• Journal of Microbiology and Biotechnology
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• v.13 no.6
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• pp.846-852
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• 2003

rpoB DNAs (279 bp) from 34 species of 5 actinomycete genera were sequenced and a phylogenetic tree was constructed based on the sequences obtained. The genera were clearly differentiated in the rpoB tree, forming clades specific to their respective genus. In addition, 2 signature amino acid residues specific to Streptomyces were found in a multiple alignment of the deduced amino acid sequences. To empirically confirm that this rpoB gene analysis system could be used to differentiate actinomycete isolates, the proposed system was used to identify 16 actinomycete isolates from Jeju Island. All isolates were successfully differentiated into the genera Streptomyces and Micromonospora. Accordingly, this is the first report that an rpoB sequence analysis has been effectively used to differentiate actinomycete strains at the genus level.

• International Journal of Oral Biology
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• v.38 no.1
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• pp.29-36
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• 2013

Mitis group streptococci (MGS) were classified based on the nucleotide sequences 16S rRNA gene (16S rDNA) and comprised 13 Streptococcus species. However, 16S rDNA homogeneity among MGS was too high to discriminate between clinical strains at the species level, notably between Streptococcus mitis, Streptococcus oralis, Streptococcus pneumoniae, and Streptococcus pseudopneumoniae. The purpose of this study was to discriminate between 37 strains of MGS isolated from Korean oral cavities using phylogenetic analysis of the DNA-dependant RNA polymerase beta-subunit gene (rpoB). 16S rDNA and rpoB from clinical strains of MGS were sequenced using the dideoxy chain termination method and analyzed using MEGA version 5 software. The resulting phylogenetic data showed that the rpoB sequences could delineate clinical strains of MGS at the species level. Phylogenetic analysis of rpoB is therefore a useful approach for identifying MGS at the species level.

• Proceedings of the Microbiological Society of Korea Conference
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• 2007.05a
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• pp.131-131
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• 2007

• Genomics & Informatics
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• v.12 no.4
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• pp.276-282
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• 2014

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.

• Biomedical Science Letters
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• v.22 no.4
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• pp.215-219
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• 2016

The problems of tuberculosis and its drug resistance are very severe. Therefore, rapid and accurate drug susceptibility assay is required. Recently, there has been an increased understanding of the genetic mechanism of Mycobacterium tuberculosis (MTB) drug resistance as well as advancement of molecular technologies. While many gene mutations correlate well with drug resistance, many genes do not show a strong correlation with drug resistance. For this reason, the current study assessed the utility of rpoB mRNA as a target to detect live mycobacteria. In this study, RT-PCR targeting of rpoB mRNA in BCG treated with rifampin was performed. Conventional RT-PCR and real-time PCR targeting rpoB mRNA as well as 85B mRNA was performed to determine whether these two methods could distinguish between viable and non-viable MTB. The levels of rpoB and 85B mRNA detected by RT- PCR were compared in parallel with colony forming unit counts of BCG that were treated with rifampin for different periods of time. The data suggests that that even though both mRNA levels of rpoB and 85B decreased gradually when rifampin-treatment increased, the rpoB mRNA seemed to represent live bacteria better than 85B mRNA. This study clearly indicates that RT-PCR is a good method to monitor viable cell counts in the liquid culture treated with the anti-tuberculosis drug.

• Journal of Microbiology
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• v.35 no.3
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• pp.177-180
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• 1997

The purpose of this study was to evaluate the efficiency of Line Probe Assay (LiPA) in detecting the rpoB gene mutation of clinically isolated Mycobacterium tuberculosis (MTB) and to compare the level of resistance to the various rifamycins with their mutation sites. The mutation in the rpoB gene was found in 84 (97.6%) out of 86 rifampicin (RMP) resistant strains as determined by LiPA. No mutation was observed in 2 RMP resistant strains and in any of 38 RMP susceptible strains tested. Only one of 3 strains with .DELTA.5/R5, one of 2 strains with .DELTA.3, and one of 3 strains with .DELTA.2/R2 LiPA profile showed a slightly lower level of resistance to the rifapentine than the other strains. Although we could not find correlations between mutation sites in the rpoB gene and the level of susceptibility to the various rifamycins, the LiPA is recommended as a fast screening tool for detection of RMP resistant MTB.

• Journal of Microbiology
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• v.34 no.3
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• pp.236-240
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• 1996

Rifampin is the most powerful drug for treating leprosy and tuberculosis today. It inhibits initiation and elongation of RNA transcription by binding to $\beta$-subunit of RNA polymerase, leading to kill mycobacteria. We isolated one variant strain of Mycobacterium leprae from 24 Korean leprosy patients who are less susceptible to rifampin or have suffered from relapse by polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP) of the rpoB gene. Direct sequencing of the rpoB region of M. leprae variant revealed missense mutations which altered the amino acids sequenceof RpoB to Ser-464, Arg-465, Arg-467 and Ala-468. This is the first finding on rpoB gene mutation of M. leprae from Korean patients ; moreover the mutant type was found to be different from the previously reported cases in other countries.

