Construction and characterization of a partial Mycobacterium tuberculosis cDNA library of genes expressed at reduced...

To determine which bacterial genes could be expressed during tuberculosis in the human body, we have prepared and characterized a collection of cDNA clones corresponding to genes that are expressed by Mycobacterium tuberculosis during in vitro growth in 5% (v/v) oxygen. These cDNA clones were obtained by purifying total RNA from M. tuberculosis and cloning small cDNA segments into Escherichia coli followed by removal of clones containing ribosomal RNA sequences. From approx. 1700 clones, a collection of 170 clones containing non-ribosomal inserts were further characterized by PCR amplification. Inserts of more than 180bp were verified by Southern hybridization to have corresponding loci in M. tuberculosis genomic DNA and their sequence was determined. We describe the genes that have been identified using this approach. Multiple independent cDNA clones were obtained for two genes, one probably encoding a stable structural RNA and the other a homologue of ferritin. RNA levels for these two genes were monitored during growth at 20% oxygen, 5% oxygen and in the nearly anaerobic culture sediments. No difference in expression levels was found at 5% oxygen compared to 20% oxygen. RNA levels for the ferritin homologue gene were significantly lower in culture sediments. The stable structural RNA, however, showed very high expression levels independently of culture conditions.