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

Microbialites from the Freshwater System of Cuatro Ciénegas, Mexico: Genomic, Molecular Organic, and Stable Isotopic Perspectives

Modern microbialites are carbonate-precipitating microbial mats and represent the closest living analogues to ancient stromatolites. These ancient carbonate formations are the oldest fossil evidence of life on Earth; however, our comprehension of their relationship to early earth ecosystems relies heavily on understanding the formation of modern microbialites. Research regarding these formation processes has suggested that chemical constraints of CaCO 3 precipitation vary on sub-millimeter spatial scales within the living microbial community. In an attempt to shed light on the importance of these chemical microenvironments, this study focused on understanding the spatial distribution of the organisms and processes involved in the formation of modern microbialites. This was accomplished by isolating five visually distinct layers from the upper 2 – 3 cm of an actively forming microbialite found in the freshwater system of Cuatro Ciénegas, Mexico. Each layer was analyzed using genomic, molecular organic, and stable isotopic techniques. Bacterial diversity was determined by 16S rRNA gene analyses, lipid biomarker content was detected by GC-MS, and carbon isotope composition of organic matter and CaCO 3 were used as indicators of specific microbial processes. Results of the 16S rRNA gene analysis showed that there is little overlap in the community composition of individual layers. Approximately 90% of the ribotypes identified in the microbialite were unique to a single layer. Furthermore, the relative accretion of CaCO 3 at each layer was used to connect the distribution of organisms and processes with two specific zones of CaCO 3 precipitation. The first zone of CaCO3 accretion, which accounted for approximately 55% of total CaCO 3 accumulation, is found in the surface two layers of the microbialites and dominated by photoautotrophic cyanobacteria and algae. The second zone of CaCO 3 precipitation, found at the interior (layers 4 and 5), is composed primarily of heterotrophic proteobacteria and dominated by sulfate-reducing !-proteobacteria. The lipid content of the microbialite reflected the community structure as determined by genomics. Numerous photosynthetic biomarkers were detected and decreased in abundance with depth, indicating the important function of heterotrophic degradation. Additionally, the detection of sulfurized phytol compounds in layer 5 highlighted an important mechanism for the preservation of biogenic signatures, and reflected both the abundance of phototrophic organisms and sulfatereducing bacteria. In combination, these interdisciplinary analyses provided an understanding of microbial community composition and metabolism while indicating the spatial relationship to CaCO 3 formation and the preservation of distinct biochemical signatures.

Synthesis and Stereochemical Determination of the Peptide Antibiotic Novo29.

This paper describes the synthesis and stereochemical determination of Novo29 (clovibactin), a new peptide antibiotic that is related to teixobactin and is active against Gram-positive bacteria. Novo29 is an eight-residue depsipeptide that contains the noncanonical amino acid hydroxyasparagine of hitherto undetermined stereochemistry in a macrolactone ring. The amino acid building blocks Fmoc-(2R,3R)-hydroxyasparagine-OH and Fmoc-(2R,3S)-hydroxyasparagine-OH were synthesized from (R,R)- and (S,S)-diethyl tartrate. Novo29 and epi-Novo29 were then prepared by solid-phase peptide synthesis using these building blocks. Correlation with an authentic sample of Novo29 through 1H NMR spectroscopy, LC-MS, and in vitro antibiotic activity established that Novo29 contains (2R,3R)-hydroxyasparagine. X-ray crystallography reveals that epi-Novo29 adopts an amphiphilic conformation, with a hydrophobic surface and a hydrophilic surface. Four sets of epi-Novo29 molecules pack in the crystal lattice to form a hydrophobic core. The macrolactone ring adopts a conformation in which the main-chain amide NH groups converge to create a cavity, which binds ordered water and acetate anion. The amphiphilic conformation of epi-Novo29 is reminiscent of the amphiphilic conformation adopted by the related antibiotic teixobactin and its derivatives, which contains a hydrophobic surface that interacts with the lipids of the bacterial cell membrane and a hydrophilic surface that interacts with the aqueous environment. Molecular modeling suggests that Novo29 can adopt an amphiphilic conformation similar to teixobactin, suggesting that Novo29 may interact with bacteria in a similar fashion to teixobactin

