ABSTRACT
Obafluorin is a Pseudomonas fluorescens antibacterial natural product that inhibits threonyl-tRNA synthetase (ThrRS). It acts as a broad-spectrum antibiotic against a range of clinically relevant pathogens and comprises a strained ß-lactone ring decorated with catechol and 4-nitro-benzyl moieties. The catechol moiety is widespread in nature and its role in the coordination of ferric iron has been well-characterised in siderophores and Trojan horse antibiotics. Here we use a combination of mutasynthesis, bioassays, enzyme assays and metal binding studies to delineate the role of the catechol moiety in the bioactivity of obafluorin. We use P. fluorescens biosynthetic mutants to generate obafluorin analogues with modified catechol moieties. We demonstrate that an intact catechol is required for both antibacterial activity and inhibition of the ThrRS molecular target. Although recent work showed that the obafluorin catechol coordinates Zn2+ in the ThrRS active site, we find that obafluorin is a weak Zn2+ binder in vitro, contrasting with a strong, specific 1 : 1 interaction with Fe3+. We use bioassays with siderophore transporter mutants to probe the role of the obafluorin catechol in Fe3+-mediated uptake. Surprisingly, obafluorin does not behave as a Trojan horse antibiotic but instead exhibits increased antibacterial activity in the presence of Fe3+. We further demonstrate that Fe3+ binding prevents the hydrolytic breakdown of the ß-lactone ring, revealing a hitherto unreported function for the catechol moiety in natural product bioactivity.
ABSTRACT
Formicamycins and their biosynthetic intermediates the fasamycins are polyketide antibiotics produced by Streptomyces formicae KY5 from a pathway encoded by the for biosynthetic gene cluster. In this work the ability of Streptomyces coelicolor M1146 and the ability of Saccharopolyspora erythraea Δery to heterologously express the for biosynthetic gene cluster were assessed. This led to the identification of eight new glycosylated fasamycins modified at different phenolic groups with either a monosaccharide (glucose, galactose, or glucuronic acid) or a disaccharide comprised of a proximal hexose (either glucose or galactose), with a terminal pentose (arabinose) moiety. In contrast to the respective aglycones, minimal inhibitory screening assays showed these glycosylated congeners lacked antibacterial activity.
Subject(s)
Galactose , Streptomyces coelicolor , Galactose/metabolism , Anti-Bacterial Agents/metabolism , Streptomyces coelicolor/genetics , Multigene Family , Glucose/metabolismABSTRACT
Prymnesium parvum is a toxin-producing microalga, which causes harmful algal blooms globally, frequently leading to massive fish kills that have adverse ecological and economic implications for natural waterways and aquaculture alike. The dramatic effects observed on fish are thought to be due to algal polyether toxins, known as the prymnesins, but their lack of environmental detection has resulted in an uncertainty about the true ichthyotoxic agents. Using qPCR, we found elevated levels of P. parvum and its lytic virus, PpDNAV-BW1, in a fish-killing bloom on the Norfolk Broads, United Kingdom, in March 2015. We also detected, for the first time, the B-type prymnesin toxins in Broads waterway samples and gill tissue isolated from a dead fish taken from the study site. Furthermore, Norfolk Broads P. parvum isolates unambiguously produced B-type toxins in laboratory-grown cultures. A 2 year longitudinal study of the Broads study site showed P. parvum blooms to be correlated with increased temperature and that PpDNAV plays a significant role in P. parvum bloom demise. Finally, we used a field trial to show that treatment with low doses of hydrogen peroxide represents an effective strategy to mitigate blooms of P. parvum in enclosed water bodies.
Subject(s)
Haptophyta , Animals , Fishes , Harmful Algal Bloom , Longitudinal Studies , United KingdomABSTRACT
Ribosomally synthesised and post-translationally modified peptides (RiPPs) are a structurally diverse class of natural product with a wide range of bioactivities. Genome mining for RiPP biosynthetic gene clusters (BGCs) is often hampered by poor annotation of the short precursor peptides that are ultimately modified into the final molecule. Here, we utilise a previously described genome mining tool, RiPPER, to identify novel RiPP precursor peptides near YcaO-domain proteins, enzymes that catalyse various RiPP post-translational modifications including heterocyclisation and thioamidation. Using this dataset, we identified a novel and diverse family of RiPP BGCs spanning over 230 species of Actinobacteria and Firmicutes. A representative BGC from Streptomyces albidoflavus J1074 (formerly known as Streptomyces albus) was characterised, leading to the discovery of streptamidine, a novel amidine-containing RiPP. This new BGC family highlights the breadth of unexplored natural products with structurally rare features, even in model organisms.
