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Gene Review

rpoB  -  DNA-directed RNA polymerase subunit beta

Mycobacterium tuberculosis CDC1551

 
 
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Disease relevance of rpoB

  • We assessed the performance of the Genotype MTBDR line probe assay that offers the simultaneous identification of Mycobacterium tuberculosis and its resistance to rifampin (RIF) and isoniazid (INH) by detecting the most commonly found mutations in the rpoB and katG genes [1].
  • In total, 16S rRNA or rpoB sequences were amplified from 15 sarcoidosis specimens (60%) but were not detected in any of the control tissues (p=0.00002, chi square) [2].
  • We describe an allele-specific PCR assay to detect mutations in three codons of the rpoB gene (516, 526, and 531) in Mycobacterium tuberculosis strains; mutations in these codons are reported to account for majority of M. tuberculosis clinical isolates resistant to rifampin (RIF), a marker of multidrug-resistant tuberculosis (MDR-TB) [3].
  • This SacI fragment also carried a portion of the rpoC gene located 43 bp downstream from the 3' end of the rpoB open reading frame; this organization is similar to that of the rpoBC operon of E. coli [4].
  • Mutations causing rifampin resistance in vegetative cells of Bacillus subtilis 168 have thus far been mapped to a rather restricted set of alterations at either Q469 or H482 within cluster I of the rpoB gene encoding the beta subunit of RNA polymerase [5].
 

High impact information on rpoB

 

Chemical compound and disease context of rpoB

  • New rpoB gene primers for detecting Rif(r) in Mycobacterium tuberculosis complex bacteria achieved 100% specificity and 88% (fresh sputa) and 92% (ethanol-preserved sputa) diagnostic sensitivity and detected up to 4 CFU/sample [9].
 

Biological context of rpoB

  • The Genotype MTBDR assay is designed to detect mutations within the 81-bp hotspot region of rpoB and mutations at katG codon 315 [10].
  • Few of the mutations at the rpoB locus could be correlated with the drug resistance levels exhibited by the M. tuberculosis isolates and occurred with frequencies different from those reported earlier [11].
  • Using PCR mutagenesis techniques, we introduced a specific rpoB point mutation (associated with clinical strains of rifampin-resistant M. tuberculosis) into the cloned M. tuberculosis rpoB gene and expressed this altered gene in the LR222 strain of M. smegmatis, which is susceptible to rifampin (MIC = 25 micrograms/ml) [4].
  • The M. tuberculosis rpoB gene was cloned into the shuttle plasmid pMV261 and electroporated into the LR223 strain of Mycobacterium smegmatis, which is highly resistant to rifampin (MIC > 200 micrograms/ml) [4].
  • The mutagenesis and expression strategy of the cloned M. tuberculosis rpoB gene that we have employed in this study will allow us to determine the rpoB mutations that are responsible for rifampin resistance in M. tuberculosis [4].
 

Associations of rpoB with chemical compounds

  • Sequencing of consecutive isolates identified by the National Tuberculosis Program showed 89% of isoniazid-resistant isolates could be detected by targeting just 2 codons, katG 315 and -15C-->T in the inhA promoter, while rifampin resistance will be more complex to detect, with many different mutation and insertion events in rpoB [12].
  • Optimized conditions included 0.3% methyl cellulose as the separation matrix, on-line staining using 1 micromol/L YOPRO1, and LIF detection for quantitative and reproducible analysis of single-base substitutions in the rifampin resistance-determining region of rpoB that give rise to the rifampin-resistant phenotype of M. tuberculosis [13].
  • The ddF protocol was performed on the amplified rpoB fragment with no preparatory steps, thus making ddF practical for laboratories equipped for polyacrylamide gel electrophoresis [14].
  • Upon RLB analysis of 315 clinical isolates from different countries, 132 (85.2%) of 155 RIF-resistant and 28 (51.0%) of 55 EMB-resistant isolates were correctly identified, showing applicability of the assay when targeting the rpoB hot-spot region and embB306 [15].
 

