Metabolite diversity among representatives of divergent Prochlorococcus ecotypes.

IMPORTANCE: Approximately half of the annual carbon fixation on Earth occurs in the surface ocean through the photosynthetic activities of phytoplankton such as the ubiquitous picocyanobacterium Prochlorococcus. Ecologically distinct subpopulations (or ecotypes) of Prochlorococcus are central conduits of organic substrates into the ocean microbiome, thus playing important roles in surface ocean production. We measured the chemical profile of three cultured ecotype strains, observing striking differences among them that have implications for the likely chemical impact of Prochlorococcus subpopulations on their surroundings in the wild. Subpopulations differ in abundance along gradients of temperature, light, and nutrient concentrations, suggesting that these chemical differences could affect carbon cycling in different ocean strata and should be considered in models of Prochlorococcus physiology and marine carbon dynamics.

Microorganisms and dissolved metabolites distinguish Florida's Coral Reef habitats.

As coral reef ecosystems experience unprecedented change, effective monitoring of reef features supports management, conservation, and intervention efforts. Omic techniques show promise in quantifying key components of reef ecosystems including dissolved metabolites and microorganisms that may serve as invisible sensors for reef ecosystem dynamics. Dissolved metabolites are released by reef organisms and transferred among microorganisms, acting as chemical currencies and contributing to nutrient cycling and signaling on reefs. Here, we applied four omic techniques (taxonomic microbiome via amplicon sequencing, functional microbiome via shotgun metagenomics, targeted metabolomics, and untargeted metabolomics) to waters overlying Florida's Coral Reef, as well as microbiome profiling on individual coral colonies from these reefs to understand how microbes and dissolved metabolites reflect biogeographical, benthic, and nutrient properties of this 500-km barrier reef. We show that the microbial and metabolite omic approaches each differentiated reef habitats based on geographic zone. Further, seawater microbiome profiling and targeted metabolomics were significantly related to more reef habitat characteristics, such as amount of hard and soft coral, compared to metagenomic sequencing and untargeted metabolomics. Across five coral species, microbiomes were also significantly related to reef zone, followed by species and disease status, suggesting that the geographic water circulation patterns in Florida also impact the microbiomes of reef builders. A combination of differential abundance and indicator species analyses revealed metabolite and microbial signatures of specific reef zones, which demonstrates the utility of these techniques to provide new insights into reef microbial and metabolite features that reflect broader ecosystem processes.

Suboxic DOM is bioavailable to surface prokaryotes in a simulated overturn of an oxygen minimum zone, Devil's Hole, Bermuda.

Oxygen minimum zones (OMZs) are expanding due to increased sea surface temperatures, subsequent increased oxygen demand through respiration, reduced oxygen solubility, and thermal stratification driven in part by anthropogenic climate change. Devil's Hole, Bermuda is a model ecosystem to study OMZ microbial biogeochemistry because the formation and subsequent overturn of the suboxic zone occur annually. During thermally driven stratification, suboxic conditions develop, with organic matter and nutrients accumulating at depth. In this study, the bioavailability of the accumulated dissolved organic carbon (DOC) and the microbial community response to reoxygenation of suboxic waters was assessed using a simulated overturn experiment. The surface inoculated prokaryotic community responded to the deep (formerly suboxic) 0.2 µm filtrate with cell densities increasing 2.5-fold over 6 days while removing 5 µmol L-1 of DOC. After 12 days, the surface community began to shift, and DOC quality became less diagenetically altered along with an increase in SAR202, a Chloroflexi that can degrade recalcitrant dissolved organic matter (DOM). Labile DOC production after 12 days coincided with an increase of Nitrosopumilales, a chemoautotrophic ammonia oxidizing archaea (AOA) that converts ammonia to nitrite based on the ammonia monooxygenase (amoA) gene copy number and nutrient data. In comparison, the inoculation of the deep anaerobic prokaryotic community into surface 0.2 µm filtrate demonstrated a die-off of 25.5% of the initial inoculum community followed by a 1.5-fold increase in cell densities over 6 days. Within 2 days, the prokaryotic community shifted from a Chlorobiales dominated assemblage to a surface-like heterotrophic community devoid of Chlorobiales. The DOM quality changed to less diagenetically altered material and coincided with an increase in the ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) gene number followed by an influx of labile DOM. Upon reoxygenation, the deep DOM that accumulated under suboxic conditions is bioavailable to surface prokaryotes that utilize the accumulated DOC initially before switching to a community that can both produce labile DOM via chemoautotrophy and degrade the more recalcitrant DOM.

