Biosynthesis of the redox cofactor mycofactocin is controlled by the transcriptional regulator MftR and induced by long-chain acyl-CoA species

Abstract

Mycofactocin (MFT) is a ribosomally synthesized and post-translationally-modified redox cofactor found in pathogenic mycobacteria. While MFT biosynthetic proteins have been extensively characterized, the physiological conditions under which MFT biosynthesis is required are not well understood. To gain insights into the mechanisms of regulation of MFT expression in Mycobacterium smegmatis mc 2 155, we investigated the DNA-binding and ligand-binding activities of the putative TetR-like transcription regulator, MftR. In this study, we demonstrated that MftR binds to the mft promoter region. We used DNase I footprinting to identify the 27 bp palindromic operator located 5′ to mftA and found it to be highly conserved in Mycobacterium tuberculosis , Mycobacterium bovis , Mycobacterium ulcerans , and Mycobacterium marinum . To determine under which conditions the mft biosynthetic gene cluster (BGC) is induced, we screened for effectors of MftR. As a result, we found that MftR binds to long-chain acyl-CoAs with low micromolar affinities. To demonstrate that oleoyl-CoA induces the mft BGC in vivo , we re-engineered a fluorescent protein reporter system to express an MftA–mCherry fusion protein. Using this mCherry fluorescent readout, we show that the mft BGC is upregulated in M. smegmatis mc 2 155 when oleic acid is supplemented to the media. These results suggest that MftR controls expression of the mft BGC and that MFT production is induced by long-chain acyl-CoAs. Since MFT-dependent dehydrogenases are known to colocalize with acyl carrier protein/CoA-modifying enzymes, these results suggest that MFT might be critical for fatty acid metabolism or cell wall reorganization.

S-Adenosylmethionine (SAM)-Dependent Methyltransferase MftM is Responsible for Methylation of the Redox Cofactor Mycofactocin

“…Criteria: (1) transcriptional upregulation in the presence of ethanol, 1 (2) proximity to oligosaccharide-modifying genes, or (3) proximity to genes associated with mycofactocin. MSMEG_6237 could be regulated by the recently characterized mycofactocin-system transcriptional regulator MftR, 6 the upstream region of MSMEG_6237 was searched for the proposed binding motif of MftR. However, no potential recognition site was found, indicating that the transcription of mftM in M. smegmatis might be regulated independently of mycofactocin biosynthesis.…”

“…tuberculosis in the absence of exogenous ethanol, thus pointing toward further roles of MFT in mycobacteria . Notably, induction of MFT biosynthesis genes by long-chain fatty acyl-CoA esters suggests an involvement in fatty acid metabolism …”

“…Curiously, the β-1,4-glucan of mycofactocins and its biosynthetic precursor AHDP-n exhibited further modification. The 2-hydroxyl group of the second glucose moiety was methylated in the majority of mycofactocin species, resulting in methylmycofactocins (MMFT- n , MMFT- n H 2 ). , This finding was surprising since no methyltransferase was encoded in the MFT biosynthetic gene cluster (BGC) of M. smegmatis.…”

“…5 Notably, induction of MFT biosynthesis genes by long-chain fatty acyl-CoA esters suggests an involvement in fatty acid metabolism. 6 The biosynthetic pathway to MFT comprises at least six gene products (MftA-F) (Figure 1A) and follows the logic of ribosomally synthesized and post-translationally modified peptide (RiPP) biosynthesis. 7,8 After translation by the ribosome, the precursor peptide MftA (MSMEG_RS06945) is bound by its chaperone MftB (MSMEG_1422) and modified at its C-terminus by the radical S-adenosylmethionine (rSAM) enzyme MftC (MSMEG_1423, Figure 1B).…”

“… 5 Notably, induction of MFT biosynthesis genes by long-chain fatty acyl-CoA esters suggests an involvement in fatty acid metabolism. 6 …”