The mycobacteria comprise both pathogenic and nonpathogenic bacteria. Although several features related to pathogenicity in various mycobacterial species, such as , have been studied in great detail, methylotrophy, i.e., the ability of an organism to utilize single-carbon (C) compounds as the sole source of carbon and energy, has remained largely unexplored in mycobacteria. Reports are available that suggest that mycobacteria, including and, are capable of utilizing alternative C compounds to meet their carbon and energy requirements. However, physiological pathways that are functional in mycobacteria to utilize such carbon compounds are only poorly understood. Here we report the identification and characterization of the gene products required for establishing methylotrophy in We present,-dimethyl--nitrosoaniline (NDMA)-dependent methanol oxidase (Mno) as the key enzyme that is essential for the growth of on methanol. We show that Mno has both methanol and formaldehyde dehydrogenase activities Further, is able to utilize methanol even in the absence of the major formaldehyde dehydrogenase MscR, which suggests that Mno is sufficient to dissimilate methanol and the resulting formaldehyde Finally, we show that devoid of phosphoenolpyruvate carboxykinase, which has been shown to fix CO in , does not grow on methanol, suggesting that the final step of methanol utilization requires CO fixation for biomass generation. Our work here thus forms the first comprehensive report that explores methylotrophy in a mycobacterial species. Methylotrophy, the ability to utilize single-carbon (C) compounds as the sole carbon and energy sources, is only poorly understood in mycobacteria. Both pathogenic and nonpathogenic mycobacteria, including , are capable of utilizing C compounds to meet their carbon and energy requirements, although the precise pathways are not well studied. Here we present a comprehensive study of methylotrophy in With several genetic knockouts, we have dissected the entire methanol metabolism pathway in We show that while methanol dissimilation in differs from that in other mycobacterial species, the concluding step of CO fixation is similar to that in It is therefore both interesting and important to examine mycobacterial physiology in the presence of alternative carbon sources.

Accumulation of formaldehyde, a highly reactive molecule, in the cell is toxic, and requires detoxification for the organism's survival. Mycothiol-dependent formaldehyde dehydrogenase or S-nitrosomycothiol reductase (MscR) from Mycobacterium smegmatis and Mycobacterium tuberculosis was previously known for detoxifying formaldehyde and protecting the cell against nitrosative stress. We here show that M. smegmatis MscR exhibits a mycothiolindependent formaldehyde dehydrogenase (FDH) activity in vitro. Presence of zinc in the reaction enhances MscR activity, thus making it a zinc-dependent FDH. Interestingly, MscR utilizes only formaldehyde and no other primary aldehydes as its substrate in vitro, and M. smegmatis lacking mscR (ΔmscR)shows sensitivity exclusively toward formaldehyde. Bioinformatics analysis of MscRs from various bacteria reveals 10 positionally conserved cysteines, whose importance in structural stability and biological activity is not yet investigated. To explore the significance of these cysteines, we generated MscR single Cys variants by systematically replacing each cysteine with serine. All of the Cys variants except C39S and C309S are unable to show a complete rescue of ΔmscR on formaldehyde, show a significant loss of enzymatic activity in vitro, pronounced structural alterations as probed by circular dichroism, and loss of homotetramerization on size exclusion chromatography. Our data thus reveal the importance of intact cysteines in the structural stability and biological activity of MscR, which is a dedicated FDH in M. smegmatis, and shows ~84% identity with M. tuberculosis MscR. We believe that this knowledge will further help in the development of FDH as a potential drug target against M. tuberculosis infections.

Transcriptional factors such as the TetR family of transcriptional regulators (TFTRs) are widely found amongst bacteria, including mycobacteria, and are accountable for their survival. Here, we characterized a novel TFTR, Ms6244, from Mycobacterium smegmatis that negatively autoregulates its expression and represses its neighbouring gene, Ms6243. We also report the binding of Ms6244 to the inverted repeats in the intergenic region of Ms6244 and Ms6243. Further, an Ms6244-deleted strain shows various morphophysiological differences compared to the wild type. We further confirmed that the deletion of Ms6244 itself and not the resultant Ms6243 overexpression is the cause of the altered physiology. Our data thus suggest that Ms6244 is an essential regulator, having far-reaching effects on M. smegmatis physiology.