Quantification of Amine- and Alcohol-Containing Metabolites in Saline Samples Using Pre-extraction Benzoyl Chloride Derivatization and Ultrahigh Performance Liquid Chromatography Tandem Mass Spectrometry (UHPLC MS/MS).

Dissolved metabolites serve as nutrition, energy, and chemical signals for microbial systems. However, the full scope and magnitude of these processes in marine systems are unknown, largely due to insufficient methods, including poor extraction of small, polar compounds using common solid-phase extraction resins. Here, we utilized pre-extraction derivatization and ultrahigh performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC-ESI-MS/MS) to detect and quantify targeted dissolved metabolites in seawater and saline culture media. Metabolites were derivatized with benzoyl chloride by their primary and secondary amine and alcohol functionalities and quantified using stable isotope-labeled internal standards (SIL-ISs) produced from 13C6-labeled benzoyl chloride. We optimized derivatization, extraction, and sample preparation for field and culture samples and evaluated matrix-derived biases. We have optimized this quantitative method for 73 common metabolites, of which 50 cannot be quantified without derivatization due to low extraction efficiencies. Of the 73 metabolites, 66 were identified in either culture media or seawater and 45 of those were quantified. This derivatization method is sensitive (detection limits = pM to nM), rapid (∼5 min per sample), and high throughput.

Hepatic metabolite profiling of polychlorinated biphenyl (PCB)-resistant and sensitive populations of Atlantic killifish (Fundulus heteroclitus).

Atlantic killifish inhabiting polluted sites along the east coast of the U.S. have evolved resistance to toxic effects of contaminants. One such contaminated site is the Acushnet River estuary, near New Bedford Harbor (NBH), Massachusetts, which is characterized by very high PCB concentrations in the sediments and in the tissues of resident killifish. Though killifish at this site appear to be thriving, the metabolic costs of survival in a highly contaminated environment are not well understood. In this study we compared the hepatic metabolite profiles of resistant (NBH) and sensitive populations (Scorton Creek (SC), Sandwich, MA) using a targeted metabolomics approach in which polar metabolites were extracted from adult fish livers and quantified. Our results revealed differences in the levels of several metabolites between fish from the two sites. The majority of these metabolites are associated with one-carbon metabolism, an important pathway that supports multiple physiological processes including DNA and protein methylation, nucleic acid biosynthesis and amino acid metabolism. We measured the gene expression of DNA methylation (DNA methyltransferase 1, dnmt1) and demethylation genes (Ten-Eleven Translocation (TET) genes) in the two populations, and observed lower levels of dnmt1 and higher levels of TET gene expression in the NBH livers, suggesting possible differences in DNA methylation profiles. Consistent with this, the two populations differed significantly in the levels of 5-methylcytosine and 5-hydroxymethylcytosine nucleotides. Overall, our results suggest that the unique hepatic metabolite signatures observed in NBH and SC reflect the adaptive mechanisms for survival in their respective habitats.

Targeted metabolomics reveals proline as a major osmolyte in the chemolithoautotroph Sulfurimonas denitrificans.

Chemoautotrophic bacteria belonging to the genus Sulfurimonas in the class Campylobacteria are widespread in many marine environments characterized by redox interfaces, yet little is known about their physiological adaptations to different environmental conditions. Here, we used liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in a targeted metabolomics approach to study the adaptations of Sulfurimonas denitrificans to varying salt concentrations that are found in its natural habitat of tidal mudflats. Proline was identified as one of the most abundant internal metabolites and its concentration showed a strong positive correlation with ionic strength, suggesting that it acts as an important osmolyte in S. denitrificans. 2,3-dihydroxypropane-1-sulfonate was also positively correlated with ionic strength, indicating it might play a previously unrecognized role in osmoregulation. Furthermore, the detection of metabolites from the reductive tricarboxylic acid cycle at high internal concentrations reinforces the importance of this pathway for carbon fixation in Campylobacteria and as a hub for biosynthesis. As the first report of metabolomic data for an campylobacterial chemolithoautotroph, this study provides data that will be useful to understand the adaptations of Campylobacteria to their natural habitat at redox interfaces.

