• Journal of Microbiology
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• 제37권1호
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• pp.45-49
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• 1999

To investigate the mechanism of cadmium tolerance in a cadmium-resistant Azomonas agilis PY101 that produces a specific fluorescent pigment promoted by cadmium, we carried out Tn5 mutagenesis and isolated four pigment-deficient mutants. In these mutants, Ppg1, Ppg2, and Ppg3 remarkably reduced the pigment production to 15.3%, 11.2%, and 13.9%, respectively. Especially, Ppg4 mutant did not produce the pigment at all. None of the mutants grew in the presence of 1500 ppm of CdCl2 in growth medium, and they exhibited differential sensitivities to cadmium. Ppg1, Ppg2, Ppg3, and Ppg4 mutants were sensitive to 900 ppm, 1100 ppm, 1000 ppm, and 800 ppm of CdCl2, respectively. These mutants also showed noticeable increase, from 8.8-fold to 13.2-fold, in the size of growth inhibition zone compared with that of the will type after treatment with cadmium. Therefore, the pigment production of A. agilis PY101 was found to decrease the toxic effects of cadmium to the bacterium.

• Journal of Microbiology and Biotechnology
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• 제16권3호
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• pp.450-456
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• 2006

Pseudomonas fluorescens MC07 is a growth-promoting rhizobacterium that suppresses mycelial growth in fungi such as Rhizoctonia solani, Pythium ultimum, Fusarium oxysporum, and Phytophthora capsici. To determine the role of the bacterium's antifungal activity in disease suppression, we screened 2,500 colonies generated by Tn5lacZ insertions, and isolated a mutant 157 that had lost antifungal activity. The EcoRI fragment carrying Tn5lacZ was cloned into pBluescript II SK(+) and used as a probe to isolate wild-type clones from a genomic library of the parent strain, MC07. Two overlapping cosmid clones, pEH4 and pEH5, that had hybridized with the mutant clone were isolated. pEH4 conferred antifungal activity to the heterologous host P.fluorescens strain 1855.344, whereas pEH5 did not. Through transposon mutagenesis of pEH4 and complementation analyses, we delineated the 14.7-kb DNA region that is responsible for the biosynthesis of an antifungal compound. DNA sequence analysis of the region identified 11 possible open reading frames (ORF), ORF1 through ORF11. A BLAST search of each putative protein implied that the proteins may be involved in an antifungal activity similar to polyketides.

• Journal of Microbiology and Biotechnology
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• 제14권2호
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• pp.302-311
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• 2004

Comamonas testosteroni (formerly Pseudomonas sp.) NCIMB 10643 can grow on biphenyl and alkylbenzenes $(C_2-C_7)$ via 3-substituted catechols. Thus, to identify the genes encoding the degradation, transposon-mutagenesis was carried out using pAG408, a promoter-probe mini-transposon with a green fluorescent protein (GFP), as a reporter. A mutant, NT-1, which was unable to grow on alkylbenzenes and biphenyl, accumulated catechols and exhibited an enhanced expression of GFP upon exposure to these substrates, indicating that the gfp had been inserted in a gene encoding a broad substrate range catechol 2,3-dioxygenase. The genes (2,826 bp) flanking the gfp cloned from an SphI-digested fragment contained three complete open reading frames that were designated bphCDorfl. The deduced amino acid sequences of bphCDorfl were identical to 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (BphD), and OrfI, respectively, that are all involved in the degradation of biphenyl/4-chlorobiphenyl (bph) by Ralstonia oxalatica A5. The deduced amino acid sequence of the orfl revealed a similarity to those of outer membrane proteins belonging to the OmpW family. The introduction of the bphCDorfl genes enabled the NT-l mutant to grow on aromatic hydrocarbons. In addition, PCR analysis indicated that the DNA sequence and gene organization of the bph operon were closely related to those in the bph operon from Tn4371 identified in strain A5. Furthermore, strain A5 was also able to grow on a similar set of alkylbenzenes as strain NCIMB 10643, demonstrating that, among the identified aromatic hydrocarbon degradation pathways, the bph degradation pathway related to Tn4371 was the most versatile in catabolizing a variety of aromatic hydrocarbons of mono- and bicyclic benzenes.

