• 미생물학회지
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• 제28권3호
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• pp.219-228
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• 1990

자연계로부터 분리한 DK1 균주(NI)가 가지고 있는 $Km^r$ plasmid를 유전자조작 기법으로 변형시킨 DKC601 균주 (GEM)를 이용하여 $Km^r$ 유전자의 전이를 무심천의 자연 수계환경에서 실험하였다. $Km^r$ 유전자의 전이 빈도는 NI 균주의 결과와 비교 연구하는 동시에, 전이과정에서 일어나는 plasmid rearrangement를 비교 분석하였다. GEM과 NI의 $Km^r$ 유전자의 전이 빈도는 5-$10^{\circ}C$의 하천수에서는 $9.1\times 10^{-12}~1.8\times 10^{-11}$로 비슷하였으나 20-$30^{\circ}C$에서는 NI 균주가 GEM 균주보다 조금 높았다. 그리고 멸균하천수나 LB broth를 이용한 실험실 환경에서의 $Km^r$ 유전자의 전이 빈도는 하천수에서 보다 다소 높게 나타났다. NI 균주가 가지고 있던 70kb인 $Km^r$ plasmid는 MTl 균주를 recipient로 했을 때에 얻은 transconjugant에서는 수계환경에 관계없이 rearrangement가 많이 일어났으나, GEM 균주에서 얻은 transconJug gant에서는 52 kb인 $Km^r$ plasmid가 안정된 상태로 발견되었다. 그러나 MT2 균주들 recipient로 했을 때에는 NI 뿐만 아니라 GEM 균주로부터 전이된 $Km^r$ plasmid가 모두 rearrangement를 나타냈다. Transconjugant들이 가지고 있는 plasmid의 수는 conjugation의 시간이 길어짐에 따라 사용한 성험균주나 수계환경에 관계없이 감소되었으며, 특히 GEM의 5 52 kb인 $Km^r$ 유전자의 크기는 24시간 후에도 그대로 유지되였다. 이와 같은 결과로 볼 때, $Km^r$ 유전자는 GEM에서나 NI로부터 전이되는 빈도는 recipient 균주에 관계없이 비슷하였으나, conjugation 과정 중 GEM의 $Km^r$ 유전자는 NI의 $Km^r$ 유전자보다 더욱 안정된 상태로 전이되었으며 이 $Km^r$ plasmid의 rearrangement는 수계환경에 관계없이 recipient 균주에 따라 다양하게 나타났다.

• 미생물학회지
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• 제45권2호
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• pp.163-169
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• 2009

본 논문은 부산소재 하수처리장 중계시설인 민락오수처리장의 하수에서 광범위 베타 락탐 분해효소(extendedspectrum $\beta$-lactamase, ESBL) 생성균주를 분리 동정하고 이들이 생성한 ESBL 유형을 조사하였다. 민락오수처리장은 부산 남구 수영구의 민락동 일대에 밀집한 횟집의 하수를 모아서 남구 용호동에 있는 하수종말처리장으로 중계시키는 시설이다. 2009 년 1 월 하수 검체로부터 항균제 이중 디스크 확산 시험(double disk synergy test)에 양성반응을 나타낸 19 균주를 1차 시험균주로 선택하였다. 인돌생성 시험 methyl-red, Voges-Proskauer, Simmon's citrate, decarboxylase-dihydrolase 및 당 발효능 생화학 시험을 통하여 Klebsiella pneumoniae (3 균주)와 Escherichia coli (16 균주)가 동정되었다. 이 균들을 공여균주로, Escherichia coli J53 (sodium $azide^r$)를 피전달균주로 하여 접합시험을 실시하여 plasmid로 매개된 4 균주 접합체를 얻었다. 이들로부터 plasmid를 추출하여 PCR로 유전자 증폭을 시켜 유전자를 분석하고 등전점을 조사한 결과 Klebsiella pneumoniae에서 생성된 ESBL 유형은 모두 SHV-12 (3 균주)였고, Escherichia coli에서 생성된 ESBL 유형은 SHV-12/TEM-1 (1 균주)였다.

• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.71.2-71
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• 2003

Ralstonia solancearum infects many solanaceous plants, however race 3 infects only potato and tomato weakly. To identify genes responsible for race specificity of R. solanacearum, we mobilized genomic library of LSD2029 (race 3) into LSD341 (race 1) and inoculated 1,000 transconjugants into hot pepper. One transconjugant that did not induce wilt symptom in hot pepper was isolated. We found that a cosmid clone, pRSl, conferred avirulence to LSD341. By deletion and mutational analyses of pRSl, we found the 0.9-kb PstI/Hindlll fragment carries avirulence functions. We sequenced the fragment and identified one possible open reading frame, a rsal gene, possibly encoding 110 amino acids. The rsal was preceded with a plant-inducible promoter (PIP) box, indicating that the gene might be regulated by HrpB. Interestingly, the promoter region of the rsal homolog in the strain GM11000 (race 1) did not have the PIP box. Rsal did not show any significant homologies with proteins in the database, indicating th e protein is different from the previously reported avirulence proteins. When we mutated the rsal gene by marker-exchange in LSD2029, the mutant was less virulent in potato.