Steroidal estrogen sources in a sewage-impacted coastal ocean.

Estrogens are known to be potent endocrine disrupting chemicals that are commonly found in wastewater effluents at ng L(-1) levels. Yet, we know very little about the distribution and fate of estrogens in coastal oceans that receive wastewater inputs. This study measured a wide range of steroidal estrogens in sewage-impacted seawater using ultra high performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) together with the method of standard addition. In Massachusetts Bay, we find conjugated, free, and halogenated estrogens at concentrations that are consistent with dilution at sites close to the sewage source. At a site 6 miles down current of the sewage source, we observe estrone (E1) concentrations (520 ± 180 pg L(-1)) that are nearly double the nearfield concentrations (320 ± 60 pg L(-1)) despite 9-fold dilution of carbamazepine, which was used as a conservative sewage tracer. Our results suggest that background E1 concentrations in Massachusetts Bay (∼270 ± 50 pg L(-1)) are derived largely from sources unrelated to wastewater effluent such as marine vertebrates.

Evidence for quorum sensing and differential metabolite production by a marine bacterium in response to DMSP.

Microbes, the foundation of the marine foodweb, do not function in isolation, but rather rely on molecular level interactions among species to thrive. Although certain types of interactions between autotrophic and heterotrophic microorganisms have been well documented, the role of specific organic molecules in regulating inter-species relationships and supporting growth are only beginning to be understood. Here, we examine one such interaction by characterizing the metabolic response of a heterotrophic marine bacterium, Ruegeria pomeroyi DSS-3, to growth on dimethylsulfoniopropionate (DMSP), an abundant organosulfur metabolite produced by phytoplankton. When cultivated on DMSP, R. pomeroyi synthesized a quorum-sensing molecule, N-(3-oxotetradecanoyl)-l-homoserine lactone, at significantly higher levels than during growth on propionate. Concomitant with the production of a quorum-sensing molecule, we observed differential production of intra- and extracellular metabolites including glutamine, vitamin B2 and biosynthetic intermediates of cyclic amino acids. Our metabolomics data indicate that R. pomeroyi changes regulation of its biochemical pathways in a manner that is adaptive for a cooperative lifestyle in the presence of DMSP, in anticipation of phytoplankton-derived nutrients and higher microbial density. This behavior is likely to occur on sinking marine particles, indicating that this response may impact the fate of organic matter.

Release of ecologically relevant metabolites by the cyanobacterium Synechococcus elongates CCMP 1631.

Photoautotrophic plankton in the surface ocean release organic compounds that fuel secondary production by heterotrophic bacteria. Here we show that an abundant marine cyanobacterium, Synechococcus elongatus, contributes a variety of nitrogen-rich and sulfur-containing compounds to dissolved organic matter. A combination of targeted and untargeted metabolomics and genomic tools was used to characterize the intracellular and extracellular metabolites of S. elongatus. Aromatic compounds, such as 4-hydroxybenzoic acid and phenylalanine, as well as nucleosides (e.g. thymidine, 5'-methylthioadenosine, xanthosine), the organosulfur compound 3-mercaptopropionate, and the plant auxin indole 3-acetic acid, were released by S. elongatus at multiple time points during its growth. Further, the amino acid kynurenine was found to accumulate in the media even though it was not present in the predicted metabolome of S. elongatus. This indicates that some metabolites, including those not predicted by an organism's genome, are likely excreted into the environment as waste; however, these molecules may have broader ecological relevance if they are labile to nearby microbes. The compounds described herein provide excellent targets for quantitative analysis in field settings to assess the source and lability of dissolved organic matter in situ.

Measuring free, conjugated, and halogenated estrogens in secondary treated wastewater effluent.

