Longnecker Krista

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Longnecker

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Krista

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0000-0002-6850-2694

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Benthic exometabolites and their ecological significance on threatened Caribbean coral reefs

(Springer, 2022-10-17) Weber, Laura ; Soule, Melissa Kido ; Longnecker, Krista ; Becker, Cynthia C. ; Huntley, Naomi ; Kujawinski, Elizabeth B. ; Apprill, Amy

Benthic organisms are the architectural framework supporting coral reef ecosystems, but their community composition has recently shifted on many reefs. Little is known about the metabolites released from these benthic organisms and how compositional shifts may influence other reef life, including prolific microorganisms. To investigate the metabolite composition of benthic exudates and their ecological significance for reef microbial communities, we harvested exudates from six species of Caribbean benthic organisms including stony corals, octocorals, and an invasive encrusting alga, and subjected these exudates to untargeted and targeted metabolomics approaches using liquid chromatography-mass spectrometry. Incubations with reef seawater microorganisms were conducted to monitor changes in microbial abundances and community composition using 16 S rRNA gene sequencing in relation to exudate source and three specific metabolites. Exudates were enriched in amino acids, nucleosides, vitamins, and indole-based metabolites, showing that benthic organisms contribute labile organic matter to reefs. Furthermore, exudate compositions were species-specific, and riboflavin and pantothenic acid emerged as significant coral-produced metabolites, while caffeine emerged as a significant invasive algal-produced metabolite. Microbial abundances and individual microbial taxa responded differently to exudates from stony corals and octocorals, demonstrating that exudate mixtures released from different coral species select for specific bacteria. In contrast, microbial communities did not respond to individual additions of riboflavin, pantothenic acid, or caffeine. This work indicates that recent shifts in benthic organisms alter exudate composition and likely impact microbial communities on coral reefs.
Extracellular reef metabolites across the protected Jardines de la Reina, Cuba Reef System

(Frontiers Media, 2020-12-14) Weber, Laura ; Armenteros, Maickel ; Kido Soule, Melissa C. ; Longnecker, Krista ; Kujawinski, Elizabeth B. ; Apprill, Amy

Coral reef ecosystems are incredibly diverse marine biomes that rely on nutrient cycling by microorganisms to sustain high productivity in generally oligotrophic regions of the ocean. Understanding the composition of extracellular reef metabolites in seawater, the small organic molecules that serve as the currency for microorganisms, may provide insight into benthic-pelagic coupling as well as the complexity of nutrient cycling in coral reef ecosystems. Jardines de la Reina (JR), Cuba is an ideal environment to examine extracellular metabolites across protected and high-quality reefs. Here, we used liquid chromatography mass spectrometry (LC-MS) to quantify specific known metabolites of interest (targeted metabolomics approach) and to survey trends in metabolite feature composition (untargeted metabolomics approach) from surface and reef depth (6 – 14 m) seawater overlying nine forereef sites in JR. We found that untargeted metabolite feature composition was surprisingly similar between reef depth and surface seawater, corresponding with other biogeochemical and physicochemical measurements and suggesting that environmental conditions were largely homogenous across forereefs within JR. Additionally, we quantified 32 of 53 detected metabolites using the targeted approach, including amino acids, nucleosides, vitamins, and other metabolic intermediates. Two of the quantified metabolites, riboflavin and xanthosine, displayed interesting trends by depth. Riboflavin concentrations were higher in reef depth compared to surface seawater, suggesting that riboflavin may be produced by reef organisms at depth and degraded in the surface through photochemical oxidation. Xanthosine concentrations were significantly higher in surface reef seawater. 5′-methylthioadenosine (MTA) concentrations increased significantly within the central region of the archipelago, displaying biogeographic patterns that warrant further investigation. Here we lay the groundwork for future investigations of variations in metabolite composition across reefs, sources and sinks of reef metabolites, and changes in metabolites over environmental, temporal, and reef health gradients.
Organic sulfur: a spatially variable and understudied component of marine organic matter

(Wiley Open Access, 2020-02-05) Longnecker, Krista ; Oswald, Lisa ; Kido Soule, Melissa C. ; Cutter, Gregory A. ; Kujawinski, Elizabeth B.

