Lingin Derived Organic Matter in Georgia Coastal Waters

The importance of riverine- and saltmarsh-derived organic matter to the food web of Georgia coastal waters has been a focus of ecological research over the past thirty years. Studies based on energy budgets, carbon flux measurements, and stable isotope ratios have generally implicated marsh detritus as an important, although not always dominant, source of organic matter within the marshes and adjacent estuaries (Teal 1962, Odum and de la Cruz 1967, Haines 1977, Peterson and Howarth 1987). However, data on the contribution of exported marsh and riverine organic matter to productivity futher offshore, in continental shelf waters off the coast of Georgia, remains equivocal (Chalmers et al. 1985, Hopkinson 1985). Dissolved lignin-derived compounds provide molecular level markers by which to determine the presence of terrestrially-derived organic matter in marine ecosystems. Lignin is found only in vascular plants and has no known oceanic sources. Thus its presence in seawater serves as unequivocal evidence of input of marsh or riverine organic matter, and indeed such lignin-derived material has been found in open ocean water a great distance from its probable point of origin (Meyers-Schulte and Hedges 1986). In this study, we present data on concentrations and distribution of lignin phenols in seawater samples collected during October and November 1987 on the continental shelf of the southeastern U.S., between Cape Hattaras and Cape Canaveral

Diel investments in metabolite production and consumption in a model microbial system

Organic carbon transfer between surface ocean photosynthetic and heterotrophic microbes is a central but poorly understood process in the global carbon cycle. In a model community in which diatom extracellular release of organic molecules sustained growth of a co-cultured bacterium, we determined quantitative changes in the diatom endometabolome and the bacterial uptake transcriptome over two diel cycles. Of the nuclear magnetic resonance (NMR) peaks in the diatom endometabolites, 38% had diel patterns with noon or mid-afternoon maxima; the remaining either increased (36%) or decreased (26%) through time. Of the genes in the bacterial uptake transcriptome, 94% had a diel pattern with a noon maximum; the remaining decreased over time (6%). Eight diatom endometabolites identified with high confidence were matched to the bacterial genes mediating their utilization. Modeling of these coupled inventories with only diffusion-based phytoplankton extracellular release could not reproduce all the patterns. Addition of active release mechanisms for physiological balance and bacterial recognition significantly improved model performance. Estimates of phytoplankton extracellular release range from only a few percent to nearly half of annual net primary production. Improved understanding of the factors that influence metabolite release and consumption by surface ocean microbes will better constrain this globally significant carbon flux

Analyzing Gene Expression from Marine Microbial Communities using Environmental Transcriptomics

Analogous to metagenomics, environmental transcriptomics (metatranscriptomics) retrieves and sequences environmental mRNAs from a microbial assemblage without prior knowledge of what genes the community might be expressing. Thus it provides the most unbiased perspective on community gene expression in situ. Environmental transcriptomics protocols are technically difficult since prokaryotic mRNAs generally lack the poly(A) tails that make isolation of eukaryotic messages relatively straightforward 1 and because of the relatively short half lives of mRNAs 2. In addition, mRNAs are much less abundant than rRNAs in total RNA extracts, thus an rRNA background often overwhelms mRNA signals. However, techniques for overcoming some of these difficulties have recently been developed. A procedure for analyzing environmental transcriptomes by creating clone libraries using random primers to reverse-transcribe and amplify environmental mRNAs was recently described was successful in two different natural environments, but results were biased by selection of the random primers used to initiate cDNA synthesis 3. Advances in linear amplification of mRNA obviate the need for random primers in the amplification step and make it possible to use less starting material decreasing the collection and processing time of samples and thereby minimizing RNA degradation 4. In vitro transcription methods for amplifying mRNA involve polyadenylating the mRNA and incorporating a T7 promoter onto the 3 end of the transcript. Amplified RNA (aRNA) can then be converted to double stranded cDNA using random hexamers and directly sequenced by pyrosequencing 5. A first use of this method at Station ALOHA demonstrated its utility for characterizing microbial community gene expression 6

Distribution of Terrestrially Derived Dissolved Organic Matter on the Southeastern United States Continental Shelf

