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 aceta mide. A DNA fragment containing the start of the M. smegmatis hisD gen e was cloned in front of the promoter, such that the antisense message was produced. When this construct was induced in vivo, the bacteria b ecame phenotypically histidine auxotrophs; this auxotrophy was restore d by histidine supplementation. Auxotrophy was not observed under non- induced conditions. Antisense mutagenesis may be useful for observing the phenotypic inactivation of specific mycobacterial genes, and an in ducible system such as that described would allow the study of essenti al genes.