10 research outputs found
In vitro biosynthetic studies of bottromycin expand the enzymatic capabilities of the YcaO superfamily
The bottromycins belong to the ribosomally synthesized and posttranslationally modified peptide (RiPP) family of natural products. Bottromycins exhibit unique structural features, including a hallmark macrolactamidine ring and thiazole heterocycle for which divergent members of the YcaO superfamily have been biosynthetically implicated. Here we report the in vitro reconstitution of two YcaO proteins, BmbD and BmbE, responsible for the ATP-dependent cyclodehydration reactions that yield thiazoline- and macrolactamidine-functionalized products, respectively. We also establish the substrate tolerance for BmbD and BmbE and systematically dissect the role of the follower peptide, which we show serves a purpose similar to canonical leader peptides in directing the biosynthetic enzymes to the substrate. Lastly, we leverage the expanded capabilities of YcaO proteins to conduct an extensive bioinformatic survey to classify known YcaO chemistry. This analysis predicts new functions remain to be uncovered within the superfamily
antiSMASH 4.0—improvements in chemistry prediction and gene cluster boundary identification
Many antibiotics, chemotherapeutics, crop protection agents and food preservatives originate from molecules produced by bacteria, fungi or plants. In recent years, genome mining methodologies have been widely adopted to identify and characterize the biosynthetic gene clusters encoding the production of such compounds. Since 2011, the ‘antibiotics and secondary metabolite analysis shell—antiSMASH’ has assisted researchers in efficiently performing this, both as a web server and a standalone tool. Here, we present the thoroughly updated antiSMASH version 4, which adds several novel features, including prediction of gene cluster boundaries using the ClusterFinder method or the newly integrated CASSIS algorithm, improved substrate specificity prediction for non-ribosomal peptide synthetase adenylation domains based on the new SANDPUMA algorithm, improved predictions for terpene and ribosomally synthesized and post-translationally modified peptides cluster products, reporting of sequence similarity to proteins encoded in experimentally characterized gene clusters on a per-protein basis and a domain-level alignment tool for comparative analysis of trans-AT polyketide synthase assembly line architectures. Additionally, several usability features have been updated and improved. Together, these improvements make antiSMASH up-to-date with the latest developments in natural product research and will further facilitate computational genome mining for the discovery of novel bioactive molecules
An antibiotic from an uncultured bacterium binds to an immutable target
Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C 55PP, lipid II, and lipid III WTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses 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. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development. </p
A new antibiotic from an uncultured bacterium binds to an immutable target
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 bacterial 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 (C 55 PP, Lipid II, Lipid WTA ). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate, but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the irreversible 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
An antibiotic from an uncultured bacterium binds to an immutable target
Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C 55PP, lipid II, and lipid III WTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses 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. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development
Bioinformatic Expansion and Discovery of Thiopeptide Antibiotics
Thiopeptides
are members of the ribosomally synthesized and post-translationally
modified peptide family of natural products. Most characterized thiopeptides
display nanomolar potency toward Gram-positive bacteria by blocking
protein translation with several being produced at the industrial
scale for veterinary and livestock applications. Employing our custom
bioinformatics program, RODEO, we expand the thiopeptide family of
natural products by a factor of four. This effort revealed many new
thiopeptide biosynthetic gene clusters with products predicted to
be distinct from characterized thiopeptides and identified gene clusters
for previously characterized molecules of unknown biosynthetic origin.
To further validate our data set of predicted thiopeptide biosynthetic
gene clusters, we isolated and characterized a structurally unique
thiopeptide featuring a central piperidine and rare thioamide moiety.
Termed saalfelduracin, this thiopeptide displayed potent antibiotic
activity toward several drug-resistant Gram-positive pathogens. A
combination of whole-genome sequencing, comparative genomics, and
heterologous expression experiments confirmed that the thioamide moiety
of saalfelduracin is installed post-translationally by the joint action
of two proteins, TfuA and YcaO. These results reconcile the previously
unknown origin of the thioamide in two long-known thiopeptides, thiopeptin
and Sch 18640. Armed with these new insights into thiopeptide chemical-genomic
space, we provide a roadmap for the discovery of additional members
of this natural product family
Bioinformatic Expansion and Discovery of Thiopeptide Antibiotics
Thiopeptides
are members of the ribosomally synthesized and post-translationally
modified peptide family of natural products. Most characterized thiopeptides
display nanomolar potency toward Gram-positive bacteria by blocking
protein translation with several being produced at the industrial
scale for veterinary and livestock applications. Employing our custom
bioinformatics program, RODEO, we expand the thiopeptide family of
natural products by a factor of four. This effort revealed many new
thiopeptide biosynthetic gene clusters with products predicted to
be distinct from characterized thiopeptides and identified gene clusters
for previously characterized molecules of unknown biosynthetic origin.
