남조세균 흔들말목(Cyanobacteria, Oscillatoriales) 해양 균주의 16S rRNA와 rpoB 유전자 변이

천주용,이민아,기장서,Cheon, Ju-Yong,Lee, Min-Ah,Ki, Jang-Seu 한국미생물학회 2012 미생물학회지 Vol.48 No.4

본 연구는 남조세균 흔들말목(Cyanobacteria, Oscillatoriales)의 16S ribosomal RNA (rRNA) 및 RNA polymerase beta subunit(rpoB) 유전자를 대상으로 염기서열 변이 및 분자계통학적 특성을 분석한 것이다. 흔들말목 rpoB 유전자는 16S rRNA보다 유전자 변이(유전거리: rpoB=0.270, 16S=0.109)가 큰 것으로 조사되었으며, 통계적으로 유의한 차이를 보였다(Student t-test, p<0.001). 흔들말목 16S rRNA와 rpoB의 계통분석에서 유사한 계통 분지형태를 보였으며, rpoB 유전자가 높은 해상도를 갖고 있어 흔들말목 분류군을 더 명확하게 구분하였다. 또한, parsimony 분석을 통해 rpoB 유전자가 16S rRNA 보다 2.40배 빠르게 진화하는 것으로 파악되었다. 본 연구결과는 rpoB 유전자가 흔들말목의 분자계통 및 종 분류 연구에 매우 유용하다는 것을 제시해 준다. In this study, we investigated molecular divergences and phylogenetic characteristics of the 16S ribosomal RNA (rRNA) and RNA polymerase beta subunit (rpoB) gene sequences from the order Oscillatoriales (Cyanobacteria). The rpoB of Oscillatoriales showed higher genetic divergence when compared with those of 16S rRNA (p-distance: rpoB=0.270, 16S=0.109), and these differences were statistically significant (Student t-test, p<0.001). Phylogenetic trees of 16S rRNA and rpoB were generally compatible; however, rpoB tree clearly separated the compared Oscillatoriales taxa, with higher phylogenetic resolution. In addition, parsimony analyses showed that rpoB gene evolved 2.40-fold faster than 16S rRNA. These results suggest that the rpoB is a useful gene for the molecular phylogenetics and species discrimination in the order Oscillatoriales.

RpoB<SUB>127-135</SUB> Peptide Derived from Mycobacterium tuberculosis is Processed and Presented to HLA-A*0201 Restricted CD8+ T Cells via an Alternate HLA-I Processing Pathway

Jang-Eun Cho,Sang-Nae Cho,Sungae Cho 대한의생명과학회 2014 Biomedical Science Letters Vol.20 No.4

Mycobacterium tuberculosis (MTB) resides and replicates inside macrophages. In our previous report, we reported that CD8+ T cell-mediated immune responses specific for the peptide derived from MTB RNA polymerase beta-subunit (RpoB127-135) could be induced in TB patients expressing HLA-A*0201 subtype. In order to examine whether RpoB127-135 specific CD8+ T cells can recognize MTB infected macrophages in vitro, CD8+ T cell lines specific for RpoB127-135 peptide were generated from peripheral blood mononuclear cells (PBMCs) of healthy HLA-A*0201 subjects by in vitro immunization technique. In this study, we observed RpoB127-135 specific CD8+ T cells could recognize and destroy macrophages infected with MTB for 2 to 4 days. RpoB127-135 specific CD8+ T cell immune response was inducible from PBMC of healthy subjects expressing HLA-A*0206 subtype, one of HLA-A2 supertype members. Next, we investigated the HLA-I processing mechanism of RpoB127-135 peptide in MTB infected macrophages. As a result, the presentation of the MTB derived epitope peptide, RpoB127-135, to CD8+ T cells was not inhibited by the treatment with brefeldin-A (ER-Golgi transport inhibitor) or lactacystin (proteasome inhibitor), which blocks the classical HLA-I processing pathway. However, RpoB127-135 specific CD8+ T cell activity was blocked either by the blocking agent for the endocytosis (cytochalasin D) or by the blocking antibody (W6/32) for HLA-I molecules. Therefore, the RpoB127-135 peptide may be processed by accessing the alternate HLA-I processing pathway. Understanding the processing and presentation mechanisms of the MTB derived proteins will help to improve the efficacy of vaccines and the efficiency of therapeutic agents for TB.