• 미생물학회지
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• 제30권5호
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• pp.347-354
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• 1992

New regulatory, loci which participate in the regulation of anaerobic inducible gene expression in Salmonella typhimurium were identified. We observed the regulatory network of new regulator mutations to various anaerobic inducible gene (1). Some anaerobic inducible lac fusions were also induced at low pH condition which was severe environment to withstand for its virulence at the place like phagolysosome. Sic oxygen-regulated regulatory mutants (oxr) isolated by Tn10 mutagenesis were divided into two groups. Five of them were found to show negative effect on the regulation of anaerobic gene expression, while on e showed positive effect on the regulation. Genetic loci of four oxr were identified with 54 Mud-P22 lysogens covering the whole chromosome of S. typhimurium, in the nearby region of map unit 87 min (oxr101), 63 min (oxr104), 97 min (oxr 105), and 57 min (oxr 106), respectively. Two oxr mutants were subjected to two-dimensional polyacrylamide electrophoretic analysis of anaerobic inducible proteins for searching the control circuitry of our oxr mutants.

• 식물병연구
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• 제16권2호
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• pp.136-140
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• 2010

Pectobacterium carotovorum subsp. carotovorum은 채소 무름병의 원인균이다. 박테리오신인 carocin D는 Pectobacterium carotovorum subsp. Carotovorum Pcc21 균주가 생산한다. Pcc21 균주의 비병원성인 돌연변이주를 선발하기 위해 Tn5을 이용하여 무작위적 삽입 돌연변이체들을 대상으로 병원성 검사를 하였다. 실험 결과 선발된 P. carotovorum subsp. carotovorum Pcc21-M15과 carocin D 유전자의 형질전환체인 E. coli(pRG3431)은 비병원성인 동시에 carocin D를 생산하여 무름병균인 P. carotovorum subsp. carotovorum Pcc3의 생장을 억제함을 확인하였다. Pcc21-M15와 E. coli (pRG3431)를 무름병균인 P. carotovorum subsp. carotovorum Pcc3과 함께 미네랄 오일법으로 상추에 처리한 결과 Pcc3 균주를 단독 처리했을 경우에는 3일만에 90%가 무름병에 걸린 반면, Pcc21-M15와 E. coli(pRG3431)를 함께 처리한 경우는 6일 후에도 25%의 상추에서만 무름병 증상을 보였다.

• The Plant Pathology Journal
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• 제35권4호
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• pp.351-361
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• 2019

In our previous study, pyrrolnitrin produced in Pseudomonas chlororaphis G05 plays more critical role in suppression of mycelial growth of some fungal pathogens that cause plant diseases in agriculture. Although some regulators for pyrrolnitrin biosynthesis were identified, the pyrrolnitrin regulation pathway was not fully constructed. During our screening novel regulator candidates, we obtained a white conjugant G05W02 while transposon mutagenesis was carried out between a fusion mutant $G05{\Delta}phz{\Delta}prn::lacZ$ and E. coli S17-1 (pUT/mini-Tn5Kan). By cloning and sequencing of the transposon-flanking DNA fragment, we found that a vfr gene in the conjugant G05W02 was disrupted with mini-Tn5Kan. In one other previous study on P. fluorescens, however, it was reported that the deletion of the vfr caused increased production of pyrrolnitrin and other antifungal metabolites. To confirm its regulatory function, we constructed the vfr-knockout mutant $G05{\Delta}vfr$ and $G05{\Delta}phz{\Delta}prn::lacZ{\Delta}vfr$. By quantifying ${\beta}-galactosidase$ activities, we found that deletion of the vfr decreased the prn operon expression dramatically. Meanwhile, by quantifying pyrrolnitrin production in the mutant $G05{\Delta}vfr$, we found that deficiency of the Vfr caused decreased pyrrolnitrin production. However, production of phenazine-1-carboxylic acid was same to that in the wild-type strain G05. Taken together, Vfr is required for pyrrolnitrin but not for phenazine-1-carboxylic acid biosynthesis in P. chlororaphis G05.

• 미생물학회지
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• 제41권1호
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• pp.67-73
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• 2005