To further validate our data set of predicted thiopeptide biosynthetic
gene clusters, we isolated and characterized a structurally unique
thiopeptide featuring a central piperidine and rare thioamide moiety.
Termed saalfelduracin, this thiopeptide displayed potent antibiotic
activity toward several drug-resistant Gram-positive pathogens. A
combination of whole-genome sequencing, comparative genomics, and
heterologous expression experiments confirmed that the thioamide moiety
of saalfelduracin is installed post-translationally by the joint action
of two proteins, TfuA and YcaO. These results reconcile the previously
unknown origin of the thioamide in two long-known thiopeptides, thiopeptin
and Sch 18640. Armed with these new insights into thiopeptide chemical-genomic
space, we provide a roadmap for the discovery of additional members
of this natural product family
Bioinformatic Expansion and Discovery of Thiopeptide Antibiotics
Thiopeptides
are members of the ribosomally synthesized and post-translationally
modified peptide family of natural products. Most characterized thiopeptides
display nanomolar potency toward Gram-positive bacteria by blocking
protein translation with several being produced at the industrial
scale for veterinary and livestock applications. Employing our custom
bioinformatics program, RODEO, we expand the thiopeptide family of
natural products by a factor of four. This effort revealed many new
thiopeptide biosynthetic gene clusters with products predicted to
be distinct from characterized thiopeptides and identified gene clusters
for previously characterized molecules of unknown biosynthetic origin.
To further validate our data set of predicted thiopeptide biosynthetic
gene clusters, we isolated and characterized a structurally unique
thiopeptide featuring a central piperidine and rare thioamide moiety.
Termed saalfelduracin, this thiopeptide displayed potent antibiotic
activity toward several drug-resistant Gram-positive pathogens. A
combination of whole-genome sequencing, comparative genomics, and
heterologous expression experiments confirmed that the thioamide moiety
of saalfelduracin is installed post-translationally by the joint action
of two proteins, TfuA and YcaO. These results reconcile the previously
unknown origin of the thioamide in two long-known thiopeptides, thiopeptin
and Sch 18640. Armed with these new insights into thiopeptide chemical-genomic
space, we provide a roadmap for the discovery of additional members
of this natural product family
Bioinformatic Expansion and Discovery of Thiopeptide Antibiotics
Thiopeptides
are members of the ribosomally synthesized and post-translationally
modified peptide family of natural products. Most characterized thiopeptides
display nanomolar potency toward Gram-positive bacteria by blocking
protein translation with several being produced at the industrial
scale for veterinary and livestock applications. Employing our custom
bioinformatics program, RODEO, we expand the thiopeptide family of
natural products by a factor of four. This effort revealed many new
thiopeptide biosynthetic gene clusters with products predicted to
be distinct from characterized thiopeptides and identified gene clusters
for previously characterized molecules of unknown biosynthetic origin.
To further validate our data set of predicted thiopeptide biosynthetic
gene clusters, we isolated and characterized a structurally unique
thiopeptide featuring a central piperidine and rare thioamide moiety.
Termed saalfelduracin, this thiopeptide displayed potent antibiotic
activity toward several drug-resistant Gram-positive pathogens. A
combination of whole-genome sequencing, comparative genomics, and
heterologous expression experiments confirmed that the thioamide moiety
of saalfelduracin is installed post-translationally by the joint action
of two proteins, TfuA and YcaO. These results reconcile the previously
unknown origin of the thioamide in two long-known thiopeptides, thiopeptin
and Sch 18640. Armed with these new insights into thiopeptide chemical-genomic
space, we provide a roadmap for the discovery of additional members
of this natural product family