Sulfur (S) is a major heteroatom in organic matter. This project evaluated spatial variability in the concentration and molecular‐level composition of organic sulfur along gradients of depth and latitude. We measured the concentration of total organic sulfur (TOS) directly from whole seawater. Our data reveal high variability in organic sulfur, relative to established variability in total organic carbon or nitrogen. The deep ocean contained significant amounts of organic sulfur, and the concentration of TOS in North Atlantic Deep Water (NADW) decreased with increasing age while total organic carbon remained stable. Analysis of dissolved organic matter extracts by ultrahigh resolution mass spectrometry revealed that 6% of elemental formulas contained sulfur. The sulfur‐containing compounds were structurally diverse, and showed higher numbers of sulfur‐containing elemental formulas as NADW moved southward. These measurements of organic sulfur in seawater provide the foundation needed to define the factors controlling organic sulfur in the global ocean.
Pathway-centric analysis of microbial metabolic potential and expression along nutrient and energy gradients in the western Atlantic Ocean

(Frontiers Media, 2022-05-19) Cavaco, Maria A. ; Bhatia, Maya P. ; Hawley, Alyse K. ; Torres-Beltrán, Mónica ; Johnson, Winifred M. ; Longnecker, Krista ; Konwar, Kishori ; Kujawinski, Elizabeth B. ; Hallam, Steven J.

Microbial communities play integral roles in driving nutrient and energy transformations in the ocean, collectively contributing to fundamental biogeochemical cycles. Although it is well known that these communities are stratified within the water column, there remains limited knowledge of how metabolic pathways are distributed and expressed. Here, we investigate pathway distribution and expression patterns from surface (5 m) to deep dark ocean (4000 m) at three stations along a 2765 km transect in the western South Atlantic Ocean. This study is based on new data, consisting of 43 samples for 16S rRNA gene sequencing, 20 samples for metagenomics and 19 samples for metatranscriptomics. Consistent with previous observations, we observed vertical zonation of microbial community structure largely partitioned between light and dark ocean waters. The metabolic pathways inferred from genomic sequence information and gene expression stratified with depth. For example, expression of photosynthetic pathways increased in sunlit waters. Conversely, expression of pathways related to carbon conversion processes, particularly those involving recalcitrant and organic carbon degradation pathways (i.e., oxidation of formaldehyde) increased in dark ocean waters. We also observed correlations between indicator taxa for specific depths with the selective expression of metabolic pathways. For example, SAR202, prevalent in deep waters, was strongly correlated with expression of the methanol oxidation pathway. From a biogeographic perspective, microbial communities along the transect encoded similar metabolic potential with some latitudinal stratification in gene expression. For example, at a station influenced by input from the Amazon River, expression of pathways related to oxidative stress was increased. Finally, when pairing distinct correlations between specific particulate metabolites (e.g., DMSP, AMP and MTA) and both the taxonomic microbial community and metatranscriptomic pathways across depth and space, we were able to observe how changes in the marine metabolite pool may be influenced by microbial function and vice versa. Taken together, these results indicate that marine microbial communities encode a core repertoire of widely distributed metabolic pathways that are differentially regulated along nutrient and energy gradients. Such pathway distribution patterns are consistent with robustness in microbial food webs and indicate a high degree of functional redundancy.
Intracellular metabolites in marine microorganisms during an experiment evaluating microbial mortality

(MDPI, 2020-03-12) Longnecker, Krista ; Kujawinski, Elizabeth B.