ABSTRACT
Correction for 'CuAAC click chemistry for the enhanced detection of novel alkyne-based natural product toxins' by Edward S. Hems et al., Chem. Commun., 2018, 54, 12234-12237.
ABSTRACT
In the context of discovering and quantifying terminal alkyne-based natural products, here we report the combination of CuAAC click chemistry with LC-MS for the detection of polyether toxins (prymnesins) associated with harmful algal blooms. The added-value of the CuAAC-based approach is evident from our ability to detect novel prymnesin-like compounds in algal species with previously uncharacterised toxins.
Subject(s)
Alkynes/chemistry , Click Chemistry , Lipoproteins/analysis , Mass Spectrometry , Toxins, Biological/analysis , Azides/chemistry , Catalysis , Chromatography, High Pressure Liquid , Copper/chemistry , Cycloaddition Reaction , Haptophyta/metabolism , Harmful Algal Bloom/physiologyABSTRACT
A suite of glycosylated glycerol derivatives representing various fragments of the glycosylated ichthyotoxins called prymnesins were chemically synthesised. Glycerol was used to represent a small fragment of the prymnesin backbone, and was glycosylated at the 2° position with the sugars currently reported to be present on prymnesin toxins. Neighbouring group participation was utilised to synthesise 1,2-trans-glycosides. SnCl2-promoted glycosylation with furanosyl fluorides gave 1,2-cis-furanosides with moderate stereocontrol, whilst TMSOTf promoted glycosylation with a furanosyl imidate gave a 1,2-cis-furanoside with good stereocontrol. The chemical synthesis of two larger glyceryl diglycoside fragments of prymnesin-1, glycosylated with α-Ê-arabinopyranose and α-á´ -ribofuranose, is also described. As the stereochemistry of the prymnesin backbones at this region is undefined, both the 2R- and 2S- glycerol isomers were synthesised. The separated diastereoisomers were distinguished by comparing NOESY NMR with computational models.
Subject(s)
Glycosides/chemical synthesis , Pyrans/chemistry , Glycosides/chemistry , Glycosylation , Models, Molecular , Molecular Structure , Polyketides , StereoisomerismABSTRACT
Prymnesium parvum is a toxin-producing microalga that causes harmful algal blooms globally, which often result in large-scale fish kills that have severe ecological and economic implications. Although many toxins have previously been isolated from P. parvum, ambiguity still surrounds the responsible ichthyotoxins in P. parvum blooms and the biotic and abiotic factors that promote bloom toxicity. A major fish kill attributed to P. parvum occurred in Spring 2015 on the Norfolk Broads, a low-lying set of channels and lakes (Broads) found on the East of England. Here, we discuss how water samples taken during this bloom have led to diverse scientific advances ranging from toxin analysis to discovery of a new lytic virus of P. parvum, P. parvum DNA virus (PpDNAV-BW1). Taking recent literature into account, we propose key roles for sialic acids in this type of viral infection. Finally, we discuss recent practical detection and management strategies for controlling these devastating blooms.
Subject(s)
Haptophyta/growth & development , Harmful Algal Bloom , Sugars , Animals , DNA/genetics , England , Fishes , Haptophyta/genetics , Haptophyta/metabolism , Haptophyta/virology , Toxins, Biological/metabolismABSTRACT
Sugar nucleotides are essential building blocks for the glycobiology of all living organisms. Detailed information on the types of sugar nucleotides present in a particular cell and how they change as a function of metabolic, developmental, or disease status is vital. The extraction, identification, and quantification of sugar nucleotides in a given sample present formidable challenges. In this chapter, currently used techniques for sugar nucleotide extraction from cells, separation from complex biological matrices, and detection by optical and mass spectrometry methods are discussed.