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

Mycobacteria produce several unusual cofactors that contribute to their metabolic versatility and capability to survive in different environments. Mycofactocin (MFT) is a redox cofactor involved in ethanol metabolism. The redox-active core moiety of mycofactocin is derived from the short precursor peptide MftA, which is modified by several maturases. Recently, it has been shown that the core moiety is decorated by a ß-1,4-glucan chain. Remarkably, the second glucose moiety of the oligosaccharide chain was found to be 2-O-methylated in Mycolicibacterium smegmatis. The biosynthetic gene responsible for this methylation, however, remained elusive, and no methyltransferase gene was part of the MFT biosynthetic gene cluster. Here, we applied reverse genetics to identify the gene product of MSMEG_6237 (mftM) as the SAM-dependent methyltransferase was responsible for methylation of the cofactor in M. smegmatis. According to metabolic analysis and comparative genomics, the occurrence of methylated MFT species was correlated with the presence of mftM homologues in the genomes of mycofactocin producers. This study revealed that the pathogen Mycobacterium tuberculosis does not methylate mycofactocins. Interestingly, mftM homologues co-occur with both mycofactocin biosynthesis genes as well as the putative mycofactocin-dependent alcohol dehydrogenase Mdo. We further showed that mftM knock-out mutants of M. smegmatis suffer from a prolonged lag phase when grown on ethanol as a carbon source. In addition, in vitro digestion of the glucose chain by cellulase suggested a protective function of glucan methylation. These results close an important knowledge gap and provide a basis for future studies into the physiological functions of this unusual cofactor modification.

Structure elucidation of the redox cofactor mycofactocin reveals oligo-glycosylation by MftF.

Mycofactocin (MFT) is a redox cofactor belonging to the family of ribosomally synthesized and post-translationally modified peptides (RiPPs) and is involved in alcohol metabolism of mycobacteria including Mycobacterium tuberculosis. A preliminary biosynthetic model had been established by bioinformatics and in vitro studies, while the structure of natural MFT and key biosynthetic steps remained elusive. Here, we report the discovery of glycosylated MFT by 13C-labeling metabolomics and establish a model of its biosynthesis in Mycolicibacterium smegmatis. Extensive structure elucidation including NMR revealed that MFT is decorated with up to nine ß-1,4-linked glucose residues including 2-O-methylglucose. Dissection of biosynthetic genes demonstrated that the oligoglycosylation is catalyzed by the glycosyltransferase MftF. Furthermore, we confirm the redox cofactor function of glycosylated MFTs by activity-based metabolic profiling using the carveol dehydrogenase LimC and show that the MFT pool expands during cultivation on ethanol. Our results will guide future studies into the biochemical functions and physiological roles of MFT in bacteria.

The antimicrobial activity of heterotrophic bacteria isolated from the marine sponge Erylus deficiens (Astrophorida, Geodiidae).

Interest in the study of marine sponges and their associated microbiome has increased both for ecological reasons and for their great biotechnological potential. In this work, heterotrophic bacteria associated with three specimens of the marine sponge Erylus deficiens, were isolated in pure culture, phylogenetically identified and screened for antimicrobial activity. The isolation of bacteria after an enrichment treatment in heterotrophic medium revealed diversity in bacterial composition with only Pseudoalteromonas being shared by two specimens. Of the 83 selected isolates, 58% belong to Proteobacteria, 23% to Actinobacteria and 19% to Firmicutes. Diffusion agar assays for bioactivity screening against four bacterial strains and one yeast, revealed that a high number of the isolated bacteria (68.7%) were active, particularly against Candida albicans and Vibrio anguillarum. Pseudoalteromonas, Microbacterium, and Proteus were the most bioactive genera. After this preliminary screening, the bioactive strains were further evaluated in liquid assays against C. albicans, Bacillus subtilis and Escherichia coli. Filtered culture medium and acetone extracts from three and 5 days-old cultures were assayed. High antifungal activity against C. albicans in both aqueous and acetone extracts as well as absence of activity against B. subtilis were confirmed. Higher levels of activity were obtained with the aqueous extracts when compared to the acetone extracts and differences were also observed between the 3 and 5 day-old extracts. Furthermore, a low number of active strains was observed against E. coli. Potential presence of type-I polyketide synthases (PKS-I) and non-ribosomal peptide synthetases (NRPSs) genes were detected in 17 and 30 isolates, respectively. The high levels of bioactivity and the likely presence of associated genes suggest that Erylus deficiens bacteria are potential sources of novel marine bioactive compounds.

Antimicrobial activity of heterotrophic bacterial communities from the marine sponge Erylus discophorus (Astrophorida, Geodiidae).

Heterotrophic bacteria associated with two specimens of the marine sponge Erylus discophorus were screened for their capacity to produce bioactive compounds against a panel of human pathogens (Staphylococcus aureus wild type and methicillin-resistant S. aureus (MRSA), Bacillus subtilis, Pseudomonas aeruginosa, Acinetobacter baumanii, Candida albicans and Aspergillus fumigatus), fish pathogen (Aliivibrio fischeri) and environmentally relevant bacteria (Vibrio harveyi). The sponges were collected in Berlengas Islands, Portugal. Of the 212 isolated heterotrophic bacteria belonging to Alpha- and Gammaproteobacteria, Actinobacteria and Firmicutes, 31% produced antimicrobial metabolites. Bioactivity was found against both Gram positive and Gram negative and clinically and environmentally relevant target microorganisms. Bioactivity was found mainly against B. subtilis and some bioactivity against S. aureus MRSA, V. harveyi and A. fisheri. No antifungal activity was detected. The three most bioactive genera were Pseudovibrio (47.0%), Vibrio (22.7%) and Bacillus (7.6%). Other less bioactive genera were Labrenzia, Acinetobacter, Microbulbifer, Pseudomonas, Gordonia, Microbacterium, Micrococcus and Mycobacterium, Paenibacillus and Staphylococcus. The search of polyketide I synthases (PKS-I) and nonribosomal peptide synthetases (NRPSs) genes in 59 of the bioactive bacteria suggested the presence of PKS-I in 12 strains, NRPS in 3 strains and both genes in 3 strains. Our results show the potential of the bacterial community associated with Erylus discophorus sponges as producers of bioactive compounds.