Am. J. Respir. Cell Mol. Biol., Vol 11, No. 6, Dec 1994, 639-643.
Isolation of the gene for the beta subunit of RNA polymerase from rifampicin-resistant Mycobacterium tuberculosis and identification of new mutations
V Donnabella, F Martiniuk, D Kinney, M Bacerdo, S Bonk, B Hanna and WN Rom
Department of Medicine and Pathology, Bellevue Hospital Center, New York University Medical Center, New York 10016.
Tuberculosis (TB) is one of the most important infections worldwide, with
an estimated incidence of 10 million active cases per year. Rifampicin is a
key component of the first-line therapy used in the treatment of
tuberculosis. In Escherichia coli and Mycobacterium leprae, rifampicin has
been shown to inhibit the beta subunit of RNA polymerase. The gene (rpoB)
encoding this enzyme has been described in both species. We report the
isolation of the homologous functional rifampicin resistance gene from M.
tuberculosis. A library was constructed with 15 to 25 kb BamHI-digested DNA
fragments from a rifampicin-resistant M. tuberculosis clinical isolate that
was ligated into an E. coli-mycobacterial shuttle plasmid. Southern
analysis of BamHI-digested DNA from 200 recombinant plasmids was performed
and filters were hybridized to a 411 bp fragment of the beta subunit of RNA
polymerase from M. tuberculosis. Only DNA from one plasmid (#86)
hybridized, which suggested that the gene is found as a single copy per
genome. This plasmid was able to transfer rifampicin resistance to
sensitive M. smegmatis and thus codes for a functional genetic unit.
Sequence analysis in the expected "hotspot" region in eight rifampicin-
resistant M. tuberculosis strains (including one multidrug-resistant
strain) revealed two novel mutations as well as others previously
described.
