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In January 2020, the US Ocean Carbon & Biogeochemistry (OCB) Project Office funded the Ocean Nucleic Acids 'omics Intercalibration and Standardization workshop held at the University of North Carolina in Chapel Hill. Thirty-two participants from across the US, along with guests from Canada and France, met to develop a framework for standardization and intercalibration (S&I) of ocean nucleic acid ‘omics (na’omics) approaches (i.e., amplicon sequencing, metagenomics and metatranscriptomics). During the three-day workshop, participants discussed numerous topics, including: a) sample biomass collection and nucleic acid preservation for downstream analysis, b) extraction protocols for nucleic acids, c) addition of standard reference material to nucleic acid isolation protocols, d) isolation methods unique to RNA, e) sequence library construction, and f ) integration of bioinformatic considerations. This report provides a summary of these and other topics covered during the workshop and a series of recommendations for future S&I activities for na’omics approaches.

Variation in the microbial cycling of nutrients and carbon in the ocean is an emergent property of complex planktonic communities. While recent findings have considerably expanded our understanding of the diversity and distribution of nitrogen (N2) fixing marine diazotrophs, knowledge gaps remain regarding ecological interactions between diazotrophs and other community members. Using quantitative 16S and 18S V4 rDNA amplicon sequencing, we surveyed eukaryotic and prokaryotic microbial communities from samples collected in August 2016 and 2017 across the Western North Atlantic. Leveraging and significantly expanding an earlier published 2015 molecular dataset, we examined microbial community structure and ecological co-occurrence relationships associated with intense hotspots of N2 fixation previously reported at sites off the Southern New England Shelf and Mid-Atlantic Bight. Overall, we observed a negative relationship between eukaryotic diversity and both N2 fixation and net community production (NCP). Maximum N2 fixation rates occurred at sites with high abundances of mixotrophic stramenopiles, notably Chrysophyceae. Network analysis revealed such stramenopiles to be keystone taxa alongside the haptophyte diazotroph host Braarudosphaera bigelowii and chlorophytes. Our findings highlight an intriguing relationship between marine stramenopiles and high N2 fixation coastal sites.

The Galápagos Archipelago lies within the Eastern Equatorial Pacific Ocean at the convergence of major ocean currents that are subject to changes in circulation. The nutrient-rich Equatorial Undercurrent upwells from the west onto the Galápagos platform, stimulating primary production, but this source of deep water weakens during El Niño events. Based on measurements from repeat cruises, the 2015/16 El Niño was associated with declines in phytoplankton biomass at most sites throughout the archipelago and reduced utilization of nitrate, particularly in large-sized phytoplankton in the western region. Protistan assemblages were identified by sequencing the V4 region of the 18S rRNA gene. Dinoflagellates, chlorophytes and diatoms dominated most sites. Shifts in dinoflagellate communities were most apparent between the years; parasitic dinoflagellates, Syndiniales, were highly detected during the El Niño (2015) while the dinoflagellate genus, Gyrodinium, increased at many sites during the neutral period (2016). Variations in protistan communities were most strongly correlated with changes in subthermocline water density. These findings indicate that marine protistan communities in this region are regimented by deep water mass sources and thus could be profoundly affected by altered ocean circulation.

International audience; The goal of the EXport Processes in the Ocean from RemoTe Sensing (EXPORTS) field campaign is to develop a predictive understanding of the export, fate, and carbon cycle impacts of global ocean net primary production. To accomplish this goal, observations of export flux pathways, plankton community composition, food web processes, and optical, physical, and biogeochemical (BGC) properties are needed over a range of ecosystem states. Here we introduce the first EXPORTS field deployment to Ocean Station Papa in the Northeast Pacific Ocean during summer of 2018, providing context for other papers in this special collection. The experiment was conducted with two ships: a Process Ship, focused on ecological rates, BGC fluxes, temporal changes in food web, and BGC and optical properties, that followed an instrumented Lagrangian float; and a Survey Ship that sampled BGC and optical properties in spatial patterns around the Process Ship. An array of autonomous underwater assets provided measurements over a range of spatial and temporal scales, and partnering programs and remote sensing observations provided additional observational context. The oceanographic setting was typical of late-summer conditions at Ocean Station Papa: a shallow mixed layer, strong vertical and weak horizontal gradients in hydrographic properties, sluggish sub-inertial currents, elevated macronutrient concentrations and low phytoplankton abundances. Although nutrient concentrations were consistent with previous observations, mixed layer chlorophyll was lower than typically observed, resulting in a deeper euphotic zone. Analyses of surface layer temperature and salinity found three distinct surface water types, allowing for diagnosis of whether observed changes were spatial or temporal. The 2018 EXPORTS field deployment is among the most comprehensive biological pump studies ever conducted. A second deployment to the North Atlantic Ocean occurred in spring 2021, which will be followed by focused work on data synthesis and modeling using the entire EXPORTS data set.

The Galápagos Archipelago lies within the eastern equatorial Pacific Ocean at the convergence of major ocean currents that are subject to changes in circulation. The nutrient-rich Equatorial Undercurrent upwells from the west onto the Galápagos platform, stimulating primary production, but this source of deep water weakens during El Niño events. From measurements collected on repeat cruises, the 2015/16 El Niño was associated with declines in phytoplankton biomass at most sites throughout the archipelago and reduced utilization of nitrate, particularly in large-sized phytoplankton in the western region. Protistan assemblages were identified by sequencing the V4 region of the 18S rRNA gene. Dinoflagellates, chlorophytes, and diatoms dominated most sites. Shifts in dinoflagellate communities were most apparent between the years; parasitic dinoflagellates, Syndiniales, were highly detected during the El Niño (2015) while the dinoflagellate genus, Gyrodinium dominated many sites during the neutral period (2016). Variations in protistan communities were most strongly correlated with changes in subthermocline water density. These findings indicate that marine protistan communities in this region are regimented by deep water mass sources and thus could be profoundly affected by altered ocean circulation.