Metabolomics is a tool with immense potential for providing insight into the impact of biological processes on the environment. Here, we used metabolomics methods to characterize intracellular metabolites within marine microorganisms during a manipulation experiment that was designed to test the impact of two sources of microbial mortality, protozoan grazing and viral lysis. Intracellular metabolites were analyzed with targeted and untargeted mass spectrometry methods. The treatment with reduced viral mortality showed the largest changes in metabolite concentrations, although there were organic compounds that shifted when the impact of protozoan grazers was reduced. Intracellular concentrations of guanine, phenylalanine, glutamic acid, and ectoine presented significant responses to changes in the source of mortality. Unexpectedly, variability in metabolite concentrations were not accompanied by increases in microbial abundance which indicates that marine microorganisms altered their internal organic carbon stores without changes in biomass or microbial growth. We used Weighted Correlation Network Analysis (WGCNA) to identify correlations between the targeted and untargeted mass spectrometry data. This analysis revealed multiple unknown organic compounds were correlated with compatible solutes, also called osmolytes or chemical chaperones, which emphasizes the dominant role of compatible solutes in marine microorganisms.
Biogeochemistry, metabolomics, and metagenomics of Florida's Coral Reef from sampling conducted over 15 days in June 2019

(Biological and Chemical Oceanography Data Management Office (BCO-DMO). Contact: bco-dmo-data@whoi.edu, 2023-03-08) Apprill, Amy ; Kujawinski, Elizabeth ; Muller, Erinn ; Sandin, Stuart ; Weber, Laura ; Clark, Abigail ; Kido Soule, Melissa C. ; Longnecker, Krista ; Zgliczynski, Brian ; Sullivan, Chris ; Becker, Cynthia Carroll

This comparative 'omics dataset was collected over 15 days in June 2019 along Florida's Coral Reef. We assessed 85 reefs for the prevalence of stony coral tissue loss disease (SCTLD), nutrients (total organic carbon (TOC), total organic nitrogen (TON), inorganic nutrients), and abundances of microbial functional groups (Prochlorococcus, Synechococcus, picoeukaryotes, and heterotrophic microbes (unpigmented bacteria and archaea)), from reef depth waters. At 45 of the reefs, high-resolution photomosaics were used to examine the composition of benthic organisms. At 13 geographically dispersed reefs, we collected seawater (1.7 liters in biological triplicates) for both targeted and untargeted metabolomics analyses. Seawater (2 liters in duplicate) was collected at 26 sites, including the 13 examined for metabolomics, for taxonomic (bacteria and archaea 16S ribosomal RNA gene) and functional (shotgun metagenome) microbiome analyses, and chlorophyll. Given the stony coral tissue loss disease outbreak, we also targeted healthy and diseased coral tissue and near-coral seawater for taxonomic microbiome (16S rRNA gene) analysis (11 sites). Significance: Microorganisms and the dissolved metabolites they process are central to the functioning of ocean ecosystems. These 'invisible' ocean components are poorly understood in biodiverse and productive coral reef ecosystems, where they contribute to nutrient cycling and signaling cues between reef organisms. Microbes and dissolved metabolites offer a new means to examine reef features and have applications for conservation, monitoring, and restoration efforts in these changing ecosystems. For a complete list of measurements, refer to the full dataset description in the supplemental file 'Dataset_description.pdf'. The most current version of this dataset is available at: https://www.bco-dmo.org/dataset/890979
Different carboxyl-rich alicyclic molecules proxy compounds select distinct bacterioplankton for oxidation of dissolved organic matter in the mesopelagic Sargasso Sea

(Wiley, 2020-01-23) Liu, Shuting ; Parsons, Rachel J. ; Opalk, Keri ; Baetge, Nicholas ; Giovannoni, Stephen J. ; Bolaños, Luis M. ; Kujawinski, Elizabeth B. ; Longnecker, Krista ; Lu, YueHan ; Halewood, Elisa ; Carlson, Craig A.

