• Journal of Microbiology and Biotechnology
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• 제17권12호
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• pp.2046-2055
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• 2007

Wild-type V. vulnificus cannot grow using lactose as the sole carbon source or take up the sugar. However, prolonged culture of this species in media containing lactose as the sole carbon source leads to the generation of a spontaneous lactose-utilizing (LU) mutant. This mutant showed strong ${\beta}$-galactosidase activity, whereas the wild-type strain showed a barely detectable level of the activity. A mutant with a lesion in a gene homologous to the lacZ of E. coli in the bacterium no longer showed ${\beta}$-galactosidase activity or generated spontaneous LU mutants, suggesting that the lacZ homolog is responsible for the catabolism of lactose, but the expression of the gene and genes for transport of lactose is tightly regulated. Genetic analysis of spontaneous LU mutants showed that all the mutations occur in a lacI homolog, which is located downstream to the lacZ and putative ABC-type lac permease genes. Consistent with this, a genomic library clone containing the lad gene, when present in trans, made the spontaneous LU mutants no longer able to utilize lactose as the sole carbon source. Taken together with the observation that excessive amounts of exogenously supplemented possible catabolic products of lactose have negative effects on the growth and survivability of V. vulnificus, we suggest that V. vulnificus has evolved to carry a repressor that tightly regulates the expression of lacZ to keep the intracellular toxic catabolic intermediates at a sublethal level.

• 대한수의학회지
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• 제43권2호
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• pp.247-253
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• 2003

Actinobacillus pleuropneumoniae causes a highly contagious pleuropneumoniae in swine. The bacterium produces several virulence factors such as exotoxin, LPS, capsular polysaccharide, etc. Among them, the exotoxin, called Apx, has been focused as the major virulence factor, and the toxin consists of 4 gene cluster. apx CABD. apxA is the structural gene of toxin and has four different types, I, II, III, and IV. As the first step of development of a new subunit vaccine, the three different types of apxA gene were amplified from A. pleuropneumoniae isolated from Korea by PCR with primer designed based on the N- and C-terminal of the toxin. The sizes of apxIA, IIA and IIIA were 3,073, 2,971 and 3,159bps, respectively. The comparison of whole DNA sequences of apxIA, IIA and IIIA genes with those of the reference strain demonstrated 98%, 99% and 98% homology, respectively. In addition, the phylogenetic analysis was performed based on the amino acid sequences compared with 12 different RTX toxin family using the neighbor-joining method. ApxA proteins of Korean isolates were identical with reference strains in this study. All ApxA proteins were expressed in E. coli with pQE expression vector and identified using Western blot with polyclonal antibodies against culture supernatants of A. pleuropneumoniae serotype 2 or 5. The sizes of each expressed ApxA protein were about 120, 110, 125 kDa (M.W.), respectively. The results obtained in this study could be used for the future study to develop a new vaccine to porcine pleuropneumoniae.

• Journal of Microbiology and Biotechnology
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• 제25권5호
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• pp.620-628
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• 2015

A fungal strain, R9, was isolated from the South Atlantic sediment sample and identified as Aspergillus fumigatus. An antifungal protein, AfAFP_R9, was purified from the culture supernatant of Aspergillus fumigatus R9. AfAFP_R9 was identified to be restrictocin, which is a member of the ribosome-inactivating proteins (RIPs), by MALDI-TOF-TOF-MS. AfAFP_R9 displayed antifungal activity against plant pathogenic Fusarium oxysporum, Alternaria longipes, Colletotrichum gloeosporioides, Paecilomyces variotii, and Trichoderma viride at minimum inhibitory concentrations of 0.6, 0.6, 1.2, 1.2, and 2.4 μg/disc, respectively. Moreover, AfAFP_R9 exhibited a certain extent of thermostability, and metal ion and denaturant tolerance. The iodoacetamide assay showed that the disulfide bridge in AfAFP_R9 was indispensable for its antifungal action. The cDNA encoding for AfAFP_R9 was cloned from A. fumigatus R9 by RT-PCR and heterologously expressed in E. coli. The recombinant AfAFP_R9 protein exhibited obvious antifungal activity against C. gloeosporioides, T. viride, and A. longipes. These results reveal the antifungal properties of a RIP member (AfAFP_R9) from marine-derived Aspergillus fumigatus and indicated its potential application in controlling plant pathogenic fungi.