Steroidal estrogens are potent endocrine-disrupting chemicals that enter natural waters through the discharge of treated and raw sewage. Because estrogens are detrimental to aquatic organisms at sub-nanogram per liter concentrations, many studies have measured so-called "free" estrogen concentrations in wastewater effluents, rivers, and lakes. Other forms of estrogens are also of potential concern because conjugated estrogens can be easily converted to potent free estrogens by bacteria in wastewater treatment plants and receiving waters and halogenated estrogens are likely produced during wastewater disinfection. However, to the best of our knowledge, no studies have concurrently characterized free, conjugated, and halogenated estrogens. We have developed a method that is capable of simultaneously quantifying free, conjugated, and halogenated estrogens in treated wastewater effluent, in which detection limits were 0.13-1.3 ng L(-1) (free), 0.11-1.0 ng L(-1) (conjugated), and 0.18-18 ng L(-1) (halogenated). An aqueous phase additive, ammonium fluoride, was used to increase the electrospray (negative mode) ionization efficiency of free and halogenated estrogens by factors of 20 and 2.6, respectively. The method was validated using treated effluent from the greater Boston metropolitan area, where conjugated and halogenated estrogens made up 60-70% of the steroidal estrogen load on a molar basis.

Fate of dispersants associated with the deepwater horizon oil spill.

Response actions to the Deepwater Horizon oil spill included the injection of ∼771,000 gallons (2,900,000 L) of chemical dispersant into the flow of oil near the seafloor. Prior to this incident, no deepwater applications of dispersant had been conducted, and thus no data exist on the environmental fate of dispersants in deepwater. We used ultrahigh resolution mass spectrometry and liquid chromatography with tandem mass spectrometry (LC/MS/MS) to identify and quantify one key ingredient of the dispersant, the anionic surfactant DOSS (dioctyl sodium sulfosuccinate), in the Gulf of Mexico deepwater during active flow and again after flow had ceased. Here we show that DOSS was sequestered in deepwater hydrocarbon plumes at 1000-1200 m water depth and did not intermingle with surface dispersant applications. Further, its concentration distribution was consistent with conservative transport and dilution at depth and it persisted up to 300 km from the well, 64 days after deepwater dispersant applications ceased. We conclude that DOSS was selectively associated with the oil and gas phases in the deepwater plume, yet underwent negligible, or slow, rates of biodegradation in the affected waters. These results provide important constraints on accurate modeling of the deepwater plume and critical geochemical contexts for future toxicological studies.

Nonlinear vibrational spectroscopic studies of the adsorption and speciation of nitric acid at the vapor/acid solution interface.

Nitric acid plays an important role in the heterogeneous chemistry of the atmosphere. Reactions involving HNO(3) at aqueous interfaces in the stratosphere and troposphere depend on the state of nitric acid at these surfaces. The vapor/liquid interface of HNO(3)-H2O binary solutions and HNO(3)-H(2)SO(4)-H2O ternary solutions are examined here using vibrational sum frequency spectroscopy (VSFS). Spectra of the NO2 group at different HNO(3) mole fractions and under different polarization combinations are used to develop a detailed picture of these atmospherically important systems. Consistent with surface tension and spectroscopic measurements from other laboratories, molecular nitric acid is identified at the surface of concentrated solutions. However, the data here reveal the adsorption of two different hydrogen-bonded species of undissociated HNO(3) in the interfacial region that differ in their degree of solvation of the nitro group. The adsorption of these undissociated nitric acid species is shown to be sensitive to the H2O:HNO(3) ratio as well as to the concentration of sulfuric acid.

Differing adsorption behavior of environmentally important cyanophenol isomers at the air-water interface.

Vibrational sum-frequency spectroscopy and surface tensiometry have been used to study the adsorption of m- and p-cyanophenol at the air-water interface. Spectra of the cyano (CN) group under different polarization schemes are utilized to determine its hydrogen bonding environment and orientation. For both isomers, it is found that the cyano group is hydrogen bonded at the interface but that the CN orientation is independent of surface density. The average CN tilt angle (theta(0)), however, is found to differ between the isomers, such that the CN group points down toward the aqueous phase for m-cyanophenol (theta(0) = 96-106 degrees ) but points up toward the vapor phase for the p-cyanophenol (theta(0) = 65-80 degrees ). In addition, this average tilt angle is distributed over a narrow range, sigma(0) < 10 degrees for the meta isomer and sigma(0) < 16 degrees for the para isomer.