Marine dissolved organic matter (DOM) varies in its recalcitrance to rapid microbial degradation. DOM of varying recalcitrance can be exported from the ocean surface to depth by subduction or convective mixing and oxidized over months to decades in deeper seawater. Carboxyl‐rich alicyclic molecules (CRAM) are characterized as a major component of recalcitrant DOM throughout the oceanic water column. The oxidation of CRAM‐like compounds may depend on specific bacterioplankton lineages with oxidative enzymes capable of catabolizing complex molecular structures like long‐chain aliphatics, cyclic alkanes, and carboxylic acids. To investigate the interaction between bacteria and CRAM‐like compounds, we conducted microbial remineralization experiments using several compounds rich in carboxyl groups and/or alicyclic rings, including deoxycholate, humic acid, lignin, and benzoic acid, as proxies for CRAM. Mesopelagic seawater (200 m) from the northwest Sargasso Sea was used as media and inoculum and incubated over 28 d. All amendments demonstrated significant DOC removal (2–11 μmol C L−1) compared to controls. Bacterioplankton abundance increased significantly in the deoxycholate and benzoic acid treatments relative to controls, with fast‐growing Spongiibacteracea, Euryarcheaota, and slow‐growing SAR11 enriched in the deoxycholate treatment and fast‐growing Alteromonas, Euryarcheaota, and Thaumarcheaota enriched in the benzoic acid treatment. In contrast, bacterioplankton grew slower in the lignin and humic acid treatments, with oligotrophic SAR202 becoming significantly enriched in the lignin treatment. Our results indicate that the character of the CRAM proxy compounds resulted in distinct bacterioplankton removal rates of DOM and affected specific lineages of bacterioplankton capable of responding.
Pangenomics analysis reveals diversification of enzyme families and niche specialization in globally abundant SAR202 bacteria

(American Society for Microbiology, 2020-01-07) Saw, Jimmy H. W. ; Nunoura, Takuro ; Hirai, Miho ; Takaki, Yoshihiro ; Parsons, Rachel ; Michelsen, Michelle ; Longnecker, Krista ; Kujawinski, Elizabeth B. ; Stepanauskas, Ramunas ; Landry, Zachary ; Carlson, Craig A. ; Giovannoni, Stephen J.

It has been hypothesized that the abundant heterotrophic ocean bacterioplankton in the SAR202 clade of the phylum Chloroflexi evolved specialized metabolisms for the oxidation of organic compounds that are resistant to microbial degradation via common metabolic pathways. Expansions of paralogous enzymes were reported and implicated in hypothetical metabolism involving monooxygenase and dioxygenase enzymes. In the proposed metabolic schemes, the paralogs serve the purpose of diversifying the range of organic molecules that cells can utilize. To further explore SAR202 evolution and metabolism, we reconstructed single amplified genomes and metagenome-assembled genomes from locations around the world that included the deepest ocean trenches. In an analysis of 122 SAR202 genomes that included seven subclades spanning SAR202 diversity, we observed additional evidence of paralog expansions that correlated with evolutionary history, as well as further evidence of metabolic specialization. Consistent with previous reports, families of flavin-dependent monooxygenases were observed mainly in the group III SAR202 genomes, and expansions of dioxygenase enzymes were prevalent in those of group VII. We found that group I SAR202 genomes encode expansions of racemases in the enolase superfamily, which we propose evolved for the degradation of compounds that resist biological oxidation because of chiral complexity. Supporting the conclusion that the paralog expansions indicate metabolic specialization, fragment recruitment and fluorescent in situ hybridization (FISH) with phylogenetic probes showed that SAR202 subclades are indigenous to different ocean depths and geographical regions. Surprisingly, some of the subclades were abundant in surface waters and contained rhodopsin genes, altering our understanding of the ecological role of SAR202 species in stratified water columns. IMPORTANCE The oceans contain an estimated 662 Pg C in the form of dissolved organic matter (DOM). Information about microbial interactions with this vast resource is limited, despite broad recognition that DOM turnover has a major impact on the global carbon cycle. To explain patterns in the genomes of marine bacteria, we propose hypothetical metabolic pathways for the oxidation of organic molecules that are resistant to oxidation via common pathways. The hypothetical schemes we propose suggest new metabolic pathways and classes of compounds that could be important for understanding the distribution of organic carbon throughout the biosphere. These genome-based schemes will remain hypothetical until evidence from experimental cell biology can be gathered to test them. Our findings also fundamentally change our understanding of the ecology of SAR202 bacteria, showing that metabolically diverse variants of these cells occupy niches spanning all depths and are not relegated to the dark ocean.
Linkages among dissolved organic matter export, dissolved metabolites, and associated microbial community structure response in the northwestern Sargasso Sea on a seasonal scale

