Development and use of a conditional antisense mutagenesis system in mycobacteria

Expression from a 2.3 kb region upstream of the inducible acetamidase gene from Mycobacterium smegmatis was shown to be upregulated by acetamide. A DNA fragment containing the start of the M. smegmatis hisD gene was cloned in front of the promoter, such that the antisense message was produced. When this construct was induced in vivo, the bacteria became phenotypically histidine auxotrophs; this auxotrophy was restored by histidine supplementation. Auxotrophy was not observed under non-reduced conditions. Antisense mutagenesis may be useful for observing the phenotypic inactivation of specific mycobacterial genes and an reducible system such as that described would allow the study of essential genes.