Mycobacterial pathogenesis is hallmarked by lipidic polyketides that decorate the cell envelope and mediate infection. However, factors mediating persistence remain largely unknown. Dynamic cell wall remodeling could facilitate the different pathogenic phases. Recent studies have implicated type III polyketide synthases (PKSs) in cell wall alterations in several bacteria. Comparative genome analysis revealed several type III pks gene clusters in mycobacteria. In this study, we report the functional characterization of two novel type III PKSs, MMAR_2470 and MMAR_2474, in Mycobacterium marinum. These type III pkss belong to a unique pks genomic cluster conserved exclusively in pathogenic mycobacteria. Cell-free reconstitution assays and high-resolution mass spectrometric analyses revealed methylated polyketide products in independent reactions of both proteins. MMAR_2474 protein exceptionally biosynthesized methylated alkyl-resorcinol and methylated acyl-phloroglucinol products from the same catalytic core. Structure-based homology modeling, product docking, and mutational studies identified residues that could facilitate the distinctive catalysis of these proteins. Functional investigations in heterologous mycobacterial strain implicated MMAR_2474 protein to be vital for mycobacterial survival in stationary biofilms. Our investigations provide new insights into type III PKSs conserved in pathogenic mycobacterial species.

O-methylation of small molecules is a common modification widely present in most organisms. Type III polyketides undergo O-methylation at hydroxyl end to play a wide spectrum of roles in bacteria, plants, algae, and fungi. Mycobacterium marinum harbours a distinctive genomic cluster with a type III pks gene and genes for several polyketide modifiers including a methyltransferase gene, mmar_2193. This study reports functional analyses of MMAR_2193 and reveals multi-methylating potential of the protein. Comparative sequence analyses revealed conservation of catalytically important motifs in MMAR_2193 protein. Homology-based structure-function and molecular docking studies suggested type III polyketide cores as possible substrates for MMAR_2193 catalysis. In vitro enzymatic characterization revealed the capability of MMAR_2193 protein to utilize diverse polyphenolic substrates to methylate several hydroxyl positions on a single substrate molecule. High-resolution mass spectrometric analyses identified multi-methylations of type III polyketides in cell-free reconstitution assays. Notably, our metabolomics analyses identified some of these methylated molecules in biofilms of wild type Mycobacterium marinum. This study characterizes a novel mycobacterial O-methyltransferase protein with multi-methylating enzymatic ability that could be exploited to generate a palette of structurally distinct bioactive molecules.