazole을 처리한 결과, 유기용매에 대해 catalase의 활성은 79%, peroxidase의 활성은 85%까지 억제됨을 알 수 있었으며, 10mM 3-amino-1,2,4-triazole에 대한 각 효소의 활성은 그대로 유지되고 있었다. 전형적인 heme단백질 효소들의 저해제로 알려진 NaCN, NaN3, NH20H를 농도별로 처리한 결과, NaCN인 경우 catalase는 8.72×10-s M의 농도에서, peroxidase는 5.1×10-s M의 농도에서, NaN3에서 catalase는 4.2×10-7 M, peroxidase는 3.2×10-7 M, NH20H에서는 catalase인 경우는 2.0×10-7 M, peroxidase인 경우는 2.5×10-7M 농도에서 억제되었으며, 이들 저해제들은 catalase활성과 peroxidase활성을 동시에 저해하는것으로 나타났다. Five different catalases have been already found in the aerobically grown photosynthetic bacterium Rhodospirillum rubrum S1. Among them, a bifunctional catalase-peroxidase, designated Cat-3 was partially purified and characterized. The enzyme was purified through four steps in 1.6% yield for catalatic activity and 5.1% yield for peroxidatic activity. On the basis of catalatic activity, the protein purification increased nearly 4.6-fold, whereas for peroxidatic activity, an approximately 14-fold purification was achieved. The optimum pHs of the catalatic and peroxidatic activities were 6 and 5, respectively, and the optimum temperature for both activities was 30℃. The catalatic activity of the enzyme maintained at 50℃ for lh was more stably than peroxidatic activity. The catalatic and peroxidatic activities were inhibited about 79 % and 85 % by exposure to organic solvent(ethanol/chloroform), respectively. Both enzymatic activities were not neally inhibited by lOmM 3-amino-1,2,4-triazole. Fifty percent enzyme inhibition of the catalatic activity was reached with 2.0×10^(-7)M, hydroxylamine, 4.2×10^(-7)M azide and 8.7×10^(-6)M cyanide, and that of the peroxidatic activity was obtained with 2.5×10^(-7)M hydroxylamine, 3.2×10^(-7)M azide and 5.1×10^(-6)M cyanide.

