Methanol production by a broad phylogenetic array of marine phytoplankton

© The Author(s), 2016. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in PLoS 11 (2016): e0150820, doi:10.1371/journal.pone.0150820.Methanol is a major volatile organic compound on Earth and serves as an important carbon and energy substrate for abundant methylotrophic microbes. Previous geochemical surveys coupled with predictive models suggest that the marine contributions are exceedingly large, rivaling terrestrial sources. Although well studied in terrestrial ecosystems, methanol sources are poorly understood in the marine environment and warrant further investigation. To this end, we adapted a Purge and Trap Gas Chromatography/Mass Spectrometry (P&T-GC/MS) method which allowed reliable measurements of methanol in seawater and marine phytoplankton cultures with a method detection limit of 120 nanomolar. All phytoplankton tested (cyanobacteria: Synechococcus spp. 8102 and 8103, Trichodesmium erythraeum, and Prochlorococcus marinus), and Eukarya (heterokont diatom: Phaeodactylum tricornutum, coccolithophore: Emiliania huxleyi, cryptophyte: Rhodomonas salina, and non-diatom heterokont: Nannochloropsis oculata) produced methanol, ranging from 0.8–13.7 micromolar in culture and methanol per total cellular carbon were measured in the ranges of 0.09–0.3%. Phytoplankton culture time-course measurements displayed a punctuated production pattern with maxima near early stationary phase. Stabile isotope labeled bicarbonate incorporation experiments confirmed that methanol was produced from phytoplankton biomass. Overall, our findings suggest that phytoplankton are a major source of methanol in the upper water column of the world’s oceans.This project was solely supported by a grant to TJM from the National Science Foundation (Award# CHE-OCE 1131415)

Evidence for strain-specific exometabolomic responses of the coccolithophore Emiliania huxleyi to grazing by the dinoflagellate oxyrrhis marina

© 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 Marine Science 3 (2016): 1, doi:10.3389/fmars.2016.00001.The coccolithophore Emiliania huxleyi forms massive blooms and plays a critical role in global elemental cycles, sequestering significant amounts of atmospheric carbon dioxide on geological time scales via production of calcium carbonate coccoliths and emitting dimethyl sulfoniopropionate (DMSP), which has the potential for increasing atmosph-eric albedo. Because grazing in pelagic systems is a major top-down force structuring microbial communities, the influence of grazers on E. huxleyi populations has been of interest to researchers. Roles of DMSP (and related metabolites) in interactions between E. huxleyi and protist grazers have been investigated, however, little is known about the release of other metabolites that may influence, or be influenced by, such grazing interactions. We used high-resolution mass spectrometry in an untargeted approach to survey the suite of low molecular weight compounds released by four different E. huxleyi strains in response to grazing by the dinoflagellate Oxyrrhis marina. Overall, a strikingly small number of metabolites were detected from E. huxleyi and O. marina cells, but these were distinctly informative to construct metabolic footprints. At most, E. huxleyi strains shared 25% of released metabolites. Furthermore, there appeared to be no unified metabolic response in E. huxleyi strains to grazing; rather, these responses were strain specific. Concentrations of several metabolites also positively correlated with grazer activities, including grazing, ingestion, and growth rates; however, no single metabolite responded uniformly across all strains of E. huxleyi tested. Regardless, grazing clearly transformed the constituents of dissolved organic matter produced by these marine microbes. This study addresses several technical challenges, and presents a platform to further study the influence of chemical cues in aquatic systems and demonstrates the impact of strain diversity and grazing on the complexity of dissolved organic matter in marine systems.Funding for this work was provided by the Gordon and Betty Moore Foundation, Grant #3301 awarded to A Vardi, BAS. Van Mooy, K Bidle, MJ, and TM. Additional funding for this work was provided by an award from the Flatley Discovery Lab to TM

Impact of prawn farming effluent on coral reef water nutrients and microorganisms

© The Author(s), 2017. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Aquaculture Environment Interactions 9 (2017): 331-346, doi:10.3354/aei00238.Tropical coral reefs are characterized by low-nutrient waters that support oligotrophic picoplankton over a productive benthic ecosystem. Nutrient-rich effluent released from aquaculture facilities into coral reef environments may potentially upset the balance of these ecosystems by altering picoplankton dynamics. In this study, we examined how effluent from a prawn (Litopenaeus vannamei) farming facility in Al Lith, Saudi Arabia, impacted the inorganic nutrients and prokaryotic picoplankton community in the waters overlying coral reefs in the Red Sea. Across 24 sites, ranging 0-21 km from the effluent point source, we measured nutrient concentrations, quantified microbial cell abundances, and sequenced bacterial and archaeal small subunit ribosomal RNA (SSU rRNA) genes to examine picoplankton phylogenetic diversity and community composition. Our results demonstrated that sites nearest to the outfall had increased concentrations of phosphate and ammonium and elevated abundances of non-pigmented picoplankton (generally heterotrophic bacteria). Shifts in the composition of the picoplankton community were observed with increasing distance from the effluent canal outfall. Waters within 500 m of the outfall harbored the most distinct picoplanktonic community and contained putative pathogens within the genus Francisella and order Rickettsiales. While our study suggests that at the time of sampling, the Al Lith aquaculture facility exhibited relatively minor influences on inorganic nutrients and microbial communities, studying the longer-term impacts of the aquaculture effluent on the organisms within the reef will be necessary in order to understand the full extent of the facility’s impact on the reef ecosystem.This research was supported by a Woods Hole Oceanographic Institution (WHOI) Ocean Life Institute postdoctoral scholar fellowship to A.A., the Semester at WHOI Program supporting C.B., and Award No. USA 00002 to K.H. made by King Abdullah University of Science and Technology (KAUST)

