• 미생물학회지
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• 제31권2호
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• pp.123-128
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• 1993

해녀콩 뿌리혹의 질소고정 공생균인 Rhizobium sp. SNU003 균주의 게놈내 7.9 kb 의 EcoRI, 6.5kb 의 SalI, 7.3 kb 의 HindIII 와 4.4 kb 의 PstI 절편에 nifHD 가 존재함을 확인하였다. 람다파아지 EMBL3-BamHI arm 을 사용하여 genomic library 를 제조하였으며 이로부터 nif-유전자를 포함하고 있는 9개의 재조합 파아지 클론을 선별하였다. 이들 중 15.3 kb 의 삽입 DNA 를 가지고 있는 Rnif-6 클론은 7.6 kb 의 BamHI/SacI 절편에 nifHD 가 위치하고 있었다. 따라서 이절편을 pUC19 에 sub cloning 하고 제한효소 지도를 작성한 결과 Rhizobium sp. SNU003 의 nifH 와 nifD 는 4.5 kb 의 BamHI/BglII 절편에 연속배열하고 있다.

• 미생물학회지
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• 제31권2호
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• pp.129-134
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• 1993

4-Chlorobiphenyl(4CB) 과 biphenyl 을 분해하는 Pseudomoas sp. DJ-12 의 pcbAB 는 분해초기 단계에 작용하는 4-chlorobiphenyl dioxygenase 와 dihydrodiol dehydrogenase 효소를 생산하는 유전자들이다. 이 유전자를 E. coli XL1-Blue 에 플로닝하여 CU101 형질전환체를 얻었다. CU101 의 pCU101 재조합 plasmid 에 클로닝된 pcbAB 유전자는 크기가 약 2.2 kb 이고 3 개의 Hind III 제한효소 위치가 있었으며, 독자적인 promoter 를 가지고 있었다. CU101 에 대하여 biphenyl 을 기질로 하여 생성된 대사산물을 resting cell assay 를 한 결과 2, 3-dihydroxybiphenyl 이 검출되어 pcbAB 유전자들이 E. coli 에서 잔 발현된다는 것을 의미하였다. 그러나 dechlorination 작용은 pcbAB 유전자와 관계없이 4AB 의 개환과정 후 생성된 4-chlorobenzoate 에서 일어나는 것으로 해석된다.

• The Plant Pathology Journal
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• 제22권4호
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• pp.323-328
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• 2006

Serratia marcescens W1, isolated from cucumber-cultivated soil in Suwon, Korea, evidenced profound antifungal activity and produced the extracellular hydrolytic enzymes, chitinase and protease. In order to isolate the antifungal genes from S. marcescens W1, a cosmid genomic library was constructed and expressed in Escherichia coli. Transformants exhibiting chitinase and protease expression were selected, as well as those transformants evidencing antifungal effects against the rice blast fungus, Magnaporthe grisea, and the cucumber leaf spot fungus, Cercospora citrullina. Cosmid clones expressing chitinase or protease exerted no inhibitory effects against the growth of fungal pathogens. However, two cosmid clones evidencing profound antifungal activities were selected for further characterization. An 8.2 kb HindIII fragment from these clones conditioned the expression of antagonistic activity, and harbored seven predicted complete open reading frames(ORFs) and two incomplete ORFs. The deduced amino acid sequences indicated that six ORFs were highly homologous with genes from S. marcescens generating pyrroloquinoline quinone(PQQ). Only subclones harboring the full set of pqq genes were shown to solubilize insoluble phosphate and inhibit fungal pathogen growth. The results of this study indicate that the functional expression of the pqq genes of S. marcescens W1 in E. coli may be involved in antifungal activity, via as-yet unknown mechanisms.