Dissolved lignin-derived compounds in seawater indicate the presence of organic matter originating from vascular plants and therefore from terrestrial (upland and coastal marsh) ecosystems. We used a hydrophobic resin to concentrate lignin-rich humic substances and to determine concentrations of lignin oxidation products (vanillyl lignin phenols) for waters of the continental shelf of the southeastern U.S. Lignin phenol concentrations ranged from 0.05 to 4.2µg liter‒1 and accounted for 0.002–0.13% of the total dissolved organic carbon (DOC) pool in continental shelf waters. Dissolved lignin concentrations were generally highest near the shore and in those areas receiving greatest river and marsh discharge. Concentrations varied on both short-term (weekly) and seasonal time scales, however, indicating that the contribution of terrestrially derived dissolved organic matter to the C budget of the shelf is quite variable. Salinity (\u3e 31‰) was significantly correlated (negatively) with lignin phenol concentrations during three of four cruises, suggesting largely conservative mixing of lignin-derived material on the shelf In selected rivers and salt marshes contributing terrestrially derived organic matter to the continental shelf, lignin phenol C accounted for 0.14–1.0% of the DOC. A simple mixing model which assumes no biological or physical sinks of lignin-derived material during transport from terrestrial sources to the shelf predicts that an average of 6–36% of nearshore DOC derives from terrestrial ecosystems, depending on whether the terrestrial end-member (lignin source) is assumed to be a river or a salt marsh, while 5–26% of inner shelf DOC and 3–18% of mid- to outer-shelf DOC is of terrestrial origin

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)

© The Author(s), 2021. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Widner, B., Kido Soule, M. C., Ferrer-González, F. X., Moran, M. A., & Kujawinski, E. B. 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). Analytical Chemistry, 93(11), (2021): 4809-4817, https://doi.org/10.1021/acs.analchem.0c03769.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.Funding included the Gordon and Betty Moore Foundation (Award GBMF5503 to M.A.M. and E.B.K.), Simons Foundation International (Award 409923 to E.B.K.), and the National Science Foundation (Award 1656311 to M.A.M.)

Bacterial transcriptional response to labile exometabolites from photosynthetic picoeukaryote Micromonas commoda

Dissolved primary production released into seawater by marine phytoplankton is a major source of carbon fueling heterotrophic bacterial production in the ocean. The composition of the organic compounds released by healthy phytoplankton is poorly known and difficult to assess with existing chemical methods. Here, expression of transporter and catabolic genes by three model marine bacteria (Ruegeria pomeroyi DSS-3, Stenotrophomonas sp. SKA14, and Polaribacter dokdonensis MED152) was used as a biological sensor of metabolites released from the picoeukaryote Micromonas commoda RCC299. Bacterial expression responses indicated that the three species together recognized 38 picoeukaryote metabolites. This was consistent with the Micromonas expression of genes for starch metabolism and synthesis of peptidoglycan-like intermediates. A comparison of the hypothesized Micromonas exometabolite pool with that of the diatom Thalassiosira pseudonana CCMP1335, analyzed previously with the same biological sensor method, indicated that both phytoplankton released organic acids, nucleosides, and amino acids, but differed in polysaccharide and organic nitrogen release. Future ocean conditions are expected to favor picoeukaryotic phytoplankton over larger-celled microphytoplankton. Results from this study suggest that such a shift could alter the substrate pool available to heterotrophic bacterioplankton

Environmental, biochemical and genetic drivers of DMSP degradation and DMS production in the Sargasso Sea

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of John Wiley & Sons for personal use, not for redistribution. The definitive version was published in Environmental Microbiology 14 (2012): 1210-1223, doi:10.1111/j.1462-2920.2012.02700.x.Dimethylsulfide (DMS) is a climatically relevant trace gas produced and cycled by the surface ocean food web. Mechanisms driving intraannual variability in DMS production and dimethylsulfoniopropionate (DMSP) degradation in open-ocean, oligotrophic regions were investigated during a 10 month time-series at the Bermuda Atlantic Time-series Study site in the Sargasso Sea. Abundance and transcription of bacterial DMSP degradation genes, DMSP lyase enzyme activity, and DMS and DMSP concentrations, consumption rates, and production rates were quantified over time and depth. This interdisciplinary dataset was used to test current hypotheses of the role of light and carbon supply in regulating upper-ocean sulfur cycling. Findings supported UV-A dependent phytoplankton DMS production. Bacterial DMSP degraders may also contribute significantly to DMS production when temperatures are elevated and UV-A dose is moderate, but may favor DMSP demethylation under low UV-A doses. Three groups of bacterial DMSP degraders with distinct intraannual variability were identified and niche differentiation was indicated. The combination of genetic and biochemical data suggest a modified ‘bacterial switch’ hypothesis where the prevalence of different bacterial DMSP degradation pathways is regulated by a complex set of factors including carbon supply, temperature, and UV-A dose.This research was funded by National Science Foundation (NSF) grants OCE- 0525928, OCE-072417, and OCE-042516. Additional funding was provided by the NSF Center for Microbial Oceanography Research and Education (CMORE), the Gordon and Betty Moore Foundation, the Scurlock Fund, the Ocean Ventures Fund, a National Defense Science and Engineering Graduate Fellowship, and an Environmental Protection Agency STAR Graduate Fellowship