(Frontiers Media, 2022-03-08) Liu, Shuting ; Longnecker, Krista ; Kujawinski, Elizabeth B. ; Vergin, Kevin ; Bolaños, Luis M. ; Giovannoni, Stephen J. ; Parsons, Rachel J. ; Opalk, Keri ; Halewood, Elisa ; Hansell, Dennis A. ; Johnson, Rodney J. ; Curry, Ruth G. ; Carlson, Craig A.

Deep convective mixing of dissolved and suspended organic matter from the surface to depth can represent an important export pathway of the biological carbon pump. The seasonally oligotrophic Sargasso Sea experiences annual winter convective mixing to as deep as 300 m, providing a unique model system to examine dissolved organic matter (DOM) export and its subsequent compositional transformation by microbial oxidation. We analyzed biogeochemical and microbial parameters collected from the northwestern Sargasso Sea, including bulk dissolved organic carbon (DOC), total dissolved amino acids (TDAA), dissolved metabolites, bacterial abundance and production, and bacterial community structure, to assess the fate and compositional transformation of DOM by microbes on a seasonal time-scale in 2016–2017. DOM dynamics at the Bermuda Atlantic Time-series Study site followed a general annual trend of DOC accumulation in the surface during stratified periods followed by downward flux during winter convective mixing. Changes in the amino acid concentrations and compositions provide useful indices of diagenetic alteration of DOM. TDAA concentrations and degradation indices increased in the mesopelagic zone during mixing, indicating the export of a relatively less diagenetically altered (i.e., more labile) DOM. During periods of deep mixing, a unique subset of dissolved metabolites, such as amino acids, vitamins, and benzoic acids, was produced or lost. DOM export and compositional change were accompanied by mesopelagic bacterial growth and response of specific bacterial lineages in the SAR11, SAR202, and SAR86 clades, Acidimicrobiales, and Flavobacteria, during and shortly following deep mixing. Complementary DOM biogeochemistry and microbial measurements revealed seasonal changes in DOM composition and diagenetic state, highlighting microbial alteration of the quantity and quality of DOM in the ocean.
Particulate and dissolved metabolite distributions along a latitudinal transect of the western Atlantic Ocean

(Association for the Sciences of Limnology and Oceanography, 2022-12-23) Johnson, Winifred M. ; Kido Soule, Melissa C. ; Longnecker, Krista ; Bhatia, Maya P. ; Hallam, Steven J. ; Lomas, Michael W. ; Kujawinski, Elizabeth B.

Metabolites, or the small organic molecules that are synthesized by cells during metabolism, comprise a complex and dynamic pool of carbon in the ocean. They are an essential currency in interactions at the population and community levels of biological organization. Characterizing metabolite distributions inside microbial cells and dissolved in seawater is essential to understanding the controls on their production and fate, as well as their roles in shaping marine microbial food webs. Here, we apply a targeted metabolomics method to quantify particulate and dissolved distributions of a suite of biologically relevant metabolites including vitamins, amino acids, nucleic acids, osmolytes, and intermediates in biosynthetic pathways along a latitudinal transect in the western Atlantic Ocean. We find that, in the upper 200 m of the water column, most particulate or intracellular metabolites positively covary with the most abundant microbial taxa. In contrast, dissolved metabolites exhibited greater variability with differences in distribution between ocean regions. Although fewer particulate metabolites were detected below 200 m, the particulate metabolites identified in the deep ocean may be linked to adaptive physiological strategies of deep‐sea microbes. Based on the identified metabolite distributions, we propose relationships between certain metabolites and microbial populations, and find that dissolved metabolite distributions are not directly related to their particulate abundances.
Optical insight into riverine influences on dissolved and particulate organic carbon in a coastal arctic lagoon system

(American Geophysical Union, 2023-04-04) Catipovic, Luka ; Longnecker, Krista ; Okkonen, Stephen R. ; Koestner, Daniel ; Laney, Samuel R.