제주연안 갯녹음(백화) 지역의 해수에 분포하는 세균군의 분자생물학적 분석

강봉조,김미란,윤병준,이동헌,오덕철,강형일 濟州大學校 基礎科學硏究所 2002 基礎科學硏究 Vol.15 No.2

본 연구에서는 갯녹음(백화)현상이 세균생태계와 어떤 관련이 있는지에 대한 기초자료 및 정보를 얻기 위하여 갯녹음 현상이 일어난 제주도 성산과 강정지역 연안의 해수에 존재하는 세균군을 16S rRNA 증폭기법을 이용하여 조사하였다. 강정지역에서는 Alcanivorax, Paracoccus, Damselae, Pseudomonas, Rhodovulum, Silicibacter, Sulfitobacter, Roseobacter 등 다양한 종류의 세균이 분포되어 있었으며, Alcanivorax가 20%의 빈도로 가장 많이 나타났다. 반면, 성산 지역에서는 Pseudomonas속 균주가 우점종으로서 존재하였으며, Pseudomonas tolaasii(혹은 Pseudomonas corrugata)와 유연관계가 가까운 세균은 44%, Pseudomonas mandeli와 가까운 세균이 24%, Verrucomicrobiales와 가까운 세균은 4%, 기타 동정되지 않은 세균은 세 group으로 구분되었으며, 각각 8%, 8%, 12%를 차지하여, 두 곳에 분포되어 있는 세균군상이 상당한 차이점이 있음을 확인하였다. 갯녹음 지역인 강정과 성산 해수의 8월(표품 채집시기)의 수온은 27℃-27.5℃, 염분의 농도는 30.24-30.60%, pH는 8.23-8.36, 용존산소량(DO)은 각각 7.20-7.28로, 두 지역에서 매우 비슷한 것으로 조사된 바 있는데, 이는 수온이나 염분의 농도, 또는 pH보다는 다른 원인에 의하여 두 갯녹음 지역에 분포하는 세균군의 차이를 가져왔음을 제시해 주었다. In this study, the bacterial communities distributed in sea water of the whitening areas of Gangjeong and Seongsan, Jeju-do have been analyzed using the PCR amplification of 16S rRNA to obtain fundamental data and information on relationship of the whitening phenomenon and microbial ecosystem. In Gangjeong, diverse bacteria such as Alcanivorax, Paracoccus, Damselae, Pseudomonas, Rhodovulum, Silicibacier, Sulfitobacter, and Roseobacier have been found, and Alcanivorax was the most abundant clone. The most abundant clone from Seongsan was Pseudoinonas, of which Pseudomonastolaasii and Pseudonionas mandeli were most abundantly occurred in the frequency of approx 44% and 24%, respectively. Approx 4% of the bacterial clones closest to firruconiicrobiales and other unidentified clones were also found in Seongsan, suggesting there is a great discrepancy between bacterial communities from the whitening areas of Seongsan and Gangjeong. The mean tem- perature, chlorine concentration, pH, and dissolved oxygen (DO) of the sea water of Gangjeong and Seongsan in August of 2001 (sampling period) was 27℃-27.5℃, 30.24-30.60%, pH 8.23-8.36,7.20-7.28 ㎎/ℓ, suggesting other environmental factors except for the factors mentioned above might result in difference of bacterial communities distributed in both areas.

Characterization and Role of tbuX in Utilization of Toluene by Ralstonia pickettii PKO1

KAHNG, HYUNG-YEEL,BYRNE, ARMANDO M.,OLSEN, RONALD H.,KUKOR, JEROME J. 濟州大學校 基礎科學硏究所 2001 基礎科學硏究 Vol.14 No.1

The tbu regulon of Ralstonia pickettii PKOl encodes enzymes involved in the catabolism of toluene, benzene, and related alkylaromatic hydrocarbons. The first operon in this regulon contains genes that encode the tbu pathway's initial catabolic enzyme, toluene-3-monooxygenase, as well as TbuT, the NtrC-like transcriptional activator for the entire regulon. It has been previously shown that the organization of tbuT, which is located immediately downstream of tbuA1UBVA2C, and the associated promoter (PtbuA1) is unique in that it results in a cascade type of up-regulation of tbuT in response to a variety of effector compounds. In our efforts to further characterize this unusual mode of gene regulation, we discovered another open reading frame, encoded on the strand opposite that of tbuT, 63 bp downstream of the tbuT stop codon. The 1,374-bp open reading frame, encoding a 458-amino-acid peptide, was designated tbuX. The predicted amino acid sequence of TbuX exhibited significant similarity to several putative outer membrane proteins from aromatic hydrocarbon-degrading bacteria, as well as to FadL, an outer membrane protein needed for uptake of long-chain fatty acids in Escherichiu coli. Based on sequence analysis, transcriptional and expression studies, and deletion analysis, TbuX seems to play an important role in the catabolism of toluene in R. pickettii PKOl. In addition, the expression of tbuX appears to be regulated in a manner such that low levels of TbuX are always present within the cell, whereas upon toluene exposure these levels dramatically increase, even more than those of toluene- 3-monooxygenase. This expression pattern may relate to the possible role of TbuX as a facilitator of toluene entry into the cell.

