Molecular characterization of AmiC,a positive regulator in acetamidase operon of <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

Mycobacterium smegmatis, a rapidly growing non-pathogenic mycobacterium, is currently used as a model organism to study mycobacterial genetics. Acetamidase of M. smegmatis is the highly inducible enzyme of Mycobacteria, which utilizes several amide compounds as sole carbon and nitrogen sources. The acetamidase operon has a complex regulatory mechanism, which involves three regulatory proteins, four promoters, and three operator elements. In our previous study, we showed that over-expression of AmiA leads to a negative regulation of acetamidase by blocking the P2 promoter. In this study, we have identified a new positive regulatory protein, AmiC that interacts with AmiA through protein-protein interaction. Gel mobility shift assay showed that AmiC protein inhibits AmiA from binding to the P2 promoter. Interaction of AmiC with cis-acting elements identified its binding ability to multiple regulatory regions of the operon such as P3, OP3, and P1 promoter/operator. Consequently, the addition of inducer acetamide to AmiC complexe trips the complexes, causing AmiC to appear to be the sensory protein for the amides. Homology modeling and molecular docking studies suggest AmiC as a member of Periplasmic binding proteins, which preferentially bind to the inducers and not to the suppressor. Over-expression of AmiC leads to down-regulation of the negative regulator, amiA, and constitutive up-regulation of acetamidase. Based on these findings, we conclude that AmiC positively regulates the acetamidase operon.     

Mutations in the Gene for a Trna That Functions as a Regulator of a Transcriptional Attenuator in Bacillus Subtilis

It has been proposed that uncharged tRNA molecules may act as positive regulatory factors to control the expression of a number of operons in Bacillus subtilis and related bacteria by interacting with leader sequences to cause antitermination. In this study we report the isolation and characterization of regulatory mutations that modify one of the tRNA molecules predicted to have such a regulatory role. Three different alleles of the B. subtilis leucine tRNA gene leuG were found that resulted in higher expression of the ilv-leu biosynthetic operon. Each resulted in a base change in the D-loop of the leucine tRNA molecule with the anticodon 5''-GAG-3'' (leucine tRNA(GAG)). Experiments with strains that are diploid for mutant and wild-type alleles suggested that both charged and uncharged tRNA molecules may interact with leader sequences to control expression of the operon.