Abstract
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.
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Acknowledgements
Mr. Arunkumar Venkatesan would like to acknowledge the Indian Council for Medical Research (ICMR) for providing a Senior Research Fellowship. We thank Dr. Sameer Hassan and Mr. Yuvaraj for their help in bioinformatics analysis. We would also like to thank Mr. R. Senthilnathan for his technical help.
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Fig. S1
Removal of AmiA from P2 promoter. (A) AmiC removes AmiA from P2 promoter: EMSA was performed for P2 promoter with His-AmiA and AmiC. Lane 1, Labeled P2 promoter alone; Lane 2, addition of His-AmiA alone; lane 3 to 9, AmiC titration with increasing concentration (50, 75, 100, 200, 300, 400 and 500 nM). (GIF 1 kb).
Fig. S2
AmiC secondary structure. Secondary structure of AmiC was predicted using PSIPRED server. Rectangle boxes indicate alpha helixes and arrows indicate beta strands. * Represents the periplasmic binding protein domain region. (GIF 883 bytes).
Fig. S3
Graphical representation of probable AmiC binding motif. AmiC DNA binding motif from MEME analyses of P1, P3 and OP3 promoter/operator regions. (GIF 2 kb).
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Venkatesan, A., Palaniyandi, K. & Narayanan, S. Molecular characterization of AmiC, a positive regulator in acetamidase operon of Mycobacterium smegmatis . Cell Stress and Chaperones 23, 539–550 (2018). https://doi.org/10.1007/s12192-017-0861-2
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DOI: https://doi.org/10.1007/s12192-017-0861-2