Introduction of Saxicolous Lichens Distributed in Coastal Rocks of U-do Islet in Jeju, Korea

Kahng Hyung-Yeel,Yoon Byoung-Jun,Kim Sung-Hyun,Shin Duck-Ja,Hur Jae-Seoun,Kim Hyun-Woo,Kang Eui-Sung,Oh Kye-Heon,Koh Young Jin The Microbiological Society of Korea 2004 The journal of microbiology Vol.42 No.4

This study reports, for the first time, the ivestigation of the distribution of Korean saxicolous lichens in the coastal rocks of U-do islet, which is known as an unpolluted zone in Jeju. More than thirty lichens were obtained and investigated from the coastal rocks frequently contacted by seawater. A molecular analysis using PCR amplification of the rRNA ITS regions revealed the coastal rock lichens could be placed into 8 families and 14 genera, Ramalinaceae (Bacidia, Ramalina), Physciaceae (Buellia, Dirinaria, Phaeophyscia, Physcia, Pyxine), Lecanoraceae (Candelaria, Lecanora), Parmeliaceae (Xan­thopannelia), Graphidaceae (Graphis), Pertusariaceae (Pertusaria), Rhizocarpaceae (Rhizocarpon), and Teloschistaceae (Caloplaca), showing a diversity of lichens, with foliose (flat leaf-like), crustose (crust­like), and fruticose (miniature shrub-like) life forms might be distributed in the coastal rocks. These findings suggested the possibility that the lichens identified in the present work might be resistant to a salty environment.

Physiological and Phylogenetic Analysis of Burkholderia sp. HY1 Capable of Aniline Degradation

KAHNG, HYUNG-YEEL,KUKOR, JEROME J.,OH, KYE-HEON 濟州大學校 基礎科學硏究所 2001 基礎科學硏究 Vol.14 No.2

A new aniline-utilizing microorganism, strain HY1 obtained from an orchard soil, was characterized by using the BIOLOG system, an analysis of the total cellular fatty acids, and a 16s rDNA sequence. Strain HY1 was identified as a Burkholderiu species, and was designated Burkholderia sp. HY1. GC and HPLC analyses revealed that Burkholderiu sp. HY1 was able to degrade aniline to produce catechol, which was subsequently converted to cis,cis-muconic acid through an orrho-ring fission pathway under aerobic conditions. Strain HY1 exhibited a drastic reduction in the rate of aniline degradation when glucose was added to the aniline media. However, the addition of peptone or nitrate to the aniline media dramatically accelerated the rate of aniline degradation. A fatty acid analysis showed that strain HY1 was able to produce lipids 16:0,16:0 ZOH, and 1 I methyl 18: 1 ω7c approximately 3.7-, 2.2-, and 6-fold more, respectively, when grown on aniline media than when grown on TSA. An analysis of the 16S rDNA sequence revealed that strain HY1 was very closely related to Burkholderia graminis with 95% similarity based on the alignment of a 1,435 bp fragment. A phylogenetic analysis of the 16S rDNA sequence based on a 1,420 bp multi-alignment showed that strain HY1 was placed among three major clonal types of β-Proteobacteria, including Burkholderiu graminis, Burkholderia phenazinium, and Burkholderia glathei. The sequence GAT(C or G)G, which is highly conserved in several locations in the 16S rDNA gene among the major clonal type strains of β-Proteobacteria, was frequently replaced with GAT(C or G)A in the 16S rDNA sequence from strain HYI.

Isolation and Characterization of Aniline-Degrading Bacteria

Kahng, Hyung-Yeel,Kim, Seung-Il,Woo, Mi-Jeong,Park, Yong-Keun,Lee, Yung-Nok The Microbiological Society of Korea 1992 미생물학회지 Vol.30 No.3

Six isolated strains degrading aniline were selected, identified and designated as pseudomonas putida K6, Pseudomonas acidovorans K82, Achromobacter gr. D. V. K24, Achromobacter xylosocidans K4, Moraxella sp. K21 and Moraxella sp. K22. All of them degraded 1000 ppm aniline completely within 30 to 36 hours. Most of these strains are resistant to antibiotics more than one, but Moraxella sp. has not any antibiotic marker tested. Most strains except for P. acidovorans K82 were shown to have resistance to the heavy metal ions such as Ni, Cu, Li, Ba, Co, etc. but not to Hg to which only P. putida K6 was resistant. M. sp. K21 was capable of degrading aniline to a maximum concentration of 2500 ppm without any repression. The incubation of the cell in limited pH ranges (4-8) had no great effect on aniline degradation. The addition of bactopeptone to the minimal media promoted the speed of aniline degradation, but the addition of glucose rather repressed the rate of aniline degradation. Through enzyme assay, A. gr. D. V. K 24 was shown to degrade aniline through artho-pathway and formed .betha.-ketoadipate as intermediate metabolite.