Resource partitioning of phytoplankton metabolites that support bacterial heterotrophy

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Ferrer-González, F. X., Widner, B., Holderman, N. R., Glushka, J., Edison, A. S., Kujawinski, E. B., & Moran, M. A. Resource partitioning of phytoplankton metabolites that support bacterial heterotrophy. ISME Journal, (2020), doi:10.1038/s41396-020-00811-y.The communities of bacteria that assemble around marine microphytoplankton are predictably dominated by Rhodobacterales, Flavobacteriales, and families within the Gammaproteobacteria. Yet whether this consistent ecological pattern reflects the result of resource-based niche partitioning or resource competition requires better knowledge of the metabolites linking microbial autotrophs and heterotrophs in the surface ocean. We characterized molecules targeted for uptake by three heterotrophic bacteria individually co-cultured with a marine diatom using two strategies that vetted the exometabolite pool for biological relevance by means of bacterial activity assays: expression of diagnostic genes and net drawdown of exometabolites, the latter detected with mass spectrometry and nuclear magnetic resonance using novel sample preparation approaches. Of the more than 36 organic molecules with evidence of bacterial uptake, 53% contained nitrogen (including nucleosides and amino acids), 11% were organic sulfur compounds (including dihydroxypropanesulfonate and dimethysulfoniopropionate), and 28% were components of polysaccharides (including chrysolaminarin, chitin, and alginate). Overlap in phytoplankton-derived metabolite use by bacteria in the absence of competition was low, and only guanosine, proline, and N-acetyl-d-glucosamine were predicted to be used by all three. Exometabolite uptake pattern points to a key role for ecological resource partitioning in the assembly marine bacterial communities transforming recent photosynthate.This work was supported by grants from the Gordon and Betty Moore Foundation (5503) and the National Science Foundation (IOS-1656311) to MAM, ASE, and EBK, and by the Simons Foundation grant 542391 to MAM within the Principles of Microbial Ecosystems (PriME) Collaborative

Transcriptional Changes Underlying Elemental Stoichiometry Shifts in a Marine Heterotrophic Bacterium

Marine bacteria drive the biogeochemical processing of oceanic dissolved organic carbon (DOC), a 750-Tg C reservoir that is a critical component of the global C cycle. Catabolism of DOC is thought to be regulated by the biomass composition of heterotrophic bacteria, as cells maintain a C:N:P ratio of ∼50:10:1 during DOC processing. Yet a complicating factor in stoichiometry-based analyses is that bacteria can change the C:N:P ratio of their biomass in response to resource composition. We investigated the physiological mechanisms of resource-driven shifts in biomass stoichiometry in continuous cultures of the marine heterotrophic bacterium Ruegeria pomeroyi (a member of the Roseobacter clade) under four element limitation regimes (C, N, P, and S). Microarray analysis indicated that the bacterium scavenged for alternate sources of the scarce element when cells were C-, N-, or P-limited; reworked the ratios of biomolecules when C- and P- limited; and exerted tighter control over import/export and cytoplasmic pools when N-limited. Under S limitation, a scenario not existing naturally for surface ocean microbes, stress responses dominated transcriptional changes. Resource-driven changes in C:N ratios of up to 2.5-fold and in C:P ratios of up to sixfold were measured in R. pomeroyi biomass. These changes were best explained if the C and P content of the cells was flexible in the face of shifting resources but N content was not, achieved through the net balance of different transcriptional strategies. The cellular-level metabolic trade-offs that govern biomass stoichiometry in R. pomeroyi may have implications for global carbon cycling if extendable to other heterotrophic bacteria. Strong homeostatic responses to N limitation by marine bacteria would intensify competition with autotrophs. Modification of cellular inventories in C- and P-limited heterotrophs would vary the elemental ratio of particulate organic matter sequestered in the deep ocean

Divergent gene expression responses in two Baltic Sea heterotrophic model bacteria to dinoflagellate dissolved organic matter

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