본 연구에서는 남세균인 Synechocystis sp. PCC 6803 (Syn6803)을 대상으로 transposable element Tn5를 이용하여 획득된 1,200여 돌연변이주로부터 모균주에 비하여 PHB 축적량이 크게 증진된 균주를 선별하고, Tn5 삽입에 의해 결함을 나타낸 유전자를 확인함으로써 Syn6803에서의 PHB 생합성에 영향을 주는 세포내 생리학적 요인을 조사하고자 하였다. 모균주인 야생형 균주의 경우 질소원이 제한된 $BG11_0$ 배지에서의 PHB 생합성량이 건체량의 $4\%$ (w/w) 수준인데 반하여, $10-34\%$의 생합성량을 보이는 25개의 돌연변이 균주를 얻을 수 있었다. Inverse PCR을 이용하여, 선별된 돌연변이 균주내 돌연변이가 일어난 유전자를 조사한 결과, 아직까지 그 기능이 규명되지 않은 유전자가 대부분이었으나, NADH-ubiquinone oxidoreductase, O-succinylbenzoic-CoA ligase 또는 photosystem II PsbT protein과 같이 광합성과 호흡에 관여하는 유전자에 돌연변이가 일어난 4 균주와 histidine kinase가 결여된 1균주가 확인되었다. 이들 균주를 대상으로pulse-amplitude modulated fluorometer를 이용하여 세포내 $NAD(P)H/NAD(P)^+$비를 측정한 결과, 에너지 대사 흐름의 차단에 의해 세포내의 $NAD(P)HNAD(P)^+$비가 모균주에 비하여 현저하게 높은 것으로 나타났다. 이는 잉여의 전자로 포화된 세포, 즉 NAD(P)H에 의해 환원적 상태를 유지하고 있는 세포의 경우 PHB 축적 이 증진될 수 있음을 시사한다. 이러한 사실은 인위적으로 광합성과 호흡 관련 유전자가 제거되어 $NAD(P)H/NAD(P)^+$비가 높아진 것으로 알려진 다수의 Syn6803 돌연변이 균주들을 대상으로 PHB 생합성량을 조사한 결과로부터 재확인되었다.

• 한국식품영양과학회지
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• 제32권2호
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• pp.211-216
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• 2003

홍경천 뿌리의 에탄을 추출물과 분획물에 대한 항산화 활성을 측정한 결과, 에틸아세테이트 분획물에서 14.3 $\mu\textrm{g}$/DL의 강한 항산화 활성을 나타내었다. 직접 변이원인 MNNG에 대한 항돌연변이 효과에서 S. typhimurium TA100 균주에 대해 홍경천 뿌리 에틸아세테이트 분획물(2000g/p1ale)에서 다른 분획물보다 높은 89.1%의 억제효과를 나타내었다. 4NQO에서도 에틸아세테이트 분획물에서 S. typhimurium TA98 균주와 TA100균주에 대해서 동일 시료농도에서 각각 89.7%와 91.5% 로 다른 분획물보다 높은 억제효과를 나타내었다. B( u )P에 대한 억 제효과에서는 TA98, TA100 두 균주에 대하여 에틸아세테이트 분획물에서 각각 94.2%와 95.7%로 다른 분획물 보다 높은 억제활성을 나타내었으며, Tn-P-1에 대해서 는 두 균주가 각각 92.3%와 93.8%로 다른 분획물 보다 높은 억제효과를 나타내었다. 암세포 성장억 제 효과를 검토한 실험에서는 에틸아세테이트 분획물이 가장 높은 억제활성을 나타내었으며, 시료농도의 증가와 함께 억제활성도 증가하는 경향을 나타내었으며 시료농도 1 mg/CL에서 A549가 90.5%, HepG2가 81.5%, AGS가 92..2% 그리고 MCF-7이 82.6%의 암세포 성장억제효과를 나타내었다.

• 한국미생물·생명공학회지
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• 제34권2호
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• pp.101-108
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• 2006

Phytophthora sp.의 균사성장을 특이적으로 저해하는 토양 미생물인 YNB54 균주의 정확한 분류적 위치를 밝히기 위해 Biolog GN2, API 20E와 같은 상업적 생화학 kit, 16S rDNA, DAN-DNA hybridization, GC함량, MIDI 등의 분석을 수행하였다. 다양한 생화학적 kit를 사용한 동정 결과는 이 균주가 다른 어떤 종보다 Enterobacter cloacae와 E. cancerogenus에 보다 더 가까움을 보여주었다. 또한 DAN-DNA hybridization, GC함량, MIDI 분석의 결과들 역시 다른 속 (Citerobacter, Klebsiella, Leclercia)보다 Enterobacter 속에 더 유사함을 암시해 주었다. 그러나 16S rDNA분석에서 이 균주는 Citrobacter freundii(99.4%)와 동일 그룹으로 구분되었지만 Enterobacter, Leclecia, Klebsiella 속 등과도 98%이상의 상동성을 보여주는 polyphyletic 특성을 보였다. 결론적으로 YNB54의 분류 동정을 위한 우리의 조사들은 이 균주가 유전적으로 다양하고 지금까지 아는 것보다 분류학적으로 더 복잡함을 암시해줌에도 불구하고 Enterobacter속임이 가장 유력하다는 것을 보여 주었다.