• Journal of Microbiology and Biotechnology
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• 제26권3호
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• pp.521-529
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• 2016

Extensive use of antibiotics over recent decades has led to bacterial resistance against antibiotics, including gentamicin, one of the most effective aminoglycosides. The emergence of resistance is problematic for hospitals, since gentamicin is an important broad-spectrum antibiotic for the control of bacterial pathogens in the clinic. Previous study to identify gentamicin resistance genes from environmental samples have been conducted using culture-dependent screening methods. To overcome these limitations, we employed a metagenome-based culture-independent protocol to identify gentamicin resistance genes. Through functional screening of metagenome libraries derived from soil samples, a fosmid clone was selected as it conferred strong gentamicin resistance. To identify a specific functioning gene conferring gentamicin resistance from a selected fosmid clone (35-40 kb), a shot-gun library was constructed and four shot-gun clones (2-3 kb) were selected. Further characterization of these clones revealed that they contained sequences similar to that of the RNA ligase, T4 rnlA that is known as a toxin gene. The overexpression of the rnlA-like gene in Escherichia coli increased gentamicin resistance, indicating that this toxin gene modulates this trait. The results of our metagenome library analysis suggest that the rnlA-like gene may represent a new class of gentamicin resistance genes in pathogenic bacteria. In addition, we demonstrate that the soil metagenome can provide an important resource for the identification of antibiotic resistance genes, which are valuable molecular targets in efforts to overcome antibiotic resistance.

• 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.

• Applied Biological Chemistry
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• 제35권5호
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• pp.361-366
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• 1992

토양전염 병원성 사상균인 Fusarium oxyporum, Rhizoctonica solani에 대하여 길항력을 갖는 균주 Bacillus subtilis를 근권 토양에서 분리하여 동정한 후 이들 균주의 사상균에 대한 길항력, 발아율 및 작물의 생육에 미치는 영향력을 검토하였다. 또한 이 균주의 chromosome에 Bacillus thuringiensis(BT) 독소유전자를 삽입하여 균주의 형질변환을 유도하였다. BT 독소 유전자는 southern blotting에 의하여 확인되었으나 이의 최종 생성물인 독소 단백질은 SDS-PAGE에 확인되지 않았다. 이들 형질변환된 균주의 생리 및 생화학적 특성을 조사한 결과 모균주와 차이는 없었으며, BT 독소유전자가 삽입된 균주는 선충의 유충에 대한 생물학적 검정에서는 효과가 인정되지 않았으나 누에에 있어서는 1X 균체 희석액에서 10내지 20% 정도의 치사율이 관찰되었다. 모균주와 BT 독소유전자에 의하여 형질변환된 균주 모두 발아율 및 작물의 생육을 향상시켰다.

• Journal of Microbiology and Biotechnology
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• 제26권1호
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• pp.44-55
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• 2016

Bacillus cereus is a gram-positive, rod-shaped, spore-forming bacterium that has been isolated from contaminated fermented soybean food products and from the environment. B. cereus produces diarrheal and emetic toxins and has caused many outbreaks of foodborne diseases. In this study, we investigated whether B. amyloliquefaciens RD7-7, isolated from rice doenjang (Korean fermented soybean paste), a traditional Korean fermented soybean food, shows antimicrobial activity against B. cereus and regulates its toxin gene expression. B. amyloliquefaciens RD7-7 exhibited strong antibacterial activity against B. cereus and inhibited the expression of B. cereus toxin-related genes (groEL, nheA, nheC, and entFM). We also found that addition of water extracts of soybean and buckwheat soksungjang (Korean fermented soybean paste made in a short time) fermented with B. amyloliquefaciens RD7-7 significantly reduced the growth and toxin expression of B. cereus. These results indicate that B. amyloliquefaciens RD7-7 could be used to control B. cereus growth and toxin production in the fermented soybean food industry. Our findings also provide a basis for the development of candidate biological control agents against B. cereus to improve the safety of fermented soybean food products.