• 한국가금학회:학술대회논문집
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• 한국가금학회 2006년도 제23차 정기총회 및 학술발표회
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• pp.92-93
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• 2006

This study was conducted to investigate O group serotyping, antimicrobial drug resistance and distribution of extended spectrum ${\beta}$-lactamase of 203 Escherichia coli(E. coli) isolated from poultry in Korea during the period from April in 2003 to December 2005. The serogroup of 69.4% of isolates was determinated ; O 78(32.5%), O88(7.9%). O15(6.9%) and O141(6.4%) were the most common. These E. coli isolates showed resistance to nalidixic acid(92.6%), streptomycin(81.8%), ampicillin(77.3%), ciprofloxacin(70.9%), sulfisoxazole(66.5%) and trimethoprim(58.1%), respectively. The bla CTX$_{-M-3\;like}$(2 strains) and bla$\;_{CMY-2}$(2 strains) genes producing extended spectrum ${\beta}$ - lactamase(ESBL) were detected in four wild strains resistant to the third generation cephalosporin, respectively. The presence of the ESBL genes was confirmed in all transconjugants by PCR analysis with primers encoding CTX-M-3 like types or CMY-2.

• 한국식물병리학회지
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• 제10권3호
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• pp.151-156
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• 1994

Pseudomonas fluorescens produces the antibiotic, 2,4-diacetylphloroglucinol (Phl), which promotes plant growth by inhibiting bacteria and fungi. Cosmids (genomic library) were mobilized into Phl-nonproducing mutants through the triparental matings with pRK2013 as the helper plasmid at the frequency of 8.37$\times$10-4. Complemented mutants that showed antibiotic activity were selected among about 2,000 transconjugants. The complemented mutants were confirmed by acquired drug resistances (kanamycin and tetracycline). The antibiotic substances of wild type and complemented mutants showed the most excellent anti-bacterial activity. Inhibitory effects of complemented P. fluorescens against phytopathogenic fungi were equal to the parental strain. Complemented mutant and wild type of P. fluorescens were causal microbes of fungal morphological abnormalities. Complemented mutants in potato dextrose agar supplemented with bromothymol blue also showed restoration of glucose utilization as wild type. Plasmids of complemented mutants were isolated from transconjugant sand transformed into competent cells of E. coli DH5$\alpha$. The plamid DNA was reisolated from transformed E. coli DH5$\alpha$.

• Journal of Microbiology and Biotechnology
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• 제13권6호
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• pp.1001-1007
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• 2003

Metal ions contamination may inhibit microorganisms involved in the biodegradation of organic compounds and affect biodegradation rates. Therefore, it is likely that bioremediation of xenobiotics-contaminated soils and waste will require inoculation with efficient biodegrading microbial communities, with capabilities of being resistant to heavy metals as well. Two different transconjugants (Pseudomonas sp. KMl2TC and P. aeruginosa TC) were constructed by conjugation experiments. Results on MIC, induction and growth inhibition strongly indicated that arsenic-resistant plasmid, pKM20, could be mobilized, and the newly acquired phenotype of pKM20 was not only expressed but also well regulated, resulting in newly acquired resistances to $As^{5+},\;As^{3+},\;and\;Sb^{3+} in\;addition\;to\;Cd^{2+},\;Zn^{2+},\;and\;Hg^{2+}$. The phenol- degradation efficiencies of Pseudomonas sp. KMl2TC were maintained significantly even at high heavy metal concentrations at which these efficiencies of P. aeruginosa TC were completely impaired. The results in this study on the effects of heavy metals on phenol degradation, especially after conjugation, are the first ever reported. All the results described in this study encourage to establish a goal of making "designer biocatalysts" which could degrade certain xenobiotics in the area contaminated with multiple heavy metals.