• Tuberculosis and Respiratory Diseases
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• v.80 no.3
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• pp.270-276
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• 2017

Background: A disputed rpoB mutation is a specific type of rpoB mutation that can cause low-level resistances to rifampin (RIF). Here, we aimed to assess the frequency and types of disputed rpoB mutations in Mycobacterium tuberculosis isolates from South Korea. Methods: Between August 2009 and December 2015, 130 patients exhibited RIF resistance on the MTBDRplus assay at Asan Medical Center. Among these cases, we identified the strains with disputed rpoB mutation by rpoB sequencing analysis, as well as among the M. tuberculosis strains from the International Tuberculosis Research Center (ITRC). Results: Among our cases, disputed rpoB mutations led to RIF resistance in at least 6.9% (9/130) of the strains that also exhibited RIF resistance on the MTBDRplus assay. Moreover, at the ITRC, sequencing of the rpoB gene of 170 strains with the rpoB mutation indicated that 23 strains (13.5%) had the disputed mutations. By combining the findings from the 32 strains from our center and the ITRC, we identified the type of disputed rpoB mutation as follows: CTG511CCG (L511P, n=8), GAC516TAC (D516Y, n=8), CTG533CCG (L533P, n=8), CAC526CTC (H526L, n=4), CAC526AAC (H526N, n=3), and ATG515GTG (M515V, n=1). Conclusion: Disputed rpoB mutations do not seem to be rare among the strains exhibiting RIF resistance in South Korea.

• Korean Journal of Microbiology
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• v.44 no.4
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• pp.333-338
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• 2008

Computational analysis of partial rpoB gene sequence (777 bp) was done in this study to identify B. anthracis and its closely related species B. cereus and B. thuringiensis. Sequence data including 17 B. anthracis strains, 9 B. cereus strains, and 7 B. thuringiensis strains were obtained by searching databases. Those sequences were aligned and used for other computational analysis. B. anthracis strains were identificated by in silico restriction enzyme digestion. B. cereus and B. thuringiensis were not segregated by this method. Those sequencing and BLAST search were required to distinguish the two. In actual identification tests, B. anthracis strains could be identified by PCR-RFLP, and B. cereus and B. thuringiensis strains were distinguished by BLAST search with reliable e-value. In this study fast and accurate method for identifying three Bacillus species, and flow chart of identification were developed.

• Tuberculosis and Respiratory Diseases
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• v.44 no.6
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• pp.1245-1255
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• 1997

Background : Rifampicin(RFP) is a key component of the antituberculous short-course chemotherapy and the RFP resistance is a marker of multi-drug resistant(MDR) tuberculosis. RPoB gene encodes the $\beta$-subunit of RNA polymerase of M. tuberculosis which is the target of RFP. And rpoB gene mutations are the cause of RFP resistance of M. tuberculosis. Although several reports showed that PCR-SSCP would be a rapid diagnostic method for identifying the RFP resistance, there were few reports Performed using direct, clinical specimens. So we Performed PCR-SSCP analysis of rpoB gene of M. tuberculosis in direct, clinical specimens. Methods : 75 clinical specimens were collected from patients at Asan Medical Center from June to August 1996. After PCR of IS 6110 fragments, 43 both AFB smear-positive and IS6110 fragment PCR-positive specimens were evaluated. The RFP susceptibility test was referred to the referral laboratory of the Korean Tuberculosis Institute. DNA was extracted by bead beater method. And heminested PCR was done using 0.1ul(1uCi) [$\alpha-^{32}P$]-dCTP. SSCP analysis was done using non-denaturating MDE gel electrophoresis. Results : The results of PCR of IS6110 fragments of M. tuberculosis were positive in 55(73%) cases of 75 AFB smear-positive clinical specimens. Of the 55 specimens, RFP susceptibility was confirmed in only 43 specimens. Of the 43 AFB smear-positive and IS6110 fragment-positive specimens, 29 were RFP susceptible and 14 were RFP resistant. All the RFP susceptible 29 strains showed the same mobility compared with that of RFP sensitive H37Rv in SSCP analysis of ropB gene. And all the other RFP resistant 13 strains showed the different mobility. In other words they showed 100% identical results between PCR-SSCP analysis and traditional susceptibility test. Conclusion : The PCR-sseP analysis of rpoB gene in direct clinical specimens could be used as a rapid diagnostic method for detecting RFP resistant M. tuberculosis.