Deciphering the role of a conserved hypothetical protein MSMEG 5850 a putative member of TETR family in the life cycle of mycobacterium smegmatis

Abstract

Mycobacteria has the ability to adapt and survive in adverse conditions. This adaptation is due to the collaborative action of transcriptional regulators enciphered by its genome. TetR Family transcriptional regulators are the most abundant HTH regulators in mycobacteria and the physiological role of the majority of them is still unspecified. In mycobacteria, TetR regulators characterized until now have been known to regulate various aspects of bacterial physiology. This study was carried out to characterize MSMEG_5850, a putative TetR family transcriptional regulator in Mycobacterium smegmatis. The gene MSMEG_5850 was knocked out using homologous recombination and its effect on local and global gene expression was studied using transcriptome analysis. Transcriptome analysis revealed the differential expression of 148 genes in the MSMEG_5850 knockout strain in comparison to the control. However, only fifty genes showed the presence of a binding motif of MSMEG_5850 in their upstream region stating that these genes might be under the regulation of MSMEG_5850. The pathway analysis of fifty target genes of MSMEG_5850 revealed them to be involved in different pathways indicating that MSMEG_5850 might be involved in global transcriptional regulation in Mycobacterium smegmatis. EMSA binding assay confirmed the specific and direct binding of 6His-MSMEG_5850 with its binding motif. Binding stoichiometry showed that MSMEG_5850 binds to its motif as a monomer. MSMEG_5850 was upregulated under nutritional, iron and oxidative stress, while no change in expression was found in the mid-log and stationary phases. Deletion of MSMEG_5850 resulted in altered colony morphology and size, growth pattern, decreased biofilm/pellicle formation and cell wall permeability. Altogether, this study indicates the plausible role of MSMEG_5850 in the global regulation in M. smegmatis and in the adaptation/survival of M. smegmatis under different stress conditions. newline