Marine Group II Euryarchaeota (Candidatus Poseidoniales), abundant but yet-uncultivated members of marine microbial communities, are thought to be (photo)heterotrophs that metabolize dissolved organic matter (DOM), such as lipids and peptides. However, little is known about their transcriptional activity. We mapped reads from a metatranscriptomic time series collected at Sapelo Island (GA, USA) to metagenome-assembled genomes to determine the diversity of transcriptionally active Ca. Poseidoniales. Summer metatranscriptomes had the highest abundance of Ca. Poseidoniales transcripts, mostly from the O1 and O3 genera within Ca. Thalassarchaeaceae (MGIIb). In contrast, transcripts from fall and winter samples were predominantly from Ca. Poseidoniaceae (MGIIa). Genes encoding proteorhodopsin, membrane-bound pyrophosphatase, peptidase/proteases, and part of the ß-oxidation pathway were highly transcribed across abundant genera. Highly transcribed genes specific to Ca. Thalassarchaeaceae included xanthine/uracil permease and receptors for amino acid transporters. Enrichment of Ca. Thalassarchaeaceae transcript reads related to protein/peptide, nucleic acid, and amino acid transport and metabolism, as well as transcript depletion during dark incubations, provided further evidence of heterotrophic metabolism. Quantitative PCR analysis of South Atlantic Bight samples indicated consistently abundant Ca. Poseidoniales in nearshore and inshore waters. Together, our data suggest that Ca. Thalassarchaeaceae are important photoheterotrophs potentially linking DOM and nitrogen cycling in coastal waters.

The Galapagos Archipelago is located at the intersection of several major oceanographic features that produce diverse environmental conditions around the islands, and thus has the potential to serve as a natural laboratory for discerning the underlying environmental factors that structure marine microbial communities. Here we used quantitative metagenomics to characterize microbial communities in relation to archipelago marine habitats, and how those populations shift due to substantial environmental changes brought on by El Nino. Environmental conditions such as temperature, salinity, inorganic dissolved nutrients, and dissolved organic carbon (DOC) concentrations varied throughout the archipelago, revealing a diversity of potential microbial niches arising from upwelling, oligotrophic to eutrophic gradients, physical isolation, and potential island mass effects. The volumetric abundances of microbial community members shifted with these environmental changes and revealed several taxonomic indicators of different water masses. This included a transition from a Synechococcus dominated system in the west to an even mix of Synechococcus and Prochlorococcus in the east, mirroring the archipelago’s mesotrophic to oligotrophic and productivity gradients. Several flavobacteria groups displayed characteristic habitat distributions, including enrichment of Polaribacter and Tenacibaculum clades in the relatively nutrient rich western waters, Leeuwenhoekiella spp. that were enriched in the more nutrient-deplete central and eastern sites, and the streamlined MS024-2A group found to be abundant across all sites. During the 2015/16 El Nino event, both environmental conditions and microbial community composition were substantially altered, primarily on the western side of the archipelago due to the reduction of upwelling from the Equatorial Undercurrent. When the equatorial upwelling resumed, concentrations of inorganic nutrients and DOC at the western surface sites were more typical of mesopelagic depths. Correspondingly, Synechococcus abundances decreased by an order of magnitude, while groups associated with deeper water masses were enriched, including streamlined roseobacters HTCC2255 and HIMB11, Thioglobacaceae, methylotrophs (Methylophilaceae), archaea (Nitrosopumilaceae), and distinct subpopulations of Pelagibaceriales (SAR11 clade). These results provide a quantitative framework to connect community-wide microbial volumetric abundances to their environmental drivers, and thus incorporation into biogeochemical and ecological models.

Marine Group IIEuryarchaeota(CandidatusPoseidoniales), abundant but yet-uncultivated members of marine microbial communities, are thought to be (photo)heterotrophs that metabolize dissolved organic matter (DOM) such as lipids and peptides. However, little is known about their transcriptional activity. We mapped reads from a metatranscriptomic time series collected at Sapelo Island (GA, USA) to metagenome-assembled genomes to determine the diversity of transcriptionally-activeCa. Poseidoniales. Summer metatranscriptomes had the highest abundance ofCa. Poseidoniales transcripts, mostly from the O1 and O3 genera withinCa. Thalassarchaeaceae (MGIIb). In contrast, transcripts from fall and winter samples were predominantly fromCa. Poseidoniaceae (MGIIa). Genes encoding proteorhodopsin, membrane-bound pyrophosphatase, peptidase/proteases, and part of the β-oxidation pathway were highly transcribed across abundant genera. Highly transcribed genes specific toCa. Thalassarchaeaceae included xanthine/uracil permease and receptors for amino acid transporters. Enrichment ofCa. Thalassarchaeaceae transcript reads related to protein/peptide, nucleic acid, and amino acid transport and metabolism, as well as transcript depletion during dark incubations, provided further evidence of heterotrophic metabolism. Quantitative PCR analysis of South Atlantic Bight samples indicated consistently abundantCa. Poseidoniales in nearshore and inshore waters. Together, our data suggestCa. Thalassarchaeaceae are important photoheterotrophs potentially linking DOM and nitrogen cycling in coastal waters.

