• 한국어병학회지
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• 제25권3호
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• pp.159-164
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• 2012

Vibrio vulnificus ghosts (VVG) were generated using a mobilizable vector including a thermosensitive expression cassette by conjugation. The vaccine potential of VVG was investigated in mouse. Mice immunized with VVG showed significantly higher antibody titer than those with formalin-killed V. vulnificus. The present study supports the conceptive usefulness of bacterial ghosts as vaccine candidates.

• 한국미생물·생명공학회지
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• 제32권1호
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• pp.1-10
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• 2004

Many bacteria monitor their population density and control the expression of specialized gene sets in response to bacterial cell density based on a mechanism referred to as quorum sensing. In all cases, quorum sensing involves the production and detection of extracellular signaling molecules, auto inducers, as which Gram-negative and Gram-positive bacteria use most prevalently acylated homoserine lactones and processed oligo-peptides, respectively. Through quorum-sensing communication circuits, bacteria regulate a diverse array of physiological functions, including virulence, symbiosis, competence, conjugation, antibiotic production, motility, sporulation, and biofilm formation. Many pathogens have evolved quorum-sensing mechanisms to mount population-density-dependent attacks to over-whelm the defense responses of plants, animals, and humans. Since these AHL-mediated signaling mechanisms are widespread and highly conserved in many pathogenic bacteria, the disruption of quorum-sensing system might be an attractive target for novel anti-infective therapy. To control AHL-mediated pathogenicity, several promising strategies to disrupt bacterial quorum sensing have been reported, and several chemicals and enzymes have been also investigated for years. These studies indicate that anti-quorum sensing strategies could be developed as possible alternatives of antibiotics.

• 한국미생물·생명공학회지
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• 제20권4호
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• pp.483-490
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• 1992

수계 환경에서 일어나는 유전자의 전이행방을 이해하기 위하여, conjugation에 의하여 전이되는 kanamycin 내성 ($Km^r$) 유전자에 대하여 DNA probe를 이용하여 Southern hybridization 방법으로 추적하였다. 자연계로부터 분리한 $Km^r$ 세균과 $Km^r$ 유전자를 유전자 조작기법으로 변형시킨 GMM 균주들을 donor로 하여 conjugation을 했을 때, $Km^r$ 유전자는 자연계 분리 균주에서보다 수질환경에 관계없이 10~00배 잘 전이되었다. LB 배지에서 GMM 균주의 $Km^r$ 유전자가 전이된 conjugant에서는 새로 생성되는 plasmid가 많이 나타났고 AW와 FW에서는 conjugation 시간에 따라 plasmid의 재배열 현상이 다양하였다. LB에서 얻은 conjugant들의 plasmid에 대하여 $Km^r$ DNA probe로 Southern analysis를 한 결과, plasmid의 재배열이 다양함에도 불구하고 conjugant들의 $Km^r$ plasmid는 donor에서와 같은 위치에서 hybridization signal이 나타났다. 그러나 AW에서 50시간 conjugation시켰을 때 DKI의 pDK101과 DKC601의 pDT529, 그리고 AW에서 30시간 conjugation 시켰을 때 DKC600의 pDK101은 전혀 나타나지 않고, 소실되었다. 또 전이된 $Km^r$ plasmid의 크기는 AW와 FW의 수질에 따라 약간 변화되어 나타났다. 그러므로 DNA probe에 의한 Southern hybridization 방법은 수질환경에서 특정 유전자의 전이행방을 추적하는데 매우 유용하다고 판단된다.