Spatially Resolved Genomic, Stable Isotopic, and Lipid Analyses of a Modern Freshwater Microbialite from Cuatro Ciénegas, Mexico

Microbialites are biologically mediated carbonate deposits found in diverse environments worldwide. To explore the organisms and processes involved in microbialite formation, this study integrated genomic, lipid, and both organic and inorganic stable isotopic analyses to examine five discrete depth horizons spanning the surface 25 mm of a modern freshwater microbialite from Cuatro Ciénegas, Mexico. Distinct bacterial communities and geochemical signatures were observed in each microbialite layer. Photoautotrophic organisms accounted for approximately 65% of the sequences in the surface community and produced biomass with distinctive lipid biomarker and isotopic (δ13C) signatures. This photoautotrophic biomass was efficiently degraded in the deeper layers by heterotrophic organisms, primarily sulfate-reducing proteobacteria. Two spatially distinct zones of carbonate precipitation were observed within the microbialite, with the first zone corresponding to the phototroph-dominated portion of the microbialite and the second zone associated with the presence of sulfate-reducing heterotrophs. The coupling of photoautotrophic production, heterotrophic decomposition, and remineralization of organic matter led to the incorporation of a characteristic biogenic signature into the inorganic CaCO3 matrix. Overall, spatially resolved multidisciplinary analyses of the microbialite enabled correlations to be made between the distribution of specific organisms, precipitation of carbonate, and preservation of unique lipid and isotopic geochemical signatures. These findings are critical for understanding the formation of modern microbialites and have implications for the interpretation of ancient microbialite records

Biosynthesis and Mechanism of Action of the Cell Wall Targeting Antibiotic Hypeptin

Hypeptin is a cyclodepsipeptide antibiotic produced by Lysobacter sp. K5869, isolated from an environmental sample by the iChip technology, dedicated to the cultivation of previously uncultured microorganisms. Hypeptin shares structural features with teixobactin and exhibits potent activity against a broad spectrum of gram-positive pathogens. Using comprehensive in vivo and in vitro analyses, we show that hypeptin blocks bacterial cell wall biosynthesis by binding to multiple undecaprenyl pyrophosphate-containing biosynthesis intermediates, forming a stoichiometric 2:1 complex. Resistance to hypeptin did not readily develop in vitro. Analysis of the hypeptin biosynthetic gene cluster (BGC) supported a model for the synthesis of the octapeptide. Within the BGC, two hydroxylases were identified and characterized, responsible for the stereoselective beta-hydroxylation of four building blocks when bound to peptidyl carrier proteins. In vitro hydroxylation assays corroborate the biosynthetic hypothesis and lead to the proposal of a refined structure for hypeptin

Biosynthesis and Mechanism of Action of the Cell Wall Targeting Antibiotic Hypeptin

Hypeptin is a cyclodepsipeptide antibiotic produced by Lysobacter sp. K5869, isolated from an environmental sample by the iChip technology, dedicated to the cultivation of previously uncultured microorganisms. Hypeptin shares structural features with teixobactin and exhibits potent activity against a broad spectrum of gram-positive pathogens. Using comprehensive in vivo and in vitro analyses, we show that hypeptin blocks bacterial cell wall biosynthesis by binding to multiple undecaprenyl pyrophosphate-containing biosynthesis intermediates, forming a stoichiometric 2:1 complex. Resistance to hypeptin did not readily develop in vitro. Analysis of the hypeptin biosynthetic gene cluster (BGC) supported a model for the synthesis of the octapeptide. Within the BGC, two hydroxylases were identified and characterized, responsible for the stereoselective beta-hydroxylation of four building blocks when bound to peptidyl carrier proteins. In vitro hydroxylation assays corroborate the biosynthetic hypothesis and lead to the proposal of a refined structure for hypeptin