Bacteria carry out critical ecological and biogeochemical processes and form the foundations of ecosystems. Identifying the factors that influence microbial community composition and the functional capabilities encoded within them is key to predicting how microbes impact an ecosystem. Because microorganisms must compete for limited space and nutrients to promote their own propagation, they have evolved diverse mechanisms to outcompete or kill competitors. However, the genes and regulatory strategies that promote such competitive abilities are largely underexplored, particularly in free-living marine bacteria. Here, genetics and omics techniques are used to investigate how a model marine bacterium is capable of quickly eliminating natural competitors in coculture. We determined that a previously uncharacterized horizontally acquired gene cluster is required for this bacterium to kill diverse competitors. This work represents an important step toward understanding the mechanisms bacterial populations can use to become dominant members in marine microbial communities., The Roseobacter clade is a group of alphaproteobacteria that have diverse metabolic and regulatory capabilities. They are abundant in marine environments and have a substantial role in marine ecology and biogeochemistry. However, interactions between roseobacters and other bacterioplankton have not been extensively explored. In this study, we identify a killing mechanism in the model roseobacter Ruegeria pomeroyi DSS-3 by coculturing it with a group of phylogenetically diverse bacteria. The killing mechanism is diffusible and occurs when cells are grown both on surfaces and in suspension and is dependent on cell density. A screen of random transposon mutants revealed that the killing phenotype, as well as resistance to killing, require genes within an ∼8-kb putative gamma-butyrolactone synthesis gene cluster, which resembles similar pheromone-sensing systems in actinomycetes that regulate secondary metabolite production, including antimicrobials. Transcriptomics revealed the gene cluster is highly upregulated in wild-type DSS-3 compared to a nonkiller mutant when grown in liquid coculture with a roseobacter target. Our findings show that R. pomeroyi has the capability to eliminate closely and distantly related competitors, providing a mechanism to alter the community structure and function in its native habitats. IMPORTANCE Bacteria carry out critical ecological and biogeochemical processes and form the foundations of ecosystems. Identifying the factors that influence microbial community composition and the functional capabilities encoded within them is key to predicting how microbes impact an ecosystem. Because microorganisms must compete for limited space and nutrients to promote their own propagation, they have evolved diverse mechanisms to outcompete or kill competitors. However, the genes and regulatory strategies that promote such competitive abilities are largely underexplored, particularly in free-living marine bacteria. Here, genetics and omics techniques are used to investigate how a model marine bacterium is capable of quickly eliminating natural competitors in coculture. We determined that a previously uncharacterized horizontally acquired gene cluster is required for this bacterium to kill diverse competitors. This work represents an important step toward understanding the mechanisms bacterial populations can use to become dominant members in marine microbial communities.

Vibrio species of the Harveyi clade are commonly found in free-living and host-associated marine habitats. Here, we report the draft genome sequence for a Harveyi clade bacterium, Vibrio sp. strain LB10LO1, which was isolated from the Atlantic brief squid Lolliguncula brevis.

Aquatic environments contain large communities of microorganisms whose synergistic interactions mediate the cycling of major and trace nutrients, including vitamins. B-vitamins are essential coenzymes that many organisms cannot synthesize. Thus, their exchange among de novo synthesizers and auxotrophs is expected to play an important role in the microbial consortia and explain some of the temporal and spatial changes observed in diversity. In this study, we analyzed metatranscriptomes of a natural marine microbial community, diel sampled quarterly over one year to try to identify the potential major B-vitamin synthesizers and consumers. Transcriptomic data showed that the best-represented taxa dominated the expression of synthesis genes for some B-vitamins but lacked transcripts for others. For instance, Rhodobacterales dominated the expression of vitamin-B12 synthesis, but not of vitamin-B7 , whose synthesis transcripts were mainly represented by Flavobacteria. In contrast, bacterial groups that constituted less than 4% of the community (e.g., Verrucomicrobia) accounted for most of the vitamin-B1 synthesis transcripts. Furthermore, ambient vitamin-B1 concentrations were higher in samples collected during the day, and were positively correlated with chlorophyll-a concentrations. Our analysis supports the hypothesis that the mosaic of metabolic interdependencies through B-vitamin synthesis and exchange are key processes that contribute to shaping microbial communities in nature.

Microbes play a dominant role in the biogeochemistry of coastal waters, which receive organic matter from diverse sources. We present metagenomes and 45 metagenome-assembled genomes (MAGs) from Sapelo Island, Georgia, to further understand coastal microbial populations., Microbes play a dominant role in the biogeochemistry of coastal waters, which receive organic matter from diverse sources. We present metagenomes and 45 metagenome-assembled genomes (MAGs) from Sapelo Island, Georgia, to further understand coastal microbial populations. Notably, four MAGs are archaea, with two Thaumarchaeota and two marine group II Euryarchaeota.