• Journal of Microbiology and Biotechnology
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• 제16권1호
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• pp.118-125
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• 2006

The region responsible for replication of the megaplasmid pAtC58 in the nopaline-type Agrobacterium tumefaciens strain C58 was determined. A derivative ofa Co1E1 vector, pBluscript SK-, incapable of autonomous replication in Agrobacterium spp, was cloned with a 7.6-kb Bg1II-HindIII fragment from a cosmid clone of pAtC58, which contains a region adjacent to the operon for the utilization of deoxyfructosyl glutamine (DFG). The resulting plasmid conferred resistance to carbenicillin on the A. tumefaciens strain UIA5 that is a plasmidfree derivative of C58. The plasmid was stably maintained in the strain even after consecutive cultures for generations. Analysis of nested deletions of the 7.6-kb fragment showed that a 4.3-kb BglII-XhoI region sufficiently confers replication of the derivative of the ColE1 vector on UIA5. The region comprises three ORFs, which have high homologies with repA, repB, and repC of plasm ids in virulent Agrobacterium spp. including pTiC58, pTiB6S3, pTi-SAKURA, and pRiA4b as well as those of symbiotic plasmids from Rhizobium spp. Phylogenie analysis showed that rep genes in pAtC58 are more closely related to those in pRiA4 than to pTi plasmids including pTiC58, suggesting that the two inborn plasmids, pTiC58 and pAtC58, harbored in C58 evolved from distinct origins.

• Journal of Microbiology and Biotechnology
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• 제5권2호
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• pp.68-73
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• 1995

Pseudomonas sp. P20 was shown to be capable of degrading biphenyl and 4-chlorobiphenyl (4CB) to produce the corresponding benzoic acids wnich were not further degraded. But the potential of the strain for biodegradation of 4CB was shown to be excellent. The pcbA, B, C and D genes responsible for the aromatic ring-cleavage of biphenyl and 4CB degradation were cloned from the chromosomal DNA of the strain. In this study, the pebC and D genes specifying degradation of 2, 3-dihydroxybiphenyl (2, 3-DHBP) produced from biphenyl by the pebAB-encoded enzymes were cloned by using pBluescript SK(＋) as a vector. From the pCK102 (9.3 kb) containing pebC and D genes, pCK1022 inserted with a EcoRI-HindIII DNA fragment (4.1 kb) carrying pebC and D and a pCK1092 inserted with EcoRI-XbaI fragment (1.95 kb) carrying pebC were constructed. The expression of pcbC and D' in E. coli CK102 and pebC in E. coli CK1092 was examined by gas chromatography and UV-vis spectrophotometry. 2.3-dihydroxybiphenyl was readily degraded to produce meta-cleavage product (MCP) by E. coli CK102 after incubation for 10 min, and then only benzoic acid(BA) was detected in the 24-h old culture. The MCP was detected in E. coli CK1022 containing pebC and 0 genes (by the resting cells assay) for up to 3 h after incubation and then diminished completely in 8 h, whereas the MCP accumulated in the E. coli CK1092 culture even after 6 h of incubation. The 2, 3-DHBP dioxygenases (product of pebC gene) produced by E. coli CK1, CK102, CK1023, and CK1092 strains were measured by native PAGE analysis to be about 250 kDa in molecular weight, which were about same as those of Pseudomonas sp. DJ-12, P. pseudoa1caligenes KF707, and P. putida OU83.

• Journal of Microbiology and Biotechnology
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• 제18권6호
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• pp.1033-1039
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• 2008

A 3-kb HindIII fragment bearing the cry6Aa2 gene and the adjacent and intergenic regions was cloned from Bacillus thuringiensis strain YBT-1518. Two open reading frames (ORFs), namely, orf1 (termed cry6Aa2) and orf2 that were separated by an inverted-repeat sequence were identified. orf1 encoded a 54-kDa protein that exhibited high toxicity to the plant-parasitic nematode Meloidogyne hapla. The orf2 expression product was not detected by SDS-PAGE, but its mRNA was detected by RT-PCR. The orf2 coexpressed with orf1 at a high level in the absence of the inverted-repeat sequence, whereas, the expression level of otfl was decreased. When orf2 was mutated, the level of orf1 expression was enhanced obviously. In conclusion, the inverted-repeat sequence disturbs orf2 expression, and the orf2 downregulates orf1 expression. This is an example of novel negative regulation in B. thuringiensis and a potential method for enhancing the expression level of cry genes.

