A bacterial quorum-sensing precursor induces mortality in the marine coccolithophore, Emiliania huxleyi

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Frontiers in Microbiology 7 (2016): 59, doi:10.3389/fmicb.2016.00059.Interactions between phytoplankton and bacteria play a central role in mediating biogeochemical cycling and food web structure in the ocean. However, deciphering the chemical drivers of these interspecies interactions remains challenging. Here, we report the isolation of 2-heptyl-4-quinolone (HHQ), released by Pseudoalteromonas piscicida, a marine gamma-proteobacteria previously reported to induce phytoplankton mortality through a hitherto unknown algicidal mechanism. HHQ functions as both an antibiotic and a bacterial signaling molecule in cell–cell communication in clinical infection models. Co-culture of the bloom-forming coccolithophore, Emiliania huxleyi with both live P. piscicida and cell-free filtrates caused a significant decrease in algal growth. Investigations of the P. piscicida exometabolome revealed HHQ, at nanomolar concentrations, induced mortality in three strains of E. huxleyi. Mortality of E. huxleyi in response to HHQ occurred slowly, implying static growth rather than a singular loss event (e.g., rapid cell lysis). In contrast, the marine chlorophyte, Dunaliella tertiolecta and diatom, Phaeodactylum tricornutum were unaffected by HHQ exposures. These results suggest that HHQ mediates the type of inter-domain interactions that cause shifts in phytoplankton population dynamics. These chemically mediated interactions, and other like it, ultimately influence large-scale oceanographic processes.This research was support through funding from the Gordon and Betty Moore Foundation through Grant GBMF3301 to MJ and TM; NIH grant from the National Institute of Allergy and Infectious Disease (NIAID – 1R21Al119311-01) to TM and KW; the National Science Foundation (OCE – 1313747) and US National Institute of Environmental Health Science (P01-ES021921) through the Oceans and Human Health Program to BM. Additional financial support was provided to TM from the Flatley Discovery Lab

Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase-thioesterase enzyme pair

Author Posting. © The Author(s), 2016. This is the author's version of the work. It is posted here by permission of National Academy of Sciences for personal use, not for redistribution. The definitive version was published in Proceedings of the National Academy of Sciences of United States of America 113 (2016): 3797-3802, doi: 10.1073/pnas.1519695113.Halogenated pyrroles (halopyrroles) are common chemical moieties found in bioactive bacterial natural products. The halopyrrole moieties of mono- and di- halopyrrole-containing compounds arise from a conserved mechanism in which a proline-derived pyrrolyl group bound to a carrier protein is first halogenated then elaborated by peptidic or polyketide extensions. This paradigm is broken during the marine pseudoalteromonad bacterial biosynthesis of the coral larval settlement cue tetrabromopyrrole (1), which arises from the substitution of the proline-derived carboxylate by a bromine atom. To understand the molecular basis for decarboxylative bromination in the biosynthesis of 1, we sequenced two Pseudoalteromonas genomes and identified a conserved four-gene locus encoding the enzymes involved its complete biosynthesis. Through total in vitro reconstitution of the biosynthesis of 1 using purified enzymes and biochemical interrogation of individual biochemical steps, we show that all four bromine atoms in 1 are installed by the action of a single flavin-dependent halogenase- Bmp2. Tetrabromination of the pyrrole induces a thioesterase-mediated offloading reaction from the carrier protein and activates the biosynthetic intermediate for decarboxylation. Insights into the tetrabrominating activity of Bmp2 were obtained from the high-resolution crystal structure of the halogenase contrasted against structurally homologous halogenase Mpy16 that forms only a dihalogenated pyrrole in marinopyrrole biosynthesis. Structure-guided mutagenesis of the proposed substrate-binding pocket of Bmp2 led to a reduction in the degree of halogenation catalyzed. Our study provides a biogenetic basis for the biosynthesis of 1, and sets a firm foundation for querying the biosynthetic potential for the production of 1 in marine (meta)genomes.This work was jointly supported by the US National Science Foundation (OCE-1313747) and the US National Institute of Environmental Health Sciences (P01-ES021921) through the Ocean and Human Health Program to B.S.M., and the US National Institute of Allergy and Infectious Disease R01-AI47818 to B.S.M. and R21- AI119311 to K.E.W. and T.J.M., the Mote Protect Our Reef Grant Program (POR-2012-3), the Dart Foundation, the Smithsonian Competitive Grants Program for Science to V.J.P., the Howard Hughes Medical Institute to J.P.N., the US National Institutes of Health (NIH) Marine Biotechnology Training Grant predoctoral fellowship to A.E. (T32-GM067550), the Helen Hay Whitney Foundation postdoctoral fellowship to V.A., and a Swiss National Science Foundation (SNF) postdoctoral Fellowship to S.D.2016-09-2