The roseobacter clade is a group of α-proteobacteria that have diverse metabolic and regulatory capabilities. They are abundant in marine environments and have a substantial role in marine ecology and biogeochemistry. However, interactions between roseobacters and other bacterioplankton have not been extensively explored. In this study, we identify a killing mechanism in the model Roseobacter Ruegeria pomeroyi DSS-3 by competing it against a group of phylogenetically diverse bacteria. The killing mechanism involves an unidentified antimicrobial compound that is produced when cells are grown on both surfaces and in suspension and is dependent on cell density. A screen of random transposon mutants revealed the killing phenotype, as well as resistance to the antimicrobial, require genes within an ~8 kb putative γ-butyrolactone synthesis gene cluster, which resembles similar pheromone-sensing systems in actinomycetes that regulate secondary metabolite production. Transcriptomics revealed the gene cluster is highly upregulated in wild-type DSS-3 compared to a non-killer mutant when grown in liquid coculture with a roseobacter target. Our findings show that R. pomeroyi has the capability to eliminate closely- and distantly-related competitors, providing a mechanism to alter the community structure and function in its native habitats.

Marine N2 fixation supports a significant portion of oceanic primary production by making N2 bioavailable to planktonic communities, in the process influencing atmosphere-ocean carbon fluxes and our global climate. However, the geographical distribution and controlling factors of marine N2 fixation remain elusive largely due to sparse observations. Here we present unprecedented high-resolution underway N2 fixation estimates across over 6000 kilometers of the western North Atlantic. Unexpectedly, we find increasing N2 fixation rates from the oligotrophic Sargasso Sea to North America coastal waters, driven primarily by cyanobacterial diazotrophs. N2 fixation is best correlated to phosphorus availability and chlorophyll-a concentration. Globally, intense N2 fixation activity in the coastal oceans is validated by a meta-analysis of published observations and we estimate the annual coastal N2 fixation flux to be 16.7 Tg N. This study broadens the biogeography of N2 fixation, highlights the interplay of regulating factors, and reveals thriving diazotrophic communities in coastal waters with potential significance to the global nitrogen and carbon cycles. The geographical distribution and controlling factors of marine N2 fixation are understudied. Here the authors find increasing rates of N2 fixation from the Sargasso Sea to the coastal waters of North America, driven primarily by cyanobacterial diazotrophs and best correlated with phosphorus availability and chlorophyll-a concentrations.

Aquatic environments contain diverse microbial communities whose complex interactions mediate the cycling of major and trace nutrients such as vitamins. B-vitamins are essential coenzymes that many organisms cannot synthesize. Thus their exchange among de-novo synthesizers and auxotrophs is expected to play an important role in the microbial consortia and explain some of the temporal and spatial changes observed in diversity. In this study, we analyzed metatranscriptomes of a natural coastal microbial community, diel sampled-quarterly over one year to try to identify the potential major B-vitamin synthesizers and consumers. Our transcriptomic data show that the best-represented taxa dominated the expression of synthesis genes for some B-vitamins but lacked transcripts for others. For instance, Rhodobacterales dominated the expression of vitamin-B12synthesis, but not of vitamin-B7, whose synthesis transcripts were mainly represented by Flavobacteria.In contrast, bacterial groups that constituted less than 4% of the community (e.g., Verrucomicrobia) accounted for most of the vitamin-B1synthesis transcripts. Furthermore, ambient vitamin-B1concentrations were higher in samples collected during the day, and were positively correlated with chlorophyll-aconcentration. Our analysis supports the hypothesis that the mosaic of metabolic interdependencies through B-vitamin synthesis and exchange are key processes that contribute to shaping microbial communities in nature.

WOS:000447661300002; International audience; Marine net community production (NCP) tracks uptake of carbon by plankton communities and its potential transport to depth. Relationships between marine microbial community composition and NCP currently remain unclear despite their importance for assessing how different taxa impact carbon export. We conducted 16 and 18S rRNA gene (rDNA) sequencing on samples collected across the Western North Atlantic in parallel with high-resolution O-2/Ar-derived NCP measurements. Using an internal standard technique to estimate in-situ prokaryotic and eukaryotic rDNA abundances per liter, we employed statistical approaches to relate patterns of microbial diversity to NCP. Taxonomic abundances calculated using internal standards provided valuable context to traditional relative abundance metrics. A bloom in the Mid-Atlantic Bight featured high eukaryote abundances with low eukaryotic diversity and was associated with the harmful algal bloom-forming Aureococcusanophagefferens, phagotrophic algae, heterotrophic flagellates, and particle-associated bacteria. These results show that coastal Aureococcus blooms host a distinct community associated with regionally significant peaks in NCP. Meanwhile, weak relationships between taxonomy and NCP in less-productive waters suggest that productivity across much of this region is not linked to specific microplankton taxa.

WOS:000459327600015; International audience; An inherent issue in high-throughput rRNA gene tag sequencing microbiome surveys is that they provide compositional data in relative abundances. This often leads to spurious correlations, making the interpretation of relationships to biogeochemical rates challenging. To overcome this issue, we quantitatively estimated the abundance of microorganisms by spiking in known amounts of internal DNA standards. Using a 3-year sample set of diverse microbial communities from the Western Antarctica Peninsula, we demonstrated that the internal standard method yielded community profiles and taxon cooccurrence patterns substantially different from those derived using relative abundances. We found that the method provided results consistent with the traditional CHEMTAX analysis of pigments and total bacterial counts by flow cytometry. Using the internal standard method, we also showed that chloroplast 16S rRNA gene data in microbial surveys can be used to estimate abundances of certain eukaryotic phototrophs such as cryptophytes and diatoms. In Phaeocystis, scatter in the 16S/18S rRNA gene ratio may be explained by physiological adaptation to environmental conditions. We conclude that the internal standard method, when applied to rRNA gene microbial community profiling, is quantitative and that its application will substantially improve our understanding of microbial ecosystems. IMPORTANCE High-throughput-sequencing-based marine microbiome profiling is rapidly expanding and changing how we study the oceans. Although powerful, the technique is not fully quantitative; it provides taxon counts only in relative abundances. In order to address this issue, we present a method to quantitatively estimate microbial abundances per unit volume of seawater filtered by spiking known amounts of internal DNA standards into each sample. We validated this method by comparing the calculated abundances to other independent estimates, including chemical markers (pigments) and total bacterial cell counts by flow cytometry. The internal standard approach allows us to quantitatively estimate and compare marine microbial community profiles, with important implications for linking environmental microbiomes to quantitative processes such as metabolic and biogeochemical rates.

