The respiratory lipoquinone, menaquinone, functions as an inducer of genes regulated by the Mycobacterium smegmatis repressor MSMEG_2295

Abstract

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.

Roles for mycobacterial DinB2 in frameshift and substitution mutagenesis

“…In addition, recent studies revealed that the dinB2 expression is under the control of at least two TetR family proteins (Msmeg_6564 and Msmeg_2295), and their deletion and/or overexpression induce dinB2 expression ( Yang et al, 2012 ; Patra et al, 2021 ). Menaquinone, a respiratory lipoquinone, induces dinB2 expression more than 10-fold, by both inhibiting the Msmeg_2295 repression and acting as a direct inducer ( Barman et al, 2022 ). Other experimental conditions will need to be tested to assess the impact of dinB2 deletion on mutagenesis in mycobacteria, including during infection or after menaquinone treatment, to confirm the phenotypes demonstrated here which are derived from induced expression of DinB2.…”

Pleiotropic roles for mycobacterial DinB2 in frameshift and substitution mutagenesis

“…In addition, recent studies revealed that the dinB2 expression is under the control of at least two TetR family proteins (Msmeg_6564 and Msmeg_2295), and their deletion and/or overexpression induce dinB2 expression (Yang et al , 2012; Patra et al , 2021). Menaquinone, a respiratory lipoquinone, induces dinB2 expression more than 10-fold, by both inhibiting the Msmeg_2295 repression and acting as a direct inducer (Barman et al , 2022). Other experimental conditions will need to be tested to assess the impact of dinB2 deletion on mutagenesis in mycobacteria, including during infection or after menaquinone treatment.…”