• Journal of Microbiology and Biotechnology
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• 제31권5호
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• pp.710-716
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• 2021

A risk analysis of Shiga toxin (Stx)-encoding bacteriophage was carried out by confirming the transduction phage to non-Stx-producing Escherichia coli (STEC) and subsequent expression of the Shiga toxin genes. The virulence factor stx1 was identified in five phages, and both stx1 and stx2 were found in four phages from a total of 19 phage isolates with seven non-O157 STEC strains. The four phages, designated as ϕNOEC41, ϕNOEC46, ϕNOEC47, and ϕNOEC49, belonged morphologically to the Myoviridae family. The stabilities of these phages to temperature, pH, ethanol, and NaClO were high with some variabilities among the phages. The infection of five non-STEC strains by nine Stx-encoding phages occurred at a rate of approximately 40%. Non-STEC strains were transduced by Stx-encoding phage to become lysogenic strains, and seven convertant strains had stx1 and/or stx2 genes. Only the stx1 gene was transferred to the receptor strains without any deletion. Gene expression of a convertant having both stx1 and stx2 genes was confirmed to be up to 32 times higher for Stx1 in 6% NaCl osmotic media and twice for Stx2 in 4% NaCl media, compared with expression in low-salt environments. Therefore, a new risk might arise from the transfer of pathogenic genes from Stx-encoding phages to otherwise harmless hosts. Without adequate sterilization of food exposed to various environments, there is a possibility that the toxicity of the phages might increase.

• 대한의생명과학회지
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• 제16권2호
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• pp.75-82
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• 2010

The molecular epidemiological characteristics of methicillin-resistant Staphylococcus aureus (MRSA) isolates have demonstrated their genetic diversity and evolution. A total of 137 strains of MRSA clinical isolates was collected from Korean healthcare facility in 2007. The MRSA clinical isolates were analyzed by molecular typings (SCCmec element and agr locus typing), virule nce factor gene detections {(Panton-Valentine leukocidin (PVL), enterotoxin, exfoliative toxin and toxic shock syndrome toxin-1), and amplified fragment length polymorphism (AFLP)}. The MRSA clinical isolates were classified as SCCmec type II-agr type 1 (2 strains), type II-agr type 2 (79 strains), type III-agr type 1 (24 strains), type III-agr type 2 (2 strains), type IV-agr type 1 (27 strains), type IV-agr type 2 (2 strains), and non-typable (1 strain, agr type 3). Based on SCCmec types, SCCmec type II (95.1%) and III (88.5%) indicated higher multidrug resistance rate than SCCmec type IV (10.3%) (P<0.001). The most common enterotoxin genes were seg (83.8%), sei (83.1%), and sec (80.2%). The tst gene was present in 86 out of 137 (62.8%) MRSA isolates. All MRSA isolates were negative for PVL and exfoliative toxin genes. The combinations of toxin genes were observed in particular SCCmec types; 97.6% of SCCmec type II strains carried sec, seg, sei and tst genes, 73.0% of SCCmec type III strains carried sea gene, and 89.7% of SCCmec type IV strains carried sec, seg and sei genes. Each of the SCCmec types of MRSA isolates had distinct AFLP profile. In conclusion, SCCmec type II, agr type 1 and 2 have demonstrated to be the most common types in Korea, and the results indicated that the virulence factors are closely associated with their molecular types (SCCmec and agr types).

• 미생물학회지
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• 제35권3호
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• pp.205-210
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• 1999

Vibrio cholerae 는 사람에게 설사를 일으키는 병원성 세규닝며 본 연구에 이용된 V.cholerae KNIH002 는 국내의 설사질환 환자로부터 분리하였다. 콜레라 독소 검출용 프라이머를 이용하여 PCR 로 증폭한 산물을 탐침자로 이용하여 Southern hybridization을 실시한 결과 PstI 및 BglII로 이중절단된 4.5-kb 절편내에서 ctx 유전자가 존재함을 확인하였다. 따라서 염색체 DNA를 PstI 및 BglII로 절단 후 V. cholerae KNIH002 의 유전자 mini-libraries를 제조하였다. 그리고 동일 탐침자를 이용하여 colony hybridization을 실시한 결과 제조된 유전자 mini-libraries 로부터 신호를 나타내는 한 개의 클론을 선발하였다. 선발된 클로닝 지니는 플라스미드를 pCTX75 라 명명하였으며, 이 클론은 CHO 세포에 대한 세포 독력이 나타남을 확인하였다. 염기서열을 결정한 결과 클로닝된 플라스미드에는 ace 와 zot 유전자들은 각각 ATG 개시코돈과 TGA 종결코돈을 포함하여 291 bp와 1,200 bp 로 구성되어져 있었다. ace 유전자의 염기서열은 V.cholerae E7946 EI Tor Ogawa strain 이 것과 100% 일치하였다. 그러나 zot 유전자의 염기서열 및 아미노산 서열은 V. cholerae 395 Classical Ogawa strain 의 것과 각각 99% 및 98.8% 의 상동성을 보였다. 특히, V.cholerae 395 Classicale Ogawa strain 의 Zot 폴리펩타이드에서 100번, 272번, 281번째 alanine 은 V.cholerae KNIH002에서 모두 valine 으로 치환되어져 있었다.

