Regulation of the inducible acetamidase gene of Mycobacterium smegmatis

Abstract

The inducible acetamidase of Mycobacterium smegmatis NCTC 81 59 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 14 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. Afier 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.1

Modulation of Mycobacterium tuberculosis proliferation by MtrA, an essential two‐component response regulator

“…We reasoned that one approach to understand the function of MtrA would be to elevate its intracellular levels in M. tuberculosis and evaluate the consequences associated with elevated protein levels on the growth of the virulent M. tuberculosis strain under different conditions. We chose Mycobacterium smegmatis -derived inducible amidase promoter to elevate the intracellular levels of MtrA (Parish et al ., 1997;Triccas et al ., 1998). Our preliminary experiments revealed that this promoter was constitutively active in M. tuberculosis (data not shown).…”

Molecular Dissection of Phage Endolysin

“…This vector was further modified by replacing hsp60 promoter cassette with an ϳ2.6-kb PCR-amplified upstream region of formamidase gene from M. smegmatis to yield pMV261Acet vector (kindly provided by Manika I. Singh, IISER Bhopal). This cloning, although followed by the published method (30), will be detailed elsewhere. This vector has been utilized for the expression of all the constructs in M. smegmatis upon induction by acetamide; acetamide-mediated induction has been shown to be very useful for the controlled expression of proteins in mycobacteria (31,32).…”

Regulation of the SigH stress response regulon by an essential protein kinase in Mycobacterium tuberculosis

“…To confirm that SigH is phosphorylated in M. tuberculosis containing native levels of PknB, we made a strain in which M. tuberculosis sigH fused to the FLAG epitope sequence at the carboxyl-terminus was expressed under the control of the inducible acetamidase promoter (15,16). Total protein was extracted from this sigH-FLAG-expressing strain, separated by 2D GE, immunoblotted with anti-FLAG antibody, stripped, and then reprobed with phospho-T specific antibody.…”

“…To determine whether RshA is phosphorylated in vivo in M. tuberculosis, we made an M. tuberculosis strain in which M. tuberculosis rshA fused to the FLAG epitope tag sequence was expressed under the control of the inducible acetamidase promoter (15,16). Total protein was extracted from this rshA-FLAG-expressing strain, separated by 2D GE using a linear pI range 5-8 isoelectric focusing (IEF) strip, and immunoblotted with anti-FLAG antibody.…”

“…To search for proteins phosphorylated by PknB, we made an M. bovis bacillus Calmette-Guérin strain in which a copy of M. tuberculosis pknB is expressed under the control of the inducible acetamidase promoter (15,16). A combination of 2 dimensional gel electrophoresis (2D GE), immunoblot analysis with a phospho-threonine (phospho-T)-specific antibody and mass spectrometry were used to identify…”