Cellular Responses of Pseudomonas sp. KK1 to Two-Ring Polycyclic Aromatic Hydrocarbon, Naphthalene

Kahng, Hyung-Yeel 濟州大學校 基礎科學硏究所 2002 基礎科學硏究 Vol.15 No.2

The strain KK1 isolated from soil contaminated with polycyclic aromatic hydrocarbons was identified as Pseudomonas sp. based on analyses by MIDI and Biolog Identification System. Cellular and physiological responses of strain KK1 to two-ring polycyclic aromatic hydrocarbon, naphthalene were evaluated using radiorespirometry, PLFAs and sequence analysis of Rieske-type iron sulfur center of dioxygenase. KK1 was found to be able to rapidly mineralize naphthalene. Notably, KKl cells pregrown on phenanthrene were able to mineralize naphthalene much more rapidly than naphthalene- pregrown cells. The total cellular fatty acids of KKl were comprised of eleven C-even and two C-odd fatty acids (fatty acids < 0.2% in abundance were not considered in this calculation). Lipids 12:0 2OH, 12:0 3OH, 16:0, 18:l 6c, 18:0 increased for naphthalene-exposed cells, while lipids 18:l 7c/15:0 iso 2OH, 17:0 cyclo, 18:l 7c, 19:0 cyclo decreased. Data from Northern hybridization using a naphthalene dixoygenase gene fragment cloned out from KKl as a probe provided the information that naphthalene dioxygenase gene was more highly expressed in cells grown on phenanthrene than naphthalene.

Physiological and Phylogenetic Analysis of Burkholderia sp.HY1 Capable of Aniline Degradation

Kahng, Hyung Yeel,KuKor, Jerome J.,Oh, Kye Heon 한국미생물 · 생명공학회 2000 Journal of microbiology and biotechnology Vol.10 No.5

A new aniline-utilizing microorganism, strain HY1 obtained from an orchard soil, was characterized by using the BIOLOG system, an analysis of the total cellular fatty acids, and a 16S rDNA sequence. Strain HY1 was identified as a Burkholderia species, and was designated Burkholderia sp. HY1. GC and HPLC analyses revealed that Burkholderia sp. HY1 was able to degrade aniline to produce catechol, which was subsequently converted to cis,cis-muconic acid through an ortho-ring fission pathway under aerobic conditions. Strain HY1 exhibited a drastic reduction in the rate of aniline degradation when glucose was added to the aniline media. However, the addition of peptone or nitrate to the aniline media dramatically accelerated the rate of aniline degradation. A fatty acid analysis showed that strain HY1 was able to produce lipids 16:0, 16:0 20H, and 11 methyl 18:1 ω7c approximately 3.7-, 2.2-, and 6-fold more, respectively, when grown on aniline media than when grown on TSA. An analysis of the 16S rDNA sequence revealed that strain HY1 was very closely related to Burkholderia graminis with 95% similarity based on the alignment of a 1,435 bp fragment. A phylogenetic analysis of the 16S rDNA sequence based on a 1,420 bp multi-alignment showed that strain HY1 was placed among three major clonal types of β-Proteobacteria, including Burkholderia graminis, Burkholderia phenazinium, and Burkholderia glathei. The sequence GAT(C or G)G__, which is highly conserved in several locations in the 16S rDNA gene among the major clonal type strains of β-Proteobacteria, was frequently replaced with GAT(C or G)A__ in the 16S rDNA sequence from strain HY1.

Molecular Characterization of PAH-Contaminated Soils at a Former Manufactured Gas Plant Site

Kahng, Hyung-Yeel 제주대학교 기초과학연구소 2002 基礎科學硏究 Vol.15 No.1