• 한국미생물·생명공학회지
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• 제23권2호
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• pp.164-169
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• 1995

An endogenous cryptic plasmid, pBL1, which has been used to construct plasmid vectors for coryneform bacteria producing amino acids, was eliminated from Brevibacterium lactofermentum. The pBL1 was partially digested with Sau3AI and the resulting DNA fragments were subcloned into a suicide vector pEM1 which contains a kanamycin-resistant (km$^{r}$) gene. KM$^{r}$ B. lactofermentum transconjugants were obtained by conjugal transfer of the pEM1 derivatives containing pBL1 DNA fragments from Escherichia coli into B. lactofermentum. A km$^{r}$ transconjugant was analyzed to contain a plasmid pEB14, which occurred in vivo by homologous recombination between pBL1 and the conjugal-transferred plasmid. The pEB14 including the pEM1-derived km$^{r}$ gene was found to be lost concomitantly with km$^{r}$ phenotype, resulting in the construction of a pBL1-free strain of B lactofermentum. Based on transformation efficiencies and plasmid stability, the resultant pBL1- free strain is more useful than wild strain as a host cell for genetic manipulation. It could be concluded that foreign plasmid DNAs are efficiently isolated and analyzed from the pBL1-free strain because of the absence of endogenous pBL1 plasmid.

• 미생물학회지
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• 제27권4호
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• pp.334-341
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• 1989

Toluate 분해 플라스미드를 pseudomonase cepacia SUB37에서 분리하여 분자량은 한천 젤 전기영동으로 측정한 결과 79. (119kb)로 확인되었다. 이 TOL플라스미드는 Pseudomonas의 다른 균주와 다른 속의 균주에 전달되었다. m-toluate 분해에서 가장 중요한 역할을 하는 catechol-2,3-oxygenase 활성을 P. cepacia SUB37과 transconjugant의 조효소액으로부터 측정한 결과, P. putida mt 2에서와 같이, meta pathway를 거쳐 m-toluate를 분해하는 유전자들이 plasmid에 암호화됨을 알수 있었다. P. cepacia SUB37 유래의 새로운 TOL plasmid는 IncP-4 불화합성군에 속하였고, 이것은 아마도 P. putida의 IncP-9 그룹의 TOL 플라스미드의 유도체로 사료된다.

• 한국식물병리학회지
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• 제13권3호
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• pp.172-178
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• 1997

십자화과 작물에 발생하는 검은썩음병(Black rot or Black vein of crucifer)의 병원성 세균인 Xanthomonas campestris pv. crucifer)의 병원성 세균인 Xanthomonas campestris pv. campestris를 분리, 동정하고 병원성을 검정하였다. 이 X. c. pv. campestris 는 3가지 종의 Chinese cabbage에 병원성을 나타내었고, 병원성과 관련된 특성을 결정하기 위하여 Tn5 mutagenesis를 실시 cellulase negative mutant를 선발하여 병원성 검정하였다. 선발된 cellulase negative mutant를 배추에 분무 접종하여 광학 현미경과 전자현미경으로 관찰한 결과 cellulase negative mutant는 wild type와 함께 기공표면과 기공하부조직에서 정착하였지만 그 밀도는 낮았다. 반면 접종 24시간 이후 wild type은 기공표면과 기공하부조직이 lysis되기 시작하여 48시간 이후에는 병원성의 진전으로 보다 많이 lysis되었다. 6일 후, wild type은 cellulase활성에 의해 식물체 조직에서 높은 증식력을 보이며 조직을 lysis 시키고 또한 조직 깊숙이 침입, 정착하는 것을 관찰하였다. 이 결과로 X. c. pv.c campestris의 cellulase는 병원성에 관여하는 중요한 요인으로 생각된다.

• 한국식물병리학회지
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• 제11권4호
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• pp.304-311
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• 1995

To determine whether chitinolytic enzymes from Chromobacterium violaceum C-61 play an important role in the suppression of Rhizoctonia damping-off, Tn5 insertion mutants deficient in chitinolytic activity (Chi a- mutants) were selected and their chitinolytic and disease suppression were compared with those of the parental strain. Four Chi a- mutants selected from about 2,000 transconjugants did not inhibit mycelial growth of Rhizoctonia solani on nutrient agar-potato dextrose agar (BA-PDA) and their abilities to suppress Rhizoctonia damping-off were much lower than the parental strain. However, population density in the eggplant rhizosphere did not differ significantly between the parental strain and four Chi a- mutants. The crude enzyme of the parental strain inhibited growth of R. solani on NA-PDA and its chitinase activity was much higher than that of Chi a- mutants. But the N,N' -diacetylchitobiase activity between these isolates were not significantly different. The chitinase of Chi a- mutants was defective in 2 isoforms of 52- and 37-kDa among four isoforms of 54-, 52-, 50- and 37-kDa. A Tn5 element was inserted into one site of 10 kb EcoRI fragment of chromosomal DNA in three Chi- mutants, C61-C1, -C2, and -C3. In C61-C4 mutant, a Tn5 element was inserted into two sites of 10 kb and 4.4 kb EcoRI fragments. These results suggest that the chitinase of C. violaceum C-61 play an important role in the suppression of Rhizoctonia damping-off of cucumber and eggplant.