Project description:The archaeal mevalonate pathway is a recently discovered modified version of the eukaryotic mevalonate pathway. This pathway is widely conserved in archaea, except for some archaeal lineages possessing the eukaryotic or other modified mevalonate pathways. Although the pathway seems almost exclusive to the domain Archaea, the whole set of homologous genes of the pathway is found in the metagenome-assembled genome sequence of an uncultivated bacterium, Candidatus Promineifilum breve, of the phylum Chloroflexota. To prove the existence of the archaea-specific pathway in the domain Bacteria, we confirmed the activities of the enzymes specific to the pathway, phosphomevalonate dehydratase and anhydromevalonate phosphate decarboxylase, because only these two enzymes are absent in closely related Chloroflexota bacteria that possess a different type of modified mevalonate pathway. The activity of anhydromevalonate phosphate decarboxylase was evaluated by carotenoid production via the archaeal mevalonate pathway reconstituted in Escherichia coli cells, whereas that of phosphomevalonate dehydratase was confirmed by an in vitro assay using the recombinant enzyme after purification and iron-sulfur cluster reconstruction. Phylogenetic analyses of some mevalonate pathway-related enzymes suggest an evolutionary route for the archaeal mevalonate pathway in Candidatus P. breve, which probably involves horizontal gene transfer events.IMPORTANCEThe recent discovery of various modified mevalonate pathways in microorganisms, such as archaea and Chloroflexota bacteria, has shed light on the complexity of the evolution of metabolic pathways, including those involved in primary metabolism. The fact that the archaeal mevalonate pathway, which is almost exclusive to the domain Archaea, exists in a Chloroflexota bacterium provides valuable insights into the molecular evolution of the mevalonate pathways and associated enzymes. Putative genes probably involved in the archaeal mevalonate pathway have also been found in the metagenome-assembled genomes of Chloroflexota bacteria. Such genes can contribute to metabolic engineering for the bioproduction of valuable isoprenoids because the archaeal mevalonate pathway is known to be an energy-saving metabolic pathway that consumes less ATP than other mevalonate pathways do.
Project description:Antimicrobial resistance is a leading mortality factor worldwide. Here we report the discovery of clovibactin, a new antibiotic, isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positivebacterial pathogens without detectable resistance. Using biochemical assays,solid-state NMR, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, Lipid II, LipidWTA). Clovibactin uses anunusual hydrophobic interface to tightly wrap aroundpyrophosphate, butbypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups.Uncultured bacteria offer a rich reservoir of antibiotics with new mechanisms of action that could replenish the antimicrobial discovery pipeline.
Project description:This experiment aims on the identification of serine hydrolases from a complex thermophile community that live in a hot vent in Kamchatka Peninsula based on in vivo labelling with FP-alkyne directly in the hot spring and subsequent analysis using metagenomics/metaproteomics. To this end, sediment samples were collected and treated using the following three conditions. DMSO- treated control FP-alkyne labelled Samples for each condition were prepared in triplicate, resulting a total number of 6 samples per spring. Labelling was performed using 4 µM of the probe FP-alkyne and incubation for 2 h in the hot spring.