REGULATION OF THE INDUCIBLE ACETAMIDASE GENE OF MYCOBACTERIUM-SMEGMATIS

The inducible acetamidase of Mycobacterium smegmatis NCTC 8159 is expr essed at high levels in the presence of a suitable inducer, such as ac etamide, The gene and 1.5 kb of upstream sequence had previously been sequenced. A further 1.4 kb of upstream sequence has now been determin ed, containing an additional ORF on the opposite strand to the acetami dase gene. This ORF has significant homologies to genes encoding regul atory proteins involved in amidase expression in other organisms, Rest riction fragments from the 4 kb region were subcloned into a promoter- probe shuttle vector to locate the approximate region of the acetamida se promoter and investigate the mechanism of regulation. An inducible promoter was found to lie in the 1.4 kb region situated 1.5 kb upstrea m from the acetamidase coding region, Expression of the acetamidase wa s studied at the protein and mRNA levels, Using immunoblotting, induct ion of the enzyme was demonstrated in minimal medium containing succin ate plus acetamide, but not in a richer medium (Lemco broth) plus acet amide, confirming that regulation of acetamidase expression is mediate d by both positive and negative control elements. after induction by a cetamide, an increase above basal level could be detected after 1 h fo r both protein levels (using ELISA) and mRNA levels (using Northern bl ot analysis), indicating that control of expression is at the mRNA lev el, The size of the mRNA transcript detected was approximately 1.2 kb, the size of the acetamidase coding region. Since no promoter was iden tified immediately upstream of the coding region, this raises the poss ibility that a larger, primary transcript (possibly polycistronic) is cleaved to produce a stable form encoding the acetamidase protein.