ISOLATION OF THE GENE FOR THE BETA-SUBUNIT OF RNA-POLYMERASE FROM RIFAMPICIN-RESISTANT MYCOBACTERIUM-TUBERCULOSIS AND IDENTIFICATION OF NEWMUTATIONS

Tuberculosis (TB) is one of the most important infections worldwide, w ith an estimated incidence of 10 million active cases per year. Rifamp icin is a key component of the first-line therapy used in the treatmen t of tuberculosis. In Escherichia coli and Mycobacterium leprae, rifam picin has been shown to inhibit the beta subunit of RNA polymerase. Th e gene (rpoB) encoding this enzyme has been described in both species. We report the isolation of the homologous functional rifampicin resis tance gene from M. tuberculosis. A library was constructed with 15 to 25 kb BamHI-digested DNA fragments from a rifampicin-resistant M. tube rculosis clinical isolate that was ligated into an E. coli-mycobacteri al shuttle plasmid. Southern analysis of BamHI-digested DNA from 200 r ecombinant plasmids was performed and filters were hybridized to a 411 bp fragment of the beta subunit of RNA polymerase from M. tuberculosi s. Only DNA from one plasmid (#86) hybridized, which suggested that th e gene is found as a single copy per genome. This plasmid was able to transfer rifampicin resistance to sensitive M. smegmatis and thus code s for a functional genetic unit. Sequence analysis in the expected ''h otspot'' region in eight rifampicin-resistant M. tuberculosis strains (including one multidrug-resistant strain) revealed two novel mutation s as well as others previously described.