• 과학수사학회지
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• 제11권3호
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• pp.210-217
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• 2017

법미생물 검사실에서는 식품의 미생물 분석이 요구되는 경우들이 있다. 국립과학수사연구원에 식품 미생물분석이 의뢰된 샘플들 중에서 Bacillus cereus 균이 분리되었던 일부 증례가 있었다. B. cereus는 식중독을 일으킬 수 있는 중요한 병원성 세균이다. 따라서 우리는 최근 미생물 검사를 위해 의뢰된 멸치액젓에서 B. cereus를 분리한 후, 16S rDNA 서열을 기반으로 하는 MSId 방법 및 real-time PCR 법에 따라 균을 동정 하였다. 또한 설사 독소 유전자 및 구토 독소 유전자 검출을 위한 PCR을 실시하고 B. cereus 분리 균주에서 nheABC, bceT 및 entFM 설사 독소 유전자의 존재를 확인하였다. 임상적으로 중요한 몇 가지 식중독 세균들은 식품 미생물 검사 시 주목해야 할 필요가 있다. 특히, 이러한 식중독 세균들 중에서 B. cereus는 환경저항성 및 내열성이 강한 내생포자를 형성하고, 내열성 독소를 형성할 수도 있기 때문에 조리된 음식을 섭취해도 식중독을 일으킬 수 있다. 본 연구를 통해서 B. cereus등의 임상적으로 중요한 세균을 구별하는 것이 식품미생물 검사 시 중요함을 강조하고, 또한 법미생물 검사실에 앞으로 의뢰될 식품미생물 분석 사례들을 위해 B. cereus의 식별 및 세균의 독소 유전자 검출을 위한 지침을 제공 하고자 한다.

• The Plant Pathology Journal
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• 제35권3호
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• pp.200-207
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• 2019

Fusarium head blight (FHB) is a devastating wheat disease with a significant economic impact. Fhb1 is the most important large effect and stable QTL for FHB resistance. A pore-forming toxin-like (PFT) gene was recently identified as an underlying gene for Fhb1 resistance. In this study, we developed and validated a PFT-based Kompetitive allele specific PCR (KASP) marker for Fhb1. The KASP marker, PFT_KASP, was used to screen 298 diverse wheat breeding lines and cultivars. The KASP clustering results were compared with gelbased gene specific markers and the widely used linked STS marker, UMN10. Eight disagreements were found between PFT_KASP and UMN10 assays among the tested lines. Based on the genotyping and sequencing of genes in the Fhb1 region, these genotypes were found to be common with a previously characterized susceptible haplotype. Therefore, our results indicate that PFT_KASP is a perfect diagnostic marker for Fhb1 and would be a valuable tool for introgression and pyramiding of FHB resistance in wheat cultivars.

• BMB Reports
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• 제41권11호
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• pp.820-825
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• 2008

Bacillus thuringiensis Cyt2Aa toxin is a mosquito-larvicidal and cytolytic $\delta$-endotoxin, which is synthesized as a protoxin and forms crystalline inclusions within the cell. These inclusions are solubilized under alkaline conditions and are activated by proteases within the larval gut. In order to assess the functions of the N-and C-terminal regions of the protoxin, several N- and C-terminal truncated forms of Cyt2Aa were constructed. It was determined that amino acid removal at the N-terminal, which disrupts the $\beta$1 structure, might critically influence toxin production and inclusion formation. The deletion of 22 amino acids from the C-terminus reduced the production and solubility of the toxin. However, the removal of more than 22 amino acids from the C-terminus or the addition of a bulky group to this region could result in the inability of the protein to adopt the proper folding. These findings directly demonstrated the critical roles of N- and C-terminal amino acids on the production and folding of the B. thuringiensis cytolytic $\delta$-endotoxin.