• Journal of Microbiology and Biotechnology
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• 제2권4호
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• pp.243-247
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• 1992

The thr operon of Escherichia coli TF427, an $\alpha$-amino-$\beta$-hydroxyvaleric acid (AHV)-resistant threonine overproducer, was cloned in a pBluescriptII $KS^＋$ plasmid by complementation of E. coli mutants. All clones contained a common 8.8 kb HindIII-generated DNA fragment and complemented the thrA, thrB, and thrC mutants by showing that these clones contained the whole thr operon. This thr operon was subcloned in the plasmid vectors pBR322, pUC18, and pECCG117, an E. coli/Corynebacterium glutamicum shuttle vector, to form recombinant plasmids pBTF11, pUTF25 and pGTF18, respectively. The subcloned thr operon was shown to be present in a 6.0 kb insert. A transformant of E. coli TF125 with pBTF11 showed an 8~11 fold higher aspartokinase I activity, and 15~20 fold higher L-threonine production than TF125, an AHV-sensitive methionine auxotroph. Also, it was found that the aspartokinase I activity of E. coli TF125 harboring pBTF11 was not inhibited by threonine and its synthesis was not repressed by threonine plus isoleucine.

• Journal of Microbiology and Biotechnology
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• 제12권1호
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• pp.176-181
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• 2002

In order to isolate and identify Bifidobacterium spp. that originated in Korea, feces were sampled from healthy Korean adults and children living in three villages, the first having a history of longevity and the other two where the diet did not include fermented milk or any pharmaceutical preparations. Through the use of Gram staining and microscopic examination for cell morphology, 23 bacterial strains presumed to be the Bifidobacterium genus were isolated from the feces of 13 out of a total of 59 Korean people. To identify the Bifidobacterium strains at the genus level, these bacteria were then analyzed by TLC and the fructose-6-phosphate phosphoketolase (F6PPK) test. The result showed that 22 of the isolated strains were confirmed to be members of the genus Bifidobacterium. All of these bifidobacteria were also identified as Bifidobacterium spp. by the fermentation test. Using a RFLP analysis, an attempt was made to identify the Bifidobacterium spp. that had been isolated from both Korean adults and children. In a genomic Southern blot analysis after digestion with two restriction enzymes (EcoRI, HindIII), all of the 14 randomly selected Korean isolates showed patterns identical to those of three different B. longum species. Another restriction enzyme, CfoI (4-bp recognition enzyme), was then used to identify the strain. Interestingly, all the Korean isolates were identified as B. longum ATCC 15708, indicating that a RFLP analysis was effective for identifying Bifidobacterium spp. at both the strain and species levels.

• Journal of Microbiology and Biotechnology
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• 제22권9호
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• pp.1237-1244
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• 2012

Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and $42.5^{\circ}C$, and the enzyme was stable under $40^{\circ}C$. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a ${\beta}$-agarase.

• 한국균학회지
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• 제25권4호통권83호
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• pp.362-368
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• 1997

재조합 DNA probe를 이용하여 국내 Fusarium oxysporum 분화형간의 분류 가능성을 탐색하고 그들의 유전적 변이를 분석하였다. F. oxysporum f. sp. lycopersici, cucumerinum, fragariae, garlic, sesami 등 5종의 분화형을 공시하여 RFLP 분석을 실시하였다. Repetitive copy clone인 세종의 재조합 클론 pFC46, pFC52, pFC57을 이용하여 HindIII로 처리한 F. oxysporum의 genomic DNA에 대해 southern hybridization한 결과 나타난 밴드의 양상은 분화형에 따라 차이가 명확히 밝혀져 이들을 이용한 분류가 가능하였다. 또한 RFLP 분석 결과 f. sp. sesami는 다른 분화형에 비해 다소 변이가 심했으나 다른 분화형들은 변이가 거의 없어 f. sp. sesami를 제외한 f. sp. lycopersici 등 4종의 Fusarium oxysporum 분화형의 균주들은 채집 지역에 관계없이 대체로 유전적으로 안정되어 있는 것으로 판단되었다. Hybridization밴드의 양상에 기초하여 유전적 유연관계를 집괴 분석한 결과 각 분화형별로 유사도가 매우 높게 별도의 유사군을 형성하였다.