Comparison Of The Promoter Mutation Of Genes Between Mycobacterium Tuberculosis Strains H37Ra And H37Rv

Objective Comparative genomic study revealed mutations at the promoter regions of six genes (sec, pabB, phoH2, sigC, nrdH, lpdA) in avirulent Mycobacterium tuberculosis strain H37Ra compared to its virulent counterpart strain H37Rv. This study used mycobacterial promoter probe vector pMC210 to construct six genes'promoter probe vectors of strains H37Ra and H37Rv. This study used lacZ reporter gene to test whether those mutations will affect promoter activity and its potential relationship to virulence attenuation of H37Ra.Methods Promoter sequences of six genes were predicted by the"Neural Network Promoter Prediction"method (http://www.fruitfly.org/seq_tools/promoter.html). Promoter regions were amplified and inserted into mycobacterial promoter probe vector pMC210. Resultant recombinant plasmids were transformed into M. smegmatis by electroporation. This study assessed the strength of the promoter through determination ofβ-galactosidase activity. The transcription activity of lacZ in the control of cloned promoters was monitored by Quantitative Real-Time RT-PCR.Results The promoter sequences of sec, phoH2, sigC, nrdH which were submitted in the GenBank were probably controversial. Quantitative Real Time PCR or determination ofβ-galactosidase activity results showed that the promoter activity of H37Ra pabB was six times more than that of H37Rv pabB (p < 0.05), while the promoter activity of the H37Rv lpdA was two fold of that of H37Ra lpdA (p < 0.05).Conclusion The mutations in pabB and lpdA promoters affect its expression activity significantly. The T-A mutation in lpdA promoter may be related to virulence attenuation of H37Ra.