Understanding Rifampicin Resistance in Tuberculosis through a Computational Approach

Satish Kumar,Lingaraja Jena 한국유전체학회 2014 Genomics & informatics Vol.12 No.4

The disease tuberculosis, caused by Mycobacterium tuberculosis (MTB), remains a major cause of morbidity and mortality indeveloping countries. The evolution of drug-resistant tuberculosis causes a foremost threat to global health. Mostdrug-resistant MTB clinical strains are showing resistance to isoniazid and rifampicin (RIF), the frontline anti-tuberculosisdrugs. Mutation in rpoB, the beta subunit of DNA-directed RNA polymerase of MTB, is reported to be a major cause of RIFresistance. Amongst mutations in the well-defined 81-base-pair central region of the rpoB gene, mutation at codon 450(S450L) and 445 (H445Y) is mainly associated with RIF resistance. In this study, we modeled two resistant mutants of rpoB(S450L and H445Y) using Modeller9v10 and performed a docking analysis with RIF using AutoDock4.2 and compared thedocking results of these mutants with the wild-type rpoB. The docking results revealed that RIF more effectively inhibited thewild-type rpoB with low binding energy than rpoB mutants. The rpoB mutants interacted with RIF with positive bindingenergy, revealing the incapableness of RIF inhibition and thus showing resistance. Subsequently, this was verified bymolecular dynamics simulations. This in silico evidence may help us understand RIF resistance in rpoB mutant strains.

Understanding Rifampicin Resistance in Tuberculosis through a Computational Approach

Kumar, Satish,Jena, Lingaraja Korea Genome Organization 2014 Genomics & informatics Vol.12 No.4

The disease tuberculosis, caused by Mycobacterium tuberculosis (MTB), remains a major cause of morbidity and mortality in developing countries. The evolution of drug-resistant tuberculosis causes a foremost threat to global health. Most drug-resistant MTB clinical strains are showing resistance to isoniazid and rifampicin (RIF), the frontline anti-tuberculosis drugs. Mutation in rpoB, the beta subunit of DNA-directed RNA polymerase of MTB, is reported to be a major cause of RIF resistance. Amongst mutations in the well-defined 81-base-pair central region of the rpoB gene, mutation at codon 450 (S450L) and 445 (H445Y) is mainly associated with RIF resistance. In this study, we modeled two resistant mutants of rpoB (S450L and H445Y) using Modeller9v10 and performed a docking analysis with RIF using AutoDock4.2 and compared the docking results of these mutants with the wild-type rpoB. The docking results revealed that RIF more effectively inhibited the wild-type rpoB with low binding energy than rpoB mutants. The rpoB mutants interacted with RIF with positive binding energy, revealing the incapableness of RIF inhibition and thus showing resistance. Subsequently, this was verified by molecular dynamics simulations. This in silico evidence may help us understand RIF resistance in rpoB mutant strains.

Application of Multilocus Sequence Analysis (MLSA) for Accurate Identification of Legionella spp. Isolated from Municipal Fountains in Chengdu, China, Based on 16S rRNA, mip, and rpoB Genes

Wang Guan,Ying Xu,Da-li Chen,Jia-nan Xu,Yu Tian,Jian-ping Chen 한국미생물학회 2012 The journal of microbiology Vol.50 No.1

Legionellosis (Legionnaires’ disease; LD) is a form of severe pneumonia caused by species of Legionella bacteria. Because inhalation of Legionella-contaminated aerosol is considered the major infection route, routine assessments of potential infection sources such as hot water systems, air-conditioner cooling water, and municipal fountains are of great importance. In this study, we utilized in vitro culture and multilocus sequence analysis (MLSA) targeting 16S rRNA, mip, rpoB, and mip-rpoB concatenation to isolate and identify Legionella spp. from 5 municipal fountains in Chengdu City, Sichuan Province, China. Our results demonstrated that 16S rRNA was useful for initial identification, as it could recognize isolates robustly at the genus level, while the genes mip, rpoB, and mip-rpoB concatenation could confidently discriminate Legionella species. Notably, the three subspecies of L. pneumophila could be distinguished by the analysis based on rpoB. The serotyping result of strain CD-1 was consistent with genetic analysis based on the concatenation of mip and rpoB. Despite regular maintenance and sanitizing methods, 4 of the 5 municipal fountains investigated in this study were positive for Legionella contamination. Thus, regularly scheduled monitoring of municipal fountains is urgently needed as well as vigilant disinfection. Although the application of MLSA for inspection of potential sites of infection in public areas is not standard procedure, further investigations may prove its usefulness.

객담에서 중합효소연쇄반응과 Line Probe Assay를 이용한 Rifampin 내성 결핵균의 직접검출법

이미애,이미애 대한임상병리학회 1998 대한임상병리학회지 Vol.18 No.1

Evaluation of the Diversity of Cyclodextrin-Producing Paenibacillus graminis Strains Isolated from Roots and Rhizospheres of Different Plants by Molecular Methods

Renata Estebanez Vollu,Rafael Fogel,Silvia Cristina Cunha dos Santos,Fabio Faria da Mota,Lucy Seldin 한국미생물학회 2006 The journal of microbiology Vol.44 No.6

To address the diversity of cyclodextrin-producing P. graminis strains isolated from wheat roots and rhizospheres of maize and sorghum sown in Australia, Brazil, and France, restriction fragment length polymorphism analysis of part of genes encoding RNA polymerase (rpoB-RFLP) and DNA gyrase subunit B (gyrB-RFLP) was used to produce genetic fingerprints. A phylogenetic tree based on rpoB gene sequences was also constructed. The isolates originated from Brazil could be separated from those from Australia and France, when data from the rpoB-based phylogenetic tree or gyrB-RFLP were considered. These analyses also allowed the separation of all P. graminis strains studied here into four clusters; one group formed by the strains GJK201 and RSA19T, second group formed by the strains MC22.02 and MC04.21, third group formed by the strains TOD61, TOD 221, TOD302, and TOD111, and forth group formed by all strains isolated from plants sown in Cerrado soil, Brazil. As this last group was formed by strains isolated from sorghum and maize sown in the same soil (Cerrado) in Brazil, our results suggest that the diversity of these P. graminis strains is more affected by the soil type than the plant from where they have been isolated.