• 한국식물병리학회지
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• 제14권2호
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• pp.177-183
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• 1998

A DNA fragment which is involved in tolassin production was cloned to obtain a molecular marker of Pseudomonas tolaasii, a casual agent of bacterial brown blotch disease of oyster mushroom (Pleurotus ostreatus). Tolaasin is a lipodepsipeptide toxin and known as a primary disease determinant of the P. tolaasii. It is responsible for formation of white line in agar when P. tolaasii were cultured against white line reacting organisms (WLROs). White line negative mutants (WL-) were generated by conjugation between rifampicin resistant strain of P. tolaasii and E. coli carrying suicidal plasmid pSUP2021 : : Tn5. The ability of tolaasin production of the WL- mutants was examined by hemolysis test, pathogenicity test, and high pressure liquid chromatography (HPLC) analysis of culture filtrate. All of the WL- mutants were lost the ability of tolaasin production (Tol-). Genomic library of the Tol- mutant was constructed in pLAFR3 and the cosmid clone containing Tn5 was selected. DNA fragment fro franking region of Tn5 was cloned from the plasmid and used as a probe in Southern blot. DNA-DNA hybridization with the probe to total DNA from group of bacteria ecologically similar to P. tolaasii including WLORs, fluorescent Pseudomonads isolated from oyster mushroom, P. agarici, P. gingeri, and some of other species of Psedomonas showed that some of the tested bacteria do not have any hybridized band and others have bands sowing RFLP. The cloned DNA fragment or its nucleotide sequence will be useful in detection and identification of the P. tolaasii.

• 한국미생물·생명공학회지
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• 제40권3호
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• pp.197-207
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• 2012

This study investigated the role of plasmids and their relationship with the multiple antibiotic resistance of 30 Vibrios sp. isolated from molluscs and crustaceans sampled from the Kerala coastal waters of India. The biochemical identification and antibiotic resistance profiles were determined, followed by the plasmid profiles, conjugation and transformation efficiencies. The results showed a considerable difference in the level of bacterial resistance to various antibiotics; while all 30 strains were found to be MAR Vibrios sp. and their resistance patterns varied. All the strains were resistant to amoxycillin, ampicillin and carbeniciliin. 87% were resistant to rifampicin; 74% to cefuroxime; 67 to streptomycin; 53% to norfloxacin and ciprofloxacin and 47% to furazolidone and nalidixic acid. In addition to their antibiotic resistance, the plasmid DNA of the MAR Vibrios strains isolated from the molluscs and crustaceans was also studied. Nine strains isolated from crustaceans and molluscs were found to harbor 1-3 plasmids with sizes varying from 5. 98 kb to 19. 36 kb. The average transformation efficiency was about $5{\times}10^{-8}$ and the conjugation efficiency varied from $2.1{\times}10^{-3}$ to $10^{-9}$. A further study of antibiotic resistance patterns may be useful to test the extent of drug resistance in seafoods and help to devise a nationwide antibiotic policy.

• Journal of Microbiology and Biotechnology
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• 제14권5호
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• pp.938-943
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• 2004

A gene responsible for the chlorothalonil-biotransformation was cloned from the chromosomal DNA of Ochrobactrum anthropi SH35B, an isolated bacterium strain from soil. We determined the nucleotide sequences and found an open reading frame for glutathione S-transferase (GST). The drug-hypersensitive Escherichia coli KAM3 cells transformed with a plasmid carrying the GST gene can grow in the presence of chlorothalonil. The GST of O. anthropi SH35B was expressed in E. coli and purified by affinity chromatography. The fungicide chlorothalonil was rapidly transformed by the purified GST in the presence of glutathione. No significant difference in the chlorothalonil-biotransformation effect was observed among the thiol compounds (cysteine, reduced glutathione, and $\beta$-mercaptoethanol). Thus, the result reported here is the first evidence on the chlorothalonil-biotransformation by conjugation with the cellular free thiol groups, especially glutathione, catalyzed by the bacterial GST.

• 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.

