The Mycobacterium tuberculosis katG promoter region contains a novel upstream activator

An Escherichia coli-mycobacterial shuttle vector, pJCluc, containing a luci ferase reporter gene, was constructed and used to analyse the Mycobacterium tuberculosis katG promoter. A 1.9 kb region immediately upstream of katG p romoted expression of the luciferase gene in E. coil and Mycobacterium smeg matis. A smaller promoter fragment (559 bp) promoted expression with equal efficiency, and was used in all further studies. Two transcription start si tes were mapped by primer extension analysis to 47 and 56 bp upstream of th e GTG initiation codon. Putative promoters associated with these show simil arity to previously identified mycobacterial promoters. Deletions in the pr omoter fragment, introduced with BAL-31 nuclease and restriction endonuclea ses, revealed that a region between 559 and 448 bp upstream of the translat ion initiation codon, designated the upstream activator region (UAR). is es sential for promoter activity in E, coli, and is required for optimal activ ity in M. smegmatis. The katG UAR was also able to increase expression from the Mycobacterium paratuberculosis P-AN promoter 15-fold in E. coil and 12 -fold in M. smegmatis. An alternative promoter is active in deletion constr ucts in which either the UAR or the katG promoters identified here are abse nt. Expression from the katG promoter peaks during late exponential phase, and declines during stationary phase. The promoter is induced by ascorbic a cid, and is repressed by oxygen limitation and growth at elevated temperatu res. The promoter constructs exhibited similar activities in Mycobacterium bovis BCC as they did in M. smegmatis.