f Regulation of the inducible acetamidase gene of Mycobacterium smegmatis
- Authors: Tanya Parish†, Eshwar Mahenthiralingam, Philip Draper*, Elaine O. Davis, Elaine O. Colston
- *Author for correspondence: Philip Draper. Tel: + 44 181 959 3666 ext. 2346. Fax: +44 181 913 8528. e-mail: firstname.lastname@example.org
- Microbiology, July 1997 143: 2267-2276, doi: 10.1099/00221287-143-7-2267
- Subject: Genetics And Molecular Biology
- Published Online:
Summary: The inducible acetamidase of Mycobacterium smegmatis NCTC 8159 is expressed at high levels in the presence of a suitable inducer, such as acetamide. The gene and 1.5 kb of upstream sequence had previously been sequenced. A further 1.4 kb of upstream sequence has now been determined, containing an additional ORF on the opposite strand to the acetamidase gene. This ORF has significant homologies to genes encoding regulatory proteins involved in amidase expression in other organisms. Restriction fragments from the 4 kb region were subcloned into a promoter-probe shuttle vector to locate the approximate region of the acetamidase promoter and investigate the mechanism of regulation. An inducible promoter was found to lie in the 1.4 kb region situated 1.5 kb upstream from the acetamidase coding region. Expression of the acetamidase was studied at the protein and mRNA levels. Using immunoblotting, induction of the enzyme was demonstrated in minimal medium containing succinate plus acetamide, but not in a richer medium (Lemco broth) plus acetamide, confirming that regulation of acetamidase expression is mediated by both positive and negative control elements. After induction by acetamide, an increase above basal level could be detected after 1 h for both protein levels (using ELISA) and mRNA levels (using Northern blot analysis), indicating that control of expression is at the mRNA level. The size of the mRNA transcript detected was approximately 1.2 kb, the size of the acetamidase coding region. Since no promoter was identified immediately upstream of the coding region, this raises the possibility that a larger, primary transcript (possibly polycistronic) is cleaved to produce a stable form encoding the acetamidase protein.
Present address: Clinical Sciences, London School of Hygiene & Tropical Medicine, Keppel Street, London WC1E 7HT, UK.
© Society for General Microbiology 1997 | Published by the Microbiology Society
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