• 한국식물병리학회지
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• 제12권2호
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• pp.156-161
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• 1996

세계적으로 구리약제에 대해서 저항성을 나타내는 균주들이 발견되있으며, 이들은 구리약제의 방제효과를 감소시켰다. 국내에서도, 구리 저항성 균주 Xanthomonas campestris. pv. vesicatoria HN94-2, -3, -4, -5, -6 들이 천안지역의 고추재배지에서 처음으로 분리되었으며, 이들 균주들의 nutrient agar에서 황산구리(CuSo\ulcorner)에 대한 최소억제농도(minimum inhibitory concentration, MIC)는 1.4~1.6 mM이었다. 이들은 모두 황산아연(ZnSO\ulcorner)에 대해서는 감수성을 보여, 구리저항성 균주에 대한 방제약제로서 아연 함유 약제를 사용할 수 있을 것이다. 분리된 균주중 HN94-2와 HN94-6을 이용하여 접합(conjugation)을 통해 구리저항성의 전파를 실험한 결과, 이들 두 균주 모두 구리감수성 균주에게 4.3$\times$10\ulcorner에서 1.0$\times$10\ulcorner(transconjugant/donor)의 정도로 구리 저항성이 전이되었다. 이들 HN94-2와 HN94-6 균주의 구리정항성 유전자들은 약 200 kb 정도의 커다란 플라스미드(plasmid)에 존재하며, 이들은 각각 pXVK9402와 pXVK9406이라 명명되었다.

• Journal of Microbiology and Biotechnology
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• 제20권5호
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• pp.871-874
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• 2010

Many studies require expression analysis of the same gene/promoter across a range of bacterial genera. However, there is currently a lack of availability of reporters based on the broad-host-range IncQ replicon, which is compatible with a popular improved IncP transfer system that is self-transfer defective. We report IncQ lacZ reporter plasmids with features including (1) compatibility with IncP, IncW, and pBHR/pBBR replicons, (2) a variety of antibiotic markers (Sp-r, Sm-r, Km-r, Cm-r), (3) convenient mobilization via a novel self-transfer-defective IncP conjugation system, and (4) GenBank DNA sequences. Utility is demonstrated using three different promoters in different Gram-negative genera.

• Journal of Microbiology and Biotechnology
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• 제28권9호
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• pp.1563-1572
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• 2018

Gold nanoparticles (AuNP) and their conjugates have been gaining a great deal of recognition in the medical field. Meanwhile, extended-spectrum ${\beta}$-lactamases (ESBL)-producing bacteria are also demonstrating a challenging problem for health care. The aim of this study was the biosynthesis of AuNP using Rosa damascenes petal extract and conjugation of ceftriaxone antibiotic (Cef-AuNP) in inhibiting ESBL-producing bacteria and study of in vitro anticancer activity. Characterization of the synthesized AuNP and Cef-AuNP was studied. ESBL-producing strains, Acinetobacter baumannii ACI1 and Pseudomonas aeruginosa PSE4 were used for testing the efficacy of Cef-AuNP. The cells of MCF-7 breast cancer were treated with previous AuNP and Cef-AuNP at different time intervals. Cytotoxicity effects of apoptosis and its molecular mechanism were evaluated. Ultraviolet-visible spectroscopy and Fourier transform infrared spectroscopy established the formation of AuNP and Cef-AuNP. Transmission electron microscope demonstrated that the formed nanoparticles were of different shapes with sizes of 15~35 nm and conjugation was established by a slight increase in size. Minimum inhibitory concentration (MIC) values of Cef-AuNP against tested strains were obtained as 3.6 and $4{\mu}g/ml$, respectively. Cef-AuNP demonstrated a decrease in the MIC of ceftriaxone down to more than 27 folds on the studied strains. The biosynthesized AuNP displayed apoptotic and time-dependent cytotoxic effects in the cells of MCF-7 at a concentration of $0.1{\mu}g/ml$ medium. The Cef-AuNP have low significant effects on MCF-7 cells. These results enhance the conjugating utility in old unresponsive ceftriaxone with AuNP to restore its efficiency against otherwise resistant bacterial pathogens. Additionally, AuNP may be used as an alternative chemotherapeutic treatment of MCF-7 cancer cells.