Satinsky, Brandon M., et al. 'Use of internal standards for quantitative metatranscriptome and metagenome analysis.' Methods in enzymology 531 (2012): 237-250.

This protocol is from: Satinsky, Brandon M., et al. "Use of internal standards for quantitative metatranscriptome and metagenome analysis." Methods in enzymology 531 (2012): 237-250. Please see the full chapter for additional details.

Microbially-mediated transformations of dissolved organic matter (DOM) in a marsh-dominated estuarine system were investigated at the molecular level using ultrahigh resolution mass spectrometry. In addition to observing spatial and temporal variability in DOM sources in the estuary, multiple incubations with endogenous microorganisms identified the influence of DOM composition on biodegradation. A clear microbial preference for degradation of compounds associated with marine DOM relative to those of terrestrial origin was observed, resulting in an overall shift of the remaining DOM toward a stronger terrigenous signature. During short, one-day long incubations of samples rich in marine DOM, the molecular formulae that were enriched had slightly smaller mass (20-30 Da) and number of carbon atoms compared to the molecular formulae that were depleted. Over longer time scales (70 days), the mean differences in molecular mass between formulae that were depleted and enriched were substantially larger (~270 Da). The differences in elemental composition over daily time scales were consistent with transformations in functional groups; over longer time scales, the differences in elemental composition may be related to progressive transformations of functional groups of intermediate products and/or other reactions. Our results infused new data toward the understanding of DOM processing by bacterioplankton in estuarine systems.

Summary Ruegeria pomeroyi DSS-3 possesses two general pathways for metabolism of dimethylsulphoniopropionate (DMSP), an osmolyte of algae and abundant carbon source for marine bacteria. In the DMSP cleavage pathway, acrylate is transformed into acryloyl-CoA by propionate-CoA ligase (SPO2934) and other unidentified acyl-CoA ligases. Acryloyl-CoA is then reduced to propionyl-CoA by AcuI or SPO1914. Acryloyl-CoA is also rapidly hydrated to 3-hydroxypropionyl-CoA by acryloyl-CoA hydratase (SPO0147). A SPO1914 mutant was unable to grow on acrylate as the sole carbon source, supporting its role in this pathway. Similarly, growth on methylmercaptopropionate, the first intermediate of the DMSP demethylation pathway, was severely inhibited by a mutation in the gene encoding crotonyl-CoA carboxylase/reductase, demonstrating that acetate produced by this pathway was metabolized by the ethylmalonyl-CoA pathway. Amino acids and nucleosides from cells grown on 13C-enriched DMSP possessed labelling patterns that were consistent with carbon from DMSP being metabolized by both the ethylmalonyl-CoA and acrylate pathways as well as a role for pyruvate dehydrogenase. This latter conclusion was supported by the phenotype of a pdh mutant, which grew poorly on electron-rich substrates. Additionally, label from [13C-methyl] DMSP only appeared in carbons derived from methyl-tetrahydrofolate, and there was no evidence for a serine cycle of C-1 assimilation.

Satinsky, Brandon M., et al. 'Use of internal standards for quantitative metatranscriptome and metagenome analysis.' Methods in enzymology 531 (2012): 237-250.

This protocol is from: Satinsky, Brandon M., et al. "Use of internal standards for quantitative metatranscriptome and metagenome analysis." Methods in enzymology 531 (2012): 237-250. Please see the full chapter for additional details.

The members of the OM43 clade of Betaproteobacteria are abundant coastal methylotrophs with a range of carbon-utilizing capabilities. However, their underlying transcriptional and metabolic responses to shifting conditions or different carbon substrates remain poorly understood. We examined the transcriptional dynamics of OM43 isolate NB0046 subjected to various inorganic nutrient, vitamin, and carbon substrate regimes over different growth phases to (i) develop a quantitative model of its mRNA content; (ii) identify transcriptional markers of physiological activity, nutritional state, and carbon and energy utilization; and (iii) identify pathways involved in methanol or naturally occurring dissolved organic matter (DOM) metabolism. Quantitative transcriptomics, achieved through addition of internal RNA standards, allowed for analyses on a transcripts-per-cell scale. This streamlined bacterium exhibited substantial shifts in total mRNA content (ranging from 1,800 to 17 transcripts cell[superscript −1] in the exponential and deep stationary phases, respectively) and gene-specific transcript abundances (>1,000-fold increases in some cases), depending on the growth phase and nutrient conditions. Carbon metabolism genes exhibited substantial dynamics, including those for ribulose monophosphate, tricarboxylic acid (TCA), and proteorhodopsin, as well as methanol dehydrogenase (xoxF), which, while always the most abundant transcript, increased from 5 to 120 transcripts cell[superscript −1] when cultures were nutrient and vitamin amended. In the DOM treatment, upregulation of TCA cycle, methylcitrate cycle, vitamin, and organic phosphorus genes suggested a metabolic route for this complex mixture of carbon substrates. The genome-wide inventory of transcript abundances produced here provides insight into a streamlined marine bacterium’s regulation of carbon metabolism and energy flow, providing benchmarks for evaluating the activity of OM43 populations in situ., National Science Foundation (U.S.) (EF0424599), Simons Foundation (329108), Gordon and Betty Moore Foundation (GBMF3298 and GBMF3777)