Analytical, diagnostic and therapeutic context of rpoB

References

  1. Performance of the Genotype MTBDR Line Probe Assay for Detection of Resistance to Rifampin and Isoniazid in Strains of Mycobacterium tuberculosis with Low- and High-Level Resistance. Brossier, F., Veziris, N., Truffot-Pernot, C., Jarlier, V., Sougakoff, W. J. Clin. Microbiol. (2006) [Pubmed]
  2. Molecular analysis of sarcoidosis tissues for mycobacterium species DNA. Drake, W.P., Pei, Z., Pride, D.T., Collins, R.D., Cover, T.L., Blaser, M.J. Emerging Infect. Dis. (2002) [Pubmed]
  3. Allele-specific rpoB PCR assays for detection of rifampin-resistant Mycobacterium tuberculosis in sputum smears. Mokrousov, I., Otten, T., Vyshnevskiy, B., Narvskaya, O. Antimicrob. Agents Chemother. (2003) [Pubmed]
  4. The rpoB gene of Mycobacterium tuberculosis. Miller, L.P., Crawford, J.T., Shinnick, T.M. Antimicrob. Agents Chemother. (1994) [Pubmed]
  5. The spectrum of spontaneous rifampin resistance mutations in the rpoB gene of Bacillus subtilis 168 spores differs from that of vegetative cells and resembles that of Mycobacterium tuberculosis. Nicholson, W.L., Maughan, H. J. Bacteriol. (2002) [Pubmed]
  6. Rapid prediction of rifampin susceptibility of Mycobacterium tuberculosis. Ohno, H., Koga, H., Kuroita, T., Tomono, K., Ogawa, K., Yanagihara, K., Yamamoto, Y., Miyamoto, J., Tashiro, T., Kohno, S. Am. J. Respir. Crit. Care Med. (1997) [Pubmed]
  7. Evolution of rifampin resistance in human immunodeficiency virus-associated tuberculosis. Nolan, C.M., Williams, D.L., Cave, M.D., Eisenach, K.D., el-Hajj, H., Hooton, T.M., Thompson, R.L., Goldberg, S.V. Am. J. Respir. Crit. Care Med. (1995) [Pubmed]
  8. Disequilibrium in distribution of resistance mutations among Mycobacterium tuberculosis Beijing and non-Beijing strains isolated from patients in Germany. Hillemann, D., Kubica, T., Rüsch-Gerdes, S., Niemann, S. Antimicrob. Agents Chemother. (2005) [Pubmed]
  9. Newly Developed Primers for Comprehensive Amplification of the rpoB Gene and Detection of Rifampin Resistance in Mycobacterium tuberculosis. Rigouts, L., Nolasco, O., de Rijk, P., Nduwamahoro, E., Van Deun, A., Ramsay, A., Arevalo, J., Portaels, F. J. Clin. Microbiol. (2007) [Pubmed]
  10. Evaluation of the Genotype MTBDR assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates. Cavusoglu, C., Turhan, A., Akinci, P., Soyler, I. J. Clin. Microbiol. (2006) [Pubmed]
  11. Molecular characterization of multidrug-resistant isolates of Mycobacterium tuberculosis from patients in North India. Siddiqi, N., Shamim, M., Hussain, S., Choudhary, R.K., Ahmed, N., Prachee, n.u.l.l., Banerjee, S., Savithri, G.R., Alam, M., Pathak, N., Amin, A., Hanief, M., Katoch, V.M., Sharma, S.K., Hasnain, S.E. Antimicrob. Agents Chemother. (2002) [Pubmed]
  12. Mutations prevalent among rifampin- and isoniazid-resistant Mycobacterium tuberculosis isolates from a hospital in Vietnam. Caws, M., Duy, P.M., Tho, D.Q., Lan, N.T., Hoa, D.V., Farrar, J. J. Clin. Microbiol. (2006) [Pubmed]
  13. Capillary electrophoresis-based heteroduplex analysis with a universal heteroduplex generator for detection of point mutations associated with rifampin resistance in tuberculosis. Thomas, G.A., Williams, D.L., Soper, S.A. Clin. Chem. (2001) [Pubmed]
  14. Genotypic detection of Mycobacterium tuberculosis rifampin resistance: comparison of single-strand conformation polymorphism and dideoxy fingerprinting. Felmlee, T.A., Liu, Q., Whelen, A.C., Williams, D., Sommer, S.S., Persing, D.H. J. Clin. Microbiol. (1995) [Pubmed]
  15. Multicenter evaluation of reverse line blot assay for detection of drug resistance in Mycobacterium tuberculosis clinical isolates. Mokrousov, I., Bhanu, N.V., Suffys, P.N., Kadival, G.V., Yap, S.F., Cho, S.N., Jordaan, A.M., Narvskaya, O., Singh, U.B., Gomes, H.M., Lee, H., Kulkarni, S.P., Lim, K.C., Khan, B.K., van Soolingen, D., Victor, T.C., Schouls, L.M. J. Microbiol. Methods (2004) [Pubmed]
  16. Rapid genotypic detection of rifampin- and isoniazid-resistant Mycobacterium tuberculosis directly in clinical specimens. Bang, D., Bengård Andersen, A., Thomsen, V.Ø. J. Clin. Microbiol. (2006) [Pubmed]
  17. Universal pattern of RpoB gene mutations among multidrug-resistant isolates of Mycobacterium tuberculosis complex from Africa. Schilke, K., Weyer, K., Bretzel, G., Amthor, B., Brandt, J., Sticht-Groh, V., Fourie, P.B., Haas, W.H. The international journal of tuberculosis and lung disease : the official journal of the International Union against Tuberculosis and Lung Disease. (1999) [Pubmed]
  18. Denaturing HPLC for high-throughput screening of rifampicin-resistant Mycobacterium tuberculosis isolates. Yip, C.W., Leung, K.L., Wong, D., Cheung, D.T., Chu, M.Y., Tang, H.S., Kam, K.M. Int. J. Tuberc. Lung Dis. (2006) [Pubmed]
  19. Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods. Isfahani, B.N., Tavakoli, A., Salehi, M., Tazhibi, M. Mem. Inst. Oswaldo Cruz (2006) [Pubmed]
 
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