한국산 둥굴레속 식물의 형태적 특성 및 엽록체 DNA 염기서열을 이용한 유연관계 분석

김정훈,서재완,변지희,안영섭,차선우,조준형 한국약용작물학회 2014 한국약용작물학회지 Vol.22 No.6

Polygonatum is a genus placed in the family Liliaceae, distributed throughout the Northern Hemisphere and16 of the species are grown naturally in Korea. In oriental medicine, the rhizomes of Polygonatum have been used as two differentmedicines, Okjuk (Polygonati odorati Rhizoma) and Hwangjeong (Polygonati Rhizoma). However, it is difficult toidentify the morphological and chemical differences between the medicinal groups and thus easy to confuse the one with theother. Therefore, a clear classification standard needs to be established so as to be able to discriminate between them. In thestudy, the morphological characteristics of the plants, Polygonatum spp., were examined. Then, the differences in SNPsamong the DNA sequences of 7 of the Polygonatum spp. and 1 of the Disporum spp. were analyzed by DNA barcoding withrpoC1, rpoB2, matK, and psbA-trnH of the cpDNA region. In the results, three regions, rpoC1, rpoB2, and matK were usefulfor discriminating the species, P. stenophyllum and P. sibiricum. Furthermore, it was possible to discriminate the individualgermplasm within the species by using the combination of the results obtained from rpoB2, rpoC1, and matK.

한국산 둥굴레속 식물의 형태적 특성 및 엽록체 DNA 염기서열을 이용한 유연관계 분석

김정훈,서재완,변지희,안영섭,차선우,조준형 한국약용작물학회 2014 韓國藥用作物學會誌 Vol.22 No.6

Polygonatum is a genus placed in the family Liliaceae, distributed throughout the Northern Hemisphere and 16 of the species are grown naturally in Korea. In oriental medicine, the rhizomes of Polygonatum have been used as two different medicines, Okjuk (Polygonati odorati Rhizoma) and Hwangjeong (Polygonati Rhizoma). However, it is difficult to identify the morphological and chemical differences between the medicinal groups and thus easy to confuse the one with the other. Therefore, a clear classification standard needs to be established so as to be able to discriminate between them. In the study, the morphological characteristics of the plants, Polygonatum spp., were examined. Then, the differences in SNPs among the DNA sequences of 7 of the Polygonatum spp. and 1 of the Disporum spp. were analyzed by DNA barcoding with rpoC1, rpoB2, matK, and psbA-trnH of the cpDNA region. In the results, three regions, rpoC1, rpoB2, and matK were useful for discriminating the species, P. stenophyllum and P. sibiricum. Furthermore, it was possible to discriminate the individual germplasm within the species by using the combination of the results obtained from rpoB2, rpoC1, and matK.

Evaluation of the Diversity of Cyclodextrin-Producing Paenibacillus graminis Strains Isolated from Roots and Rhizospheres of Different Plants by Molecular Methods

Vollu Renata Estebanez,Fogel Rafael,Santos Silvia Cristina Cunha dos,Mota Fabio Faria da,Seldin Lucy The Microbiological Society of Korea 2006 The journal of microbiology Vol.44 No.6

To address the diversity of cyclodextrin-producing P. graminis strains isolated from wheat roots and rhizospheres of maize and sorghum sown in Australia, Brazil, and France, restriction fragment length polymorphism analysis of part of genes encoding RNA polymerase (rpoB-RFLP) and DNA gyrase subunit B (gyrB-RFLP) was used to produce genetic fingerprints. A phylogenetic tree based on rpoB gene sequences was also constructed. The isolates originated from Brazil could be separated from those from Australia and France, when data from the rpoB-based phylogenetic tree or gyrB-RFLP were considered. These analyses also allowed the separation of all P. graminis strains studied here into four clusters; one group formed by the strains GJK201 and $RSA19^T$, second group formed by the strains MC22.02 and MC04.21, third group formed by the strains TOD61, TOD 221, TOD302, and TOD111, and forth group formed by all strains isolated from plants sown in Cerrado soil, Brazil. As this last group was formed by strains isolated from sorghum and maize sown in the same soil (Cerrado) in Brazil, our results suggest that the diversity of these P. graminis strains is more affected by the soil type than the plant from where they have been isolated.