Resolving the ecological niches of coexisting marine microbial taxa is challenging due to the high species richness of microbial communities and the apparent functional redundancy in bacterial genomes and metagenomes. Here, we generated over 11 million Illumina reads of protein-encoding transcripts collected from well-mixed southeastern US coastal waters to characterize gene expression patterns distinguishing the ecological roles of hundreds of microbial taxa sharing the same environment. The taxa with highest in situ growth rates (based on relative abundance of ribosomal protein transcripts) were typically not the greatest contributors to community transcription, suggesting strong top-down ecological control, and their diverse transcriptomes indicated roles as metabolic generalists. The taxa with low in situ growth rates typically had low diversity transcriptomes dominated by specialized metabolisms. By identifying protein-encoding genes with atypically high expression for their level of conservation, unique functional roles of community members emerged related to substrate use (such as complex carbohydrates, fatty acids, methanesulfonate, taurine, tartrate, ectoine), alternative energy-conservation strategies (proteorhodopsin, AAnP, V-type pyrophosphatases, sulfur oxidation, hydrogen oxidation) and mechanisms for negotiating a heterogeneous environment (flagellar motility, gliding motility, adhesion strategies). On average, the heterotrophic bacterioplankton dedicated 7% of their transcriptomes to obtaining energy by non-heterotrophic means. This deep sequencing of a coastal bacterioplankton transcriptome provides the most highly resolved view of bacterioplankton niche dimensions yet available, uncovering a spectrum of unrecognized ecological strategies.

Decline of native pelagic species in estuarine systems is an increasing problem, especially for native fishes in the San Francisco Estuary and Delta (SFE-D). Addressing these losses depends on understanding trophodynamics in the food web that supports threatened species. We quantified the role of microzooplankton (heterotrophic-mixotrophic protists

Quantitative PCR (qPCR) analysis revealed elevated relative abundance (1.8% of prokaryotes) of marine group 1 Crenarchaeota (MG1C) in two samples of southeastern US coastal bacterioplankton, collected in August 2008, compared with samples collected from the same site at different times (mean 0.026%). We analyzed the MG1C sequences in metatranscriptomes from these samples to gain an insight into the metabolism of MG1C population growing in the environment, and for comparison with ammonia-oxidizing bacteria (AOB) in the same samples. Assemblies revealed low diversity within sequences assigned to most individual MG1C open reading frames (ORFs) and high homology with ‘Candidatus Nitrosopumilus maritimus' strain SCM1 genome sequences. Reads assigned to ORFs for ammonia uptake and oxidation accounted for 37% of all MG1C transcripts. We did not recover any reads for Nmar_1354–Nmar_1357, proposed to encode components of an alternative, nitroxyl-based ammonia oxidation pathway; however, reads from Nmar_1259 and Nmar_1667, annotated as encoding a multicopper oxidase with homology to nirK, were abundant. Reads assigned to two homologous ORFs (Nmar_1201 and Nmar_1547), annotated as hypothetical proteins were also abundant, suggesting that their unknown function is important to MG1C. Superoxide dismutase and peroxiredoxin-like transcripts were more abundant in the MG1C transcript pool than in the complete metatranscriptome, suggesting an enhanced response to oxidative stress by the MG1C population. qPCR indicated low AOB abundance (0.0010% of prokaryotes), and we found no transcripts related to ammonia oxidation and only one RuBisCO transcript among the transcripts assigned to AOB, suggesting they were not responding to the same environmental cues as the MG1C population.

Summary The fraction of dissolved dimethylsulfoniopropionate (DMSPd) converted by marine bacterioplankton into the climate-active gas dimethylsulfide (DMS) varies widely in the ocean, with the factors that determine this value still largely unknown. One current hypothesis is that the ratio of DMS formation : DMSP demethylation is determined by DMSP availability, with ‘availability’ in both an absolute sense (i.e. concentration in seawater) and in a relative sense (i.e. proportionally to other labile organic S compounds) proposed as the critical factor. We investigated these models during an experimentally induced phytoplankton bloom using a taxon-specific microarray targeting DMSP-related gene transcription in members of the Roseobacter clade, a group hypothesized to play an important role in the surface ocean sulfur cycle and well represented by genome sequences. The array consisted of 1578 probes to 431 genes and was designed to target diverse Roseobacter communities in natural seawater by using hierarchical probe design based on 13 genome sequences. The prevailing pattern of Roseobacter gene transcription showed relative depletion of DMSP-related transcripts during the peak of the bloom, despite increasing absolute concentrations and flux of DMSP-related compounds. DMSPd thus appeared to be assimilated by Roseobacter populations in proportion to its relative abundance in the organic matter pool (the ‘relative sense’ hypothesis), rather than assimilated in preference to other labile organic sulfur or carbon compounds produced during the bloom. The relative investment of the Roseobacter community in DMSP demethylation was not useful for predicting the formation of DMS, however, suggesting a complex regulatory process that may involve multiple taxa and alternative fates of DMSPd.