Biosynthesis of polybrominated aromatic molecules by marine bacteria

Nature produces a plethora of bioactive secondary metabolites with diverse hydrocarbon scaffolds and bioactivities. These natural products serve as signaling and defense molecules in their physiological settings, and as inspiration for therapeutics in the clinic. Natural product scaffolds are elaborated with functional groups that influence their biological activities. One such chemical functionality found in thousands of natural product molecules is halogenation. Reflective of the structural diversity of halogenated natural products (halometabolites), biology has evolved an equally diverse set of enzyme catalysts using numerous strategies for activating halides for addition onto various oxidation states of carbon. The last two decades have witnessed a renaissance in the discovery of mechanistically diverse halogenating enzymes due to the unprecedented opportunity to study them in the context of halometabolite biosynthetic pathways afforded by advancements in nucleic acid sequencing technologies and computational biology. Despite the fact that the majority of halometabolites are brominated compounds derived from the marine environment, the majority of halogenting enzymes characterized to date are involved in chlorination of terrestrial microbial metabolites. Chapter 2 of this thesis describes the biosynthesis of the highly brominated pyrrole-phenol marine bacterial metabolite pentabromopseudilin by the brominated marine pyrrole/phenol (Bmp) biosynthetic pathway, which revealed the first two examples of brominating enzymes—pyrrole and phenol halogenases—from a confirmed biosynthetic context. Chapter 3 provides an in-depth investigation of regiopromiscuous pyrrole halogenase Bmp2 in contrast to canonical regioselective pyrrole chlorinases. In addition to enzymes involved in the addition of halogen atoms to the aromatic building blocks, the Bmp pathway was found to contain a dehalogenating enzyme (Bmp8) that partially undoes the work of the Bmp2 to allow a free bromopyrrole moiety to participate in the final step of pentabromopseudilin biosynthesis. Chapter 4 describes the activity and mechanism of Bmp8 as the first example of a dehalogenating tailoring enzyme from the confirmed context of a halometabolite biosynthetic pathway. Chapter 5 of this thesis concludes with an exploration of additional opportunities in the study of natural products containing halogenated pyrrole moieties, describing a strategy for the elucidation of the biosynthesis of the bioactive pyrrole-imidazole alkaloid class of sponge secondary metabolites