The biogeography of the Plastisphere : implications for policy

Author Posting. © Ecological Society of America, 2015. This article is posted here by permission of Ecological Society of America for personal use, not for redistribution. The definitive version was published in Frontiers in Ecology and the Environment 13 (2015): 541–546, doi:10.1890/150017.Microplastics (particles less than 5 mm) numerically dominate marine debris and occur from coastal waters to mid-ocean gyres, where surface circulation concentrates them. Given the prevalence of plastic marine debris (PMD) and the rise in plastic production, the impacts of plastic on marine ecosystems will likely increase. Microscopic life (the “Plastisphere”) thrives on these tiny floating “islands” of debris and can be transported long distances. Using next-generation DNA sequencing, we characterized bacterial communities from water and plastic samples from the North Pacific and North Atlantic subtropical gyres to determine whether the composition of different Plastisphere communities reflects their biogeographic origins. We found that these communities differed between ocean basins – and to a lesser extent between polymer types – and displayed latitudinal gradients in species richness. Our research reveals some of the impacts of microplastics on marine biodiversity, demonstrates that the effects and fate of PMD may vary considerably in different parts of the global ocean, and suggests that PMD mitigation will require regional management efforts.This work was supported by a US National Science Foundation (NSF) collaborative grant to LAA-Z (OCE-1155571), ERZ (OCE-1155379), and TJM (OCE-1155671), and was partially funded by an NSF TUES grant (DUE-1043468) to LAA-Z and ERZ, and by the Richard Saltonstall Charitable Foundation to TJM. GP was funded through the OCE-1155379 grant and assisted with identification of plastic resins via ATR-FTIR

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

Pathways of Carbon Assimilation and Ammonia Oxidation Suggested by Environmental Genomic Analyses of Marine Crenarchaeota

Marine Crenarchaeota represent an abundant component of oceanic microbiota with potential to significantly influence biogeochemical cycling in marine ecosystems. Prior studies using specific archaeal lipid biomarkers and isotopic analyses indicated that planktonic Crenarchaeota have the capacity for autotrophic growth, and more recent cultivation studies support an ammonia-based chemolithoautotrophic energy metabolism. We report here analysis of fosmid sequences derived from the uncultivated marine crenarchaeote, Cenarchaeum symbiosum, focused on the reconstruction of carbon and energy metabolism. Genes predicted to encode multiple components of a modified 3-hydroxypropionate cycle of autotrophic carbon assimilation were identified, consistent with utilization of carbon dioxide as a carbon source. Additionally, genes predicted to encode a near complete oxidative tricarboxylic acid cycle were also identified, consistent with the consumption of organic carbon and in the production of intermediates for amino acid and cofactor biosynthesis. Therefore, C. symbiosum has the potential to function either as a strict autotroph, or as a mixotroph utilizing both carbon dioxide and organic material as carbon sources. From the standpoint of energy metabolism, genes predicted to encode ammonia monooxygenase subunits, ammonia permease, urease, and urea transporters were identified, consistent with the use of reduced nitrogen compounds as energy sources fueling autotrophic metabolism. Homologues of these genes, recovered from ocean waters worldwide, demonstrate the conservation and ubiquity of crenarchaeal pathways for carbon assimilation and ammonia oxidation. These findings further substantiate the likely global metabolic importance of Crenarchaeota with respect to key steps in the biogeochemical transformation of carbon and nitrogen in marine ecosystems

Culture-Dependent and Culture-Independent Diversity within the Obligate Marine Actinomycete Genus Salinispora

Salinispora is the first obligate marine genus within the order Actinomycetales and a productive source of biologically active secondary metabolites. Despite a worldwide, tropical or subtropical distribution in marine sediments, only two Salinispora species have thus far been cultivated, suggesting limited species-level diversity. To further explore Salinispora diversity and distributions, the phylogenetic diversity of more than 350 strains isolated from sediments collected around the Bahamas was examined, including strains cultured using new enrichment methods. A culture-independent method, using a Salinispora-specific seminested PCR technique, was used to detect Salinispora from environmental DNA and estimate diversity. Overall, the 16S rRNA gene sequence diversity of cultured strains agreed well with that detected in the environmental clone libraries. Despite extensive effort, no new species level diversity was detected, and 97% of the 105 strains examined by restriction fragment length polymorphism belonged to one phylotype (S. arenicola). New intraspecific diversity was detected in the libraries, including an abundant new phylotype that has yet to be cultured, and a new depth record of 1,100 m was established for the genus. PCR-introduced error, primarily from Taq polymerase, significantly increased clone library sequence diversity and, if not masked from the analyses, would have led to an overestimation of total diversity. An environmental DNA extraction method specific for vegetative cells provided evidence for active actinomycete growth in marine sediments while indicating that a majority of sediment samples contained predominantly Salinispora spores at concentrations that could not be detected in environmental clone libraries. Challenges involved with the direct sequence-based detection of spore-forming microorganisms in environmental samples are discussed

Time-course plot of <i>N</i>. <i>oculata</i> batch culture grown in biological triplicate showing methanol production (y-axis) over time (x-axis) in micromolar amounts in upper panel, and cell abundances and corresponding F_v/F_m in lower panel.

<p>Note, highest methanol production where cell abundance levels off.</p

Isotope ratio measurements of axenic phytoplankton cultivars grown in defined medium containing 10% of the total bicarbonate as the isotopically labeled form (¹³C).

<p>Isotope ratio measurements of axenic phytoplankton cultivars grown in defined medium containing 10% of the total bicarbonate as the isotopically labeled form (¹³C).</p