While many studies have examined mesozooplankton feeding in coastal environments, less attention has been given to this subject in estuaries. We used bottle incubation experiments to measure the feeding rates of a cladoceran (Daphnia sp.), a calanoid copepod (Acartia spp.), and 2 cyclopoid copepods (Oithona davisae and Limnoithona tetraspina) on the protist plankton ( 2 ml predator (pred.) -1 h -1 . O. davisae consumed only ciliates in September 2004, while in Novem- ber 2004 it cleared both ciliates and diatoms at similar rates (0.8 ml pred. -1 h -1 ). L. tetraspina cleared only aloricate ciliates and flagellates (0.8 to 1.0 ml pred. -1 h -1 ). Acartia spp. had the highest clearance rates on diatoms of all the predators examined (mean 2.0 ml pred. -1 h -1 ). With respect to biomass ingestion, in every experiment mesozoo- plankton were found to ingest ciliate carbon at the highest rates (3 to 29 ng C pred. -1 h -1 ). Our results indicate that while estuarine mesozooplankton are often omnivorous, important species-specific dif- ferences exist, and microzooplankton, especially ciliates, are an important component of the upper SFE food web.

This protocol is from: Satinsky, Brandon M., et al. "Use of internal standards for quantitative metatranscriptome and metagenome analysis." Methods in enzymology 531 (2012): 237-250. Please see the full chapter for additional details.

The role of bacterioplankton in the cycling of marine dissolved organic matter (DOM) is central to the carbon and energy balance in the ocean, yet there are few model organisms available to investigate the genes, metabolic pathways, and biochemical mechanisms involved in the degradation of this globally important carbon pool. To obtain microbial isolates capable of degrading semi-labile DOM for growth, we conducted dilution to extinction cultivation experiments using seawater enriched with high molecular weight (HMW) DOM. In total, 93 isolates were obtained. Amendments using HMW DOM to increase the dissolved organic carbon concentration 4x (280 μM) or 10x (700 μM) the ocean surface water concentrations yielded positive growth in 4–6% of replicate dilutions, whereas, Gordon and Betty Moore Foundation (Grant GBMF3298), Gordon and Betty Moore Foundation (Grant GBMF #3777), National Science Foundation (U.S.). Science and Technology Center (Grant ER0424599), Simons Foundation

© 2015 International Society for Microbial Ecology. The 'bacterial switch' is a proposed regulatory point in the global sulfur cycle that routes dimethylsulfoniopropionate (DMSP) to two fundamentally different fates in seawater through genes encoding either the cleavage or demethylation pathway, and affects the flux of volatile sulfur from ocean surface waters to the atmosphere. Yet which ecological or physiological factors might control the bacterial switch remains a topic of considerable debate. Here we report the first field observations of dynamic changes in expression of DMSP pathway genes by a single marine bacterial species in its natural environment. Detection of taxon-specific gene expression in Roseobacter species HTCC2255 during a month-long deployment of an autonomous ocean sensor in Monterey Bay, CA captured in situ regulation of the first gene in each DMSP pathway (dddP and dmdA) that corresponded with shifts in the taxonomy of the phytoplankton community. Expression of the cleavage pathway was relatively greater during a high-DMSP-producing dinoflagellate bloom, and expression of the demethylation pathway was greater in the presence of a mixed diatom and dinoflagellate community. These field data fit the prevailing hypothesis for bacterial DMSP gene regulation based on bacterial sulfur demand, but also suggest a modification involving oxidative stress response, evidenced as upregulation of catalase via katG, when DMSP is demethylated.

Oscillating diurnal rhythms of gene transcription, metabolic activity, and behavior are found in all three domains of life. However, diel cycles in naturally occurring heterotrophic bacteria and archaea have rarely been observed. Here, we report time-resolved whole-genome transcriptome profiles of multiple, naturally occurring oceanic bacterial populations sampled in situ over 3 days. As anticipated, the cyanobacterial transcriptome exhibited pronounced diel periodicity. Unexpectedly, several different heterotrophic bacterioplankton groups also displayed diel cycling in many of their gene transcripts. Furthermore, diel oscillations in different heterotrophic bacterial groups suggested population-specific timing of peak transcript expression in a variety of metabolic gene suites. These staggered multispecies waves of diel gene transcription may influence both the tempo and the mode of matter and energy transformation in the sea.

The direct retrieval and sequencing of environmental RNA is emerging as a powerful technique to elucidate the in situ activities of microbial communities. Here we provide a metatranscriptomic protocol describing environmental sample collection, rRNA depletion, mRNA amplification, cDNA synthesis, and bioinformatic analysis. In addition, the preparation of internal RNA standards and their addition to the sample are discussed, providing a method by which transcript numbers can be expressed as absolute abundances in the environment and more readily compared to other biogeochemical and ecological measurements.

Oscillating diurnal rhythms of gene transcription, metabolic activity, and behavior are found in all three domains of life. However, diel cycles in naturally occurring heterotrophic bacteria and archaea have rarely been observed. Here, we report time-resolved whole-genome transcriptome profiles of multiple, naturally occurring oceanic bacterial populations sampled in situ over 3 days. As anticipated, the cyanobacterial transcriptome exhibited pronounced diel periodicity. Unexpectedly, several different heterotrophic bacterioplankton groups also displayed diel cycling in many of their gene transcripts. Furthermore, diel oscillations in different heterotrophic bacterial groups suggested population-specific timing of peak transcript expression in a variety of metabolic gene suites. These staggered multispecies waves of diel gene transcription may influence both the tempo and the mode of matter and energy transformation in the sea., Gordon and Betty Moore Foundation (GBMF 492.01), Gordon and Betty Moore Foundation (GBMF 3777), National Science Foundation (U.S.) (Grant EF0424599), David & Lucile Packard Foundation

Next generation sequencing-enabled metatranscriptomic and metagenomic datasets are providing unprecedented insights into the functional diversity of microbial communities, allowing detection of the genes present in a community as well as differentiation of those being actively transcribed. An emerging challenge of meta-omics approaches is how to quantitatively compare metagenomes and metatranscriptomes collected across spatial and temporal scales, or among treatments in experimental manipulations. Here, we describe the use of internal DNA and mRNA standards in meta-omics methodologies, and highlight how data collected in an absolute framework (per L or per cell) provides increased comparative power and insight into underlying causes of differences between samples.