Biosynthesis of polybrominated aromatic molecules by marine bacteria

Nature produces a plethora of bioactive secondary metabolites with diverse hydrocarbon scaffolds and bioactivities. These natural products serve as signaling and defense molecules in their physiological settings, and as inspiration for therapeutics in the clinic. Natural product scaffolds are elaborated with functional groups that influence their biological activities. One such chemical functionality found in thousands of natural product molecules is halogenation. Reflective of the structural diversity of halogenated natural products (halometabolites), biology has evolved an equally diverse set of enzyme catalysts using numerous strategies for activating halides for addition onto various oxidation states of carbon. The last two decades have witnessed a renaissance in the discovery of mechanistically diverse halogenating enzymes due to the unprecedented opportunity to study them in the context of halometabolite biosynthetic pathways afforded by advancements in nucleic acid sequencing technologies and computational biology. Despite the fact that the majority of halometabolites are brominated compounds derived from the marine environment, the majority of halogenting enzymes characterized to date are involved in chlorination of terrestrial microbial metabolites. Chapter 2 of this thesis describes the biosynthesis of the highly brominated pyrrole-phenol marine bacterial metabolite pentabromopseudilin by the brominated marine pyrrole/phenol (Bmp) biosynthetic pathway, which revealed the first two examples of brominating enzymes—pyrrole and phenol halogenases—from a confirmed biosynthetic context. Chapter 3 provides an in-depth investigation of regiopromiscuous pyrrole halogenase Bmp2 in contrast to canonical regioselective pyrrole chlorinases. In addition to enzymes involved in the addition of halogen atoms to the aromatic building blocks, the Bmp pathway was found to contain a dehalogenating enzyme (Bmp8) that partially undoes the work of the Bmp2 to allow a free bromopyrrole moiety to participate in the final step of pentabromopseudilin biosynthesis. Chapter 4 describes the activity and mechanism of Bmp8 as the first example of a dehalogenating tailoring enzyme from the confirmed context of a halometabolite biosynthetic pathway. Chapter 5 of this thesis concludes with an exploration of additional opportunities in the study of natural products containing halogenated pyrrole moieties, describing a strategy for the elucidation of the biosynthesis of the bioactive pyrrole-imidazole alkaloid class of sponge secondary metabolites

Enzymatic Reductive Dehalogenation Controls the Biosynthesis of Marine Bacterial Pyrroles

Enzymes capable of performing dehalogenating reactions have attracted tremendous contemporary attention due to their potential application in the bioremediation of anthropogenic polyhalogenated persistent organic pollutants. Nature, in particular the marine environment, is also a prolific source of polyhalogenated organic natural products. The study of the biosynthesis of these natural products has furnished a diverse array of halogenation biocatalysts, but thus far no examples of dehalogenating enzymes have been reported from a secondary metabolic pathway. Here we show that the penultimate step in the biosynthesis of the highly brominated marine bacterial product pentabromopseudilin is catalyzed by an unusual debrominase Bmp8 that utilizes a redox thiol mechanism to remove the C-2 bromine atom of 2,3,4,5-tetrabromopyrrole to facilitate oxidative coupling to 2,4-dibromophenol. To the best of our knowledge, Bmp8 is first example of a dehalogenating enzyme from the established genetic and biochemical context of a natural product biosynthetic pathway

Bacterial Tetrabromopyrrole Debrominase Shares a Reductive Dehalogenation Strategy with Human Thyroid Deiodinase

Enzymatic dehalogenation is an important and well-studied biological process in both the detoxification and catabolism of small molecules, many of which are anthropogenic in origin. However, dedicated dehalogenation reactions that replace a halogen atom with a hydrogen are rare in the biosynthesis of natural products. In fact, the debrominase Bmp8 is the only known example. It catalyzes the reductive debromination of the coral settlement cue and the potential human toxin 2,3,4,5-tetrabromopyrrole as part of the biosynthesis of the antibiotic pentabromopseudilin. Using a combination of protein crystallography, mutagenesis, and computational modeling, we propose a catalytic mechanism for Bmp8 that is reminiscent of that catalyzed by human deiodinases in the maintenance of thyroid hormones. The identification of the key catalytic residues enabled us to recognize divergent functional homologues of Bmp8. Characterization of one of these homologues demonstrated its debromination activity even though it is found in a completely distinct genomic context. This observation suggests that additional enzymes outside those associated with the tetrabromopyrrole biosynthetic pathway may be able to alter the lifetime of this compound in the environment

Correction to Organohalogens Naturally Biosynthesized in Marine Environments and Produced As Disinfection Byproducts Alter Sarco/Endoplasmic Reticulum Ca2+ Dynamics.

The chemical syntheses with structures in the original Supporting Information was inadvertently truncated. The correct and complete Supporting Information is available here