DNA binding and gene regulatory functions of MSMEG_2295, a repressor encoded by the dinB2 operon of Mycobacterium smegmatis

Abstract

MSMEG_2295 is a TetR family protein encoded by the first gene of a Mycobacterium smegmatis (Msm) operon that expresses the gene for DinB2 (MSMEG_2294), a translesion DNA repair enzyme. We have carried out investigations to understand its function by performing DNA binding studies and gene knockout experiments. We found that the protein binds to a conserved inverted repeat sequence located upstream of the dinB2 operon and several other genes. Using a knockout of MSMEG_2295, we show that MSMEG_2295 controls the expression of at least five genes, the products of which could potentially influence carbohydrate and fatty acid metabolism as well as antibiotic and oxidative stress resistance. We have demonstrated that MSMEG_2295 is a repressor by performing complementation analysis. Knocking out of MSMEG_2295 had a significant impact on pyruvate metabolism. Pyruvate dehydrogenase activity was virtually undetectable in ΔMSMEG_2295, although in the complemented strain, it was high. We also show that knocking out of MSMEG_2295 causes resistance to H2O2, reversed in the complemented strain. We have further found that the mycobacterial growth inhibitor plumbagin, a compound of plant origin, acts as an inducer of MSMEG_2295 regulated genes. We, therefore, establish that MSMEG_2295 functions by exerting its role as a repressor of multiple Msm genes and that by doing so, it plays a vital role in controlling pyruvate metabolism and response to oxidative stress.

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

“…2b, c). These two genes have been previously reported to be tightly regulated by MSMEG_2295 [12]. This result indicates that reduced expression of MSMEG_5125 leads to induced expression of genes under the control of the repressor MSMEG_2295.…”

“…Therefore, the induction mechanism of MSMEG_2296 expression may be different from that of dinB2 , although it is a part of the MSMEG_2295 regulon. We also note that MSMEG_0089 was moderately induced () in this assay although using an MSMEG_2295 knockout mutant we had previously observed about a 100-fold increase [12]…”

“…To test this hypothesis, we examined how the presence of vitamin K2 affects the operator binding activity of MSMEG_2295. An EMSA experiment () was performed using the MSMEG_2295 binding site between the dinB2 operon and the divergently expressed gene MSMEG_2295 [12] (). The results show that in the presence of vitamin K2 the formation of the DNA bound complex was abolished, indicating that vitamin K2 can bind MSMEG_2295, thereby inactivating it ().…”

“…MSMEG_2295 is a TetR family protein encoded by the dinB2 operon of Mycobacterium smegmatis (Msm) [12, 13]. In Escherichia coli , DinB, the counterpart of Msm DinB2, functions as a translesion DNA repair enzyme.…”

Mycobacteriophage D29 induced association of Mycobacterial RNA polymerase with ancillary factors leads to increased transcriptional activity

“…The promoter P hsp60 controls the expression from the hsp60 operon and P tetR that from the dinB2 operon of Msm. The promoter is referred here as P tetR as the first gene of the operon MSMEG_2295 is a TetR family protein [31]. Similarly, D29 promoters like P site1 and P site7 were end labelled with Cy5 (the primer sequences are mentioned in Table S2a and Cy5 labelled amplicons in Table S2b).…”