Ruegeria pomeroyi DSS-3 possesses two general pathways for metabolism of dimethylsulphoniopropionate (DMSP), an osmolyte of algae and abundant carbon source for marine bacteria. In the DMSP cleavage pathway, acrylate is transformed into acryloyl-CoA by propionate-CoA ligase (SPO2934) and other unidentified acyl-CoA ligases. Acryloyl-CoA is then reduced to propionyl-CoA by AcuI or SPO1914. Acryloyl-CoA is also rapidly hydrated to 3-hydroxypropionyl-CoA by acryloyl-CoA hydratase (SPO0147). A SPO1914 mutant was unable to grow on acrylate as the sole carbon source, supporting its role in this pathway. Similarly, growth on methylmercaptopropionate, the first intermediate of the DMSP demethylation pathway, was severely inhibited by a mutation in the gene encoding crotonyl-CoA carboxylase/reductase, demonstrating that acetate produced by this pathway was metabolized by the ethylmalonyl-CoA pathway. Amino acids and nucleosides from cells grown on (13) C-enriched DMSP possessed labelling patterns that were consistent with carbon from DMSP being metabolized by both the ethylmalonyl-CoA and acrylate pathways as well as a role for pyruvate dehydrogenase. This latter conclusion was supported by the phenotype of a pdh mutant, which grew poorly on electron-rich substrates. Additionally, label from [(13) C-methyl] DMSP only appeared in carbons derived from methyl-tetrahydrofolate, and there was no evidence for a serine cycle of C-1 assimilation.

We used a combination of metatranscriptomic analyses and quantitative PCR (qPCR) to study seasonal changes in Thaumarchaeota populations from a salt marsh-dominated estuary. Surface waters (0.5 m depth) were sampled quarterly at Marsh Landing, Sapelo Island, GA, USA over a 3-year period. We found a mid-summer peak in Thaumarchaeota abundance measured by qPCR of either 16S rRNA or amoA genes in each of the 3 years. Thaumarchaeota were 100–1000-fold more abundant during the peak than at other times of the year, whereas the abundance of ammonia- and nitrite-oxidizing Bacteria varied

Next generation sequencing-enabled metatranscriptomic and metagenomic datasets are providing unprecedented insights into the functional diversity of microbial communities, allowing detection of the genes present in a community as well as differentiation of those being actively transcribed. An emerging challenge of meta-omics approaches is how to quantitatively compare metagenomes and metatranscriptomes collected across spatial and temporal scales, or among treatments in experimental manipulations. Here, we describe the use of internal DNA and mRNA standards in meta-omics methodologies, and highlight how data collected in an absolute framework (per L or per cell) provides increased comparative power and insight into underlying causes of differences between samples.

A typical marine bacterial cell in coastal seawater contains only ∼200 molecules of mRNA, each of which lasts only a few minutes before being degraded. Such a surprisingly small and dynamic cellular mRNA reservoir has important implications for understanding the bacterium’s responses to environmental signals, as well as for our ability to measure those responses. In this perspective, we review the available data on transcript dynamics in environmental bacteria, and then consider the consequences of a small and transient mRNA inventory for functional metagenomic studies of microbial communities.

Dimethylsulfoniopropionate (DMSP) is an organic sulfur compound that is rapidly metabolized by marine bacteria either by cleavage to dimethylsulfide (DMS) or demethylation to 3-methiolpropionate. The abundance and diversity of genes encoding bacterial DMS production ( dddP ) and demethylation ( dmdA ) were measured in the North Pacific subtropical gyre (NPSG) between May 2008 and February 2009 at Station ALOHA (22°45′N, 158°00′W) at two depths: 25 m and the deep chlorophyll maximum (DCM; ∼100 m). The highest abundance of dmdA genes was in May 2008 at 25 m, with ∼16.5% of cells harboring a gene in one of the eight subclades surveyed, while the highest abundance of dddP genes was in July 2008 at 25 m, with ∼2% of cells harboring a gene. The dmdA gene pool was consistently dominated by homologs from SAR11 subclades, which was supported by findings in metagenomic data sets derived from Station ALOHA. Expression of the SAR11 dmdA genes was low, with typical transcript:gene ratios between 1:350 and 1:1,400. The abundance of DMSP genes was statistically different between 25 m and the DCM and correlated with a number of environmental variables, including primary production, photosynthetically active radiation, particulate DMSP, and DMS concentrations. At 25 m, dddP abundance was positively correlated with pigments that are diagnostic of diatoms; at the DCM, dmdA abundance was positively correlated with temperature. Based on gene abundance, we hypothesize that SAR11 bacterioplankton dominate DMSP cycling in the oligotrophic NPSG, with lesser but consistent involvement of other members of the bacterioplankton community.