A previous study reported that the Mycobacterium smegmatis (Msm) protein MSMEG_2295 is a repressor controlling the expression of several genes, including that for MSMEG_5125, a putative isoprenoid binding protein belonging to the YceI family, and DinB2, a DNA damage repair enzyme. This repressor is encoded by the first gene of the operon that also expresses the gene for DinB2. Targeted inhibition of MSMEG_5125 using CRISPRi technology resulted in a significant loss of Msm’s respiratory activity and viability. Since this protein has been predicted to be an isoprenoid binding protein, we suspected a role of menaquinones, which are isoprenoid naphthoquinones, in the observed phenomenon. Accordingly, we tested whether MSMEG_5125’s deficiency-induced lethality could be reversed by adding menaquinone. The result was positive, implying cooperation between MSMEG_5125 and menaquinone in bringing about respiration. Inhibition of MSMEG_5125 expression led to the induction of MSMEG_0089 and 2296, two hallmark genes of the MSMEG_2295 regulon. This result suggests that when MSMEG_5125 becomes limiting, a feedback-loop derepresses the MSMEG_2295 regulon genes, including its own. Interestingly, menaquinone functioned as an inducer of MSMEG_5125, indicating that it is likely to mediate the feedback mechanism. This result also strengthens our hypothesis that the functions of menaquinone and MSMEG_5125 are interrelated. Menaquinone also induced the MSMEG_2295-controlled operon MSMEG_2295–2294 (dinB2) not induced following the inactivation of MSMEG_5125. Therefore, the activation mechanism of MSMEG_2295-regulated genes may not be the same for all, although derepression is likely to be a common feature. In vitro, menaquinone abolished MSMEG_2295’s DNA binding activity by interacting with it, confirming its role as an inducer. Therefore, a menaquinone–MSMEG_5125-regulated gene expression circuit controls Msm respiration and possibly oxidative stress-induced DNA damage repair.

In Mycobacterium smegmatis (renamed Mycolicibacterium smegmatis ), glucose 6-phosphate (G6P) level is exceptionally high as compared to other bacteria, E. coli for example. Earlier investigations have indicated that G6P protects M. smegmatis (Msm) against oxidative stress-inducing agents. G6P is a glycolytic intermediate formed either directly through the phosphorylation of glucose or indirectly via the gluconeogenic pathway. Its consumption is catalysed by several enzymes, one of which being the NADPH dependent G6P dehydrogenase (G6PDH) encoded by zwf (msmeg_0314). While investigating the extent to which the carbon sources glucose and glycerol influence Msm growth, we observed that intracellular concentration of G6P was lower in the former’s presence than the latter. We could correlate this difference with that in the growth rate, which was higher in glycerol than glucose. We also found that lowering of G6P content in glucose-grown cells was triggered by the induced expression of zwf and the resultant increase in G6PDH activity. When we silenced zwf using CRISPR-Cas9 technology, we observed a significant rise in the growth rate of Msm. Therefore, we have found that depletion of G6P in glucose-grown cells due to increased G6PDH activity is at least one reason why the growth rate of Msm in glucose is less than glycerol. However, we could not establish a similar link-up between slow growth in glucose and lowering of G6P level in the case of Mycobacterium tuberculosis (Mtb). Mycobacteria, therefore, may have evolved diverse mechanisms to ensure that they use glycerol preferentially over glucose for their growth.

A unique feature found in the genomes of mycobacteriophages such as L5 belonging to the A cluster is the presence of multiple dispersed repeated elements known as stoperators. The phage repressor binds these repeat elements, shutting off transcription globally and thereby promoting lysogeny. Interestingly, the sequence of these stoperators closely matches that of the consensus −35 region of prokaryotic promoters, leading us to propose that they may have a role to play in the initiation of transcription by serving as RNA polymerase binding sites. Mycobacteriophage D29 is closely related to phage L5, and their genome organizations are very similar. As in L5, there are multiple stoperators in the genome of D29. The positions occupied by the stoperators in the two genomes are almost identical. The significant difference between the two phages is that D29 lacks the gene encoding the equivalent of the L5 repressor. Since phage D29 does not produce a repressor, we considered it to be a suitable model for testing our hypothesis that the stoperators function as promoters in the absence of the repressor. To prove our point, we targeted CRISPR guide RNAs against six stoperators. In the case of five out of the six, we found a significant reduction in downstream gene expression and phage growth. Based on this observation and primer extension assays, we conclude that promoting gene expression is likely to be the primary function of stoperators.