Arctic coastal margins receive organic material input from rivers, melted sea ice, and coastal erosion, phenomena that are all undergoing changes related to global climate. The optical properties of coastal Arctic waters contain information on this organic material, and we examined three optical properties of seawater (absorption, backscatter, and fluorescence) for their relationships to variability in dissolved and particulate organic carbon (DOC and POC) in Stefansson Sound, Alaska, a coastal Arctic embayment. During open water periods in 2018 and 2019, DOC was inversely correlated with salinity (r2 = 0.97) and positively correlated with dissolved organic matter fluorescence (fDOM; r2 = 0.67). DOC showed strong correlation with the nonparticulate absorption coefficient at 440 nm (ag(440)) only in 2018 (r2 = 0.95). The vertical structure of fDOM in Stefansson Sound aligned with density profiles more strongly in 2018 than in 2019, and higher levels of fDOM, ag(440), and backscatter seen near the bottom in 2019 suggest wind‐driven mixing and/or bottom resuspension events. In both years, DOC correlated strongly with the spectral slope of the absorption coefficient between 412 and 550 nm (r2 = 0.70), and POC was well correlated with spectral backscattering at 470, 532, and 660 nm (r2 = 0.90, 0.71, and 0.59). These interannual differences in the spatial and vertical distributions of DOC and POC, and their respective correlations with optical proxies, likely reflect regional climatological factors such as precipitation over the adjacent watersheds, wind patterns, and residual sea ice in late summer.
Chitin utilization by marine picocyanobacteria and the evolution of a planktonic lifestyle

(National Academy of Sciences, 2023-05-16) Capovilla, Giovanna ; Braakman, Rogier ; Fournier, Gregory P. ; Hackl, Thomas ; Schwartzman, Julia ; Lu, Xinda ; Yelton, Alexis ; Longnecker, Krista ; Soule, Melissa C. Kido ; Thomas, Elaina ; Swarr, Gretchen ; Mongera, Alessandro ; Payette, Jack G. ; Castro, Kurt G. ; Waldbauer, Jacob R. ; Kujawinski, Elizabeth B. ; Cordero, Otto X. ; Chisholm, Sallie W.

Marine picocyanobacteria Prochlorococcus and Synechococcus, the most abundant photosynthetic cells in the oceans, are generally thought to have a primarily single-celled and free-living lifestyle. However, while studying the ability of picocyanobacteria to supplement photosynthetic carbon fixation with the use of exogenous organic carbon, we found the widespread occurrence of genes for breaking down chitin, an abundant source of organic carbon that exists primarily as particles. We show that cells that encode a chitin degradation pathway display chitin degradation activity, attach to chitin particles, and show enhanced growth under low light conditions when exposed to chitosan, a partially deacetylated soluble form of chitin. Marine chitin is largely derived from arthropods, which underwent major diversifications 520 to 535 Mya, close to when marine picocyanobacteria are inferred to have appeared in the ocean. Phylogenetic analyses confirm that the chitin utilization trait was acquired at the root of marine picocyanobacteria. Together this leads us to postulate that attachment to chitin particles allowed benthic cyanobacteria to emulate their mat-based lifestyle in the water column, initiating their expansion into the open ocean, seeding the rise of modern marine ecosystems. Subsequently, transitioning to a constitutive planktonic life without chitin associations led to cellular and genomic streamlining along a major early branch within Prochlorococcus. Our work highlights how the emergence of associations between organisms from different trophic levels, and their coevolution, creates opportunities for colonizing new environments. In this view, the rise of ecological complexity and the expansion of the biosphere are deeply intertwined processes.

Longnecker Krista

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