• Journal of Microbiology and Biotechnology
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• 제10권6호
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• pp.865-872
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• 2000

Helocobacter phylori is the major cause of gastritis, peptic ulcer, and a principal risk factor for gastric cancer. As the firs step towards a vaccine against H. pylori infection, Hy.pylori urease was expressed and purified as a recombinant apoenzyme (rUrease) in E. coli. In order to develop an effective immunization protocol using rUrease, the host immune responses were evaluated after the oral immunization of mice with rUrease preparations plus cholera toxin relative to various conditions, such as the physical nature of the antigen, the frequency of the booster immunization, the dose of the antigen, and the route of administration. The protective efficacy was assessed using a quantitative culture following an H. pylori SS1 challenge. It was demonstrated that rUrease, due to its particulated nature, was more superior than the UreB subunit as a vaccine antigen. The oral immunization of rUrease elicited significant systemic and secretory antibody responses, and activated predominantly Th2-type cellular responses. The bacterial colonization was significantly reduced (~100-fold) in those mice immunized with three or four weekly oran doses of rUrease plus cholera toxin (p<0.05), when compared to the non-immunized/challenged controls. The protection correlated well with the elicited secretory IgA level against rUrease, and these secretory antibody responses were highly dependent on the frequency of the booster immunization, yet unaffected by the dose of the antigen (25-200$\mu\textrm{g}$). These results demonstrate the remarkable potential of rUrease as a vaccine antigen, thereby strengthening the possibility of developing an H. pylori vaccine for humans.

• 식물조직배양학회지
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• 제24권2호
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• pp.93-97
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• 1997

Linoleic acid를 linolenic acid로 전환시키는 지방산불포화효소의 유전자(fad3)를 유채의 fad3 DNA probe를 이용한 plaque hybridization방법으로 $\lambda$ZAPII Arabidopsis thaliana cDNA expression library로부터 분리하였으며 1.8 kb-EcoRI DNA조각을 지니는 lambda clone을 pGEM7으로 subcloning하여 염기서열을 분석하였다. 그 결과로부터의 아미노산서열분석에 의하면 fad3 유전자는 open reading frame이 386개의 아미노산으로 이루어졌으며 44,075 Da의 분자량이 예측되고 있다. 엽록체의 $\omega$-3 지방산불포화효소(fad7)와 endoplasmic reticulum의 지방산불포화효소(fad2)와의 비교시 각각 70%와 58%의 유사성이 나타났다. 특히 82-151의 아미노산서열과 276-333의 아미노산지역은 보존성이 높았으며 이는 불포화지방산효소의 기능에 필수적인 지역으로 보여진다. 한편 대장균을 이용한 IPTG에 의한 Fad3단백질의 유도생성은 대장균의 생육에 독성효과를 나타내는 것으로 나타났다.

• 한국환경보건학회지
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• 제42권3호
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• pp.189-195
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• 2016

Objectives: To ensure the microbiological safety of groundwater, it was confirmed whether waterborne pathogenic bacteria in groundwater samples tested positive for total coliforms in the Chungcheongnam-do Province region. Methods: Total colony counts, total coliforms and fecal coliforms were tested according to the process mandated by the drinking water quality testing standards of Korea. DNA was extracted from the samples, tested positive for total coliforms, and then subjected to real-time PCR to detect waterborne pathogenic bacteria. Results: A total of 115 samples were inadequate for drinking water. Thirty-one cases (27%) showed positive for fecal coliforms and nine cases (7.8%) showed total colony counts exceeding drinking water standards. Twenty-seven cases (23.5%) showed three items (total colony counts, total coliforms and fecal coliforms). Using the real-time PCR method, waterborne pathogens were detected in 57 cases (49.6%) in 115 samples. Seventy-eight cases of waterborne pathogenic bacteria were detected (including duplications): 27 cases of pathogenic E. coli (EPEC (19), ETEC (5), EHEC (1), EAEC (1) and EIEC (1)); 45 of Bacillus cereus; two of Yersinia spp.; two of Salmonella spp.; one of Staphylococcus aureus; one of Clostridium perfringens. Conclusion: The real-time PCR method can offer rapid and accurate detection of waterborne pathogenic bacteria. Therefore, this assay could be an alternative to conventional culture methods and can further ensure the microbiological safety of groundwater.

• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.650-655
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• 1998

The chromosomal DNA fragment from Phaffia rhodozyma CBS 6938 which is able to autonomously replicate in the yeast Saccharomyces cerevisiae was cloned on an integrative URA3 plasmid. Its minimal fragment exhibiting autonomously replicating activiy in the S. cerevisiae gave a higher frequency transformation efficiency than that found for centromere-based plasmid, and enabled extrachromosoma1ly stable transmission of the plasmids in one copy per yeast cell under non-selective culture condition. The 836-bp DNA element lacked an ORF and did not contain any acceptable match to an ARS core consensus. Sequence analysis, however, displayed a cluster of three hairpin-Ioop-sequences with individual $\triangle {G_{25}}^{\circ}C$ free energy value of -10.0, -17.5, and -17.0 kcal. $mor^{-l}$as well as a 9-bp sequence with two base pair mismatches to the S. cerevisiae/E. coli gyrase-binding site. This 836-bp sequence also included one 7-bp sequence analogous to the core consensus of centromeric DNA element III (CDEIII) of S. cerevisiae, but CDEIII-like 7 bp sequence alone did not give a replicative function in this yeast.

• Applied Biological Chemistry
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• 제36권6호
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• pp.562-566
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• 1993

제라니움(Pelargonium zenale hybrids)의 원형질체에 외래유전자를 전기충격에 의해 직접도입시키는 조건을 methylene blue 염색법을 이용하여 조사하였다. 그 결과 1.77 kV/cm의 직류전압을 $40{\mu}\;sec$ 동안 가해 주는 것이 제일 효과적이었으며 이때의 원형질체 생존율은 70% 염색율은 58%이었다. 정제한 pBin19 plasmid를 전기충격법으로 원형질체에 삽입시킨 뒤 kanamycin이 포함된 배지에서 배양하였으며 원형질체의 세포분열은 KM8P 액체배지에서 제일 높았다. 윈형질체의 최적 전기융합 조건은 교류 주파수 1 MHz를 40 V/cm에서 15 sec 동안 가한 후 직류전압 0.5 kV/cm를 $60{\mu}\;sec$ 동안 처리해 준 결과 이때의 융합율과 생존율은 각각 13%, 81%이었다.

• Journal of Microbiology and Biotechnology
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• 제28권5호
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• pp.663-670
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• 2018

The present study focused on the production, characterization, and in vitro prebiotic evaluation of an exopolysaccharides (EPS) from Bacillus sonorensis MJM60135 isolated from ganjang (fermented soy sauce). Strain MJM60135 showed the highest production ($8.4{\pm}0.8g/l$) of EPSs compared with other isolates that were screened for EPS production based on ropy culture morphology. Furthermore, MJM60135 was cultured in 5 L of medium and the EPS was extracted by ethanol precipitation. The emulsification activity of the EPS was higher in toluene than in o-xylene. Fourier transform infrared spectroscopy analysis showed the presence of hydroxyl and carboxyl groups and glycosidic linkages. The isolated EPS contained mannose and glucose, as observed by thin-layer chromatography analysis of the EPS hydrolysate. Lactic acid bacteria (LAB) and pathogenic E. coli K99 and Salmonella enterica serovar Typhimurium were tested for their growth utilizing the EPS from B. sonorensis MJM60135 as the sole carbon source for its possible use as a prebiotic. All the tested LAB exhibited growth in the EPS-supplied medium compared with glucose as carbon source, whereas the pathogenic strains did not grow in the EPS-supplied medium. These findings indicate that the EPS from B. sonorensis MJM60135 has potential application in the bioremediation of hydrocarbons and could also be used as a prebiotic.

• 한국미생물·생명공학회지
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• 제30권3호
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• pp.206-209
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• 2002

Bacillus stearothermophilus의 CCTase 유전자(cgtS) 대장균과 효모의 shuttle vector로서 항구적 promoter인 adh l promoter를 함유한 pVT103-U(6.9Kb)에 도입하여 재조합 plasmid pVT-CCTS (9.0Kb)을 구축하고 효모 숙주 S. cerevisiae 2805에서 발현시켰다. 재조합 균주의 항구적 발현계인 2805/pv7-CGTS의 최적 발현조건은 YP배지에 dextrose 2%, pH 5.5, 30"C에서 최적 발효조건이었으며, CCTase의 최대 발현량은 48시간 배양시 0.624unit/mL을 나타내었다. B. stearothermophilus의 signal peptide가 재조합 효모에서도 높은 분비효율을 나타내어서 발현된 효소의 87%가 세포 외로 분비 생산되었다.산되었다.