New chemical and microbial perspectives on vitamin B1 and vitamer dynamics of a coastal system.

Vitamin B1 (thiamin, B1) is an essential micronutrient for cells, yet intriguingly in aquatic systems most bacterioplankton are unable to synthesize it de novo (auxotrophy), requiring an exogenous source. Cycling of this valuable metabolite in aquatic systems has not been fully investigated and vitamers (B1-related compounds) have only begun to be measured and incorporated into the B1 cycle. Here, we identify potential key producers and consumers of B1 and gain new insights into the dynamics of B1 cycling through measurements of B1 and vitamers (HMP: 4-amino-5-hydroxymethyl-2-methylpyrimidine, HET: 4-methyl-5-thiazoleethanol, FAMP: N-formyl-4-amino-5-aminomethyl-2-methylpyrimidine) in the particulate and dissolved pool in a temperate coastal system. Dissolved B1 was not the primary limiting nutrient for bacterial production and was relatively stable across seasons with concentrations ranging from 74-117 pM, indicating a balance of supply and demand. However, vitamer concentration changed markedly with season as did transcripts related to vitamer salvage and transport suggesting use of vitamers by certain bacterioplankton, e.g. Pelagibacterales. Genomic and transcriptomic analyses showed that up to 78% of the bacterioplankton taxa were B1 auxotrophs. Notably, de novo B1 production was restricted to a few abundant bacterioplankton (e.g. Vulcanococcus, BACL14 (Burkholderiales), Verrucomicrobiales) across seasons. In summer, abundant picocyanobacteria were important putative B1 sources, based on transcriptional activity, leading to an increase in the B1 pool. Our results provide a new dynamic view of the players and processes involved in B1 cycling over time in coastal waters, and identify specific priority populations and processes for future study.

Short-term acidification promotes diverse iron acquisition and conservation mechanisms in upwelling-associated phytoplankton.

Coastal upwelling regions are among the most productive marine ecosystems but may be threatened by amplified ocean acidification. Increased acidification is hypothesized to reduce iron bioavailability for phytoplankton thereby expanding iron limitation and impacting primary production. Here we show from community to molecular levels that phytoplankton in an upwelling region respond to short-term acidification exposure with iron uptake pathways and strategies that reduce cellular iron demand. A combined physiological and multi-omics approach was applied to trace metal clean incubations that introduced 1200 ppm CO2 for up to four days. Although variable, molecular-level responses indicate a prioritization of iron uptake pathways that are less hindered by acidification and reductions in iron utilization. Growth, nutrient uptake, and community compositions remained largely unaffected suggesting that these mechanisms may confer short-term resistance to acidification; however, we speculate that cellular iron demand is only temporarily satisfied, and longer-term acidification exposure without increased iron inputs may result in increased iron stress.

Use and detection of a vitamin B1 degradation product yields new views of the marine B1 cycle and plankton metabolite exchange.

Vitamin B1 (thiamin) is a vital nutrient for most cells in nature, including marine plankton. Early and recent experiments show that B1 degradation products instead of B1 can support the growth of marine bacterioplankton and phytoplankton. However, the use and occurrence of some degradation products remains uninvestigated, namely N-formyl-4-amino-5-aminomethyl-2-methylpyrimidine (FAMP), which has been a focus of plant oxidative stress research. We investigated the relevance of FAMP in the ocean. Experiments and global ocean meta-omic data indicate that eukaryotic phytoplankton, including picoeukaryotes and harmful algal bloom species, use FAMP while bacterioplankton appear more likely to use deformylated FAMP, 4-amino-5-aminomethyl-2-methylpyrimidine. Measurements of FAMP in seawater and biomass revealed that it occurs at picomolar concentrations in the surface ocean, heterotrophic bacterial cultures produce FAMP in the dark-indicating non-photodegradation of B1 by cells, and B1-requiring (auxotrophic) picoeukaryotic phytoplankton produce intracellular FAMP. Our results require an expansion of thinking about vitamin degradation in the sea, but also the marine B1 cycle where it is now crucial to consider a new B1-related compound pool (FAMP), as well as generation (dark degradation-likely via oxidation), turnover (plankton uptake), and exchange of the compound within the networks of plankton. IMPORTANCE Results of this collaborative study newly show that a vitamin B1 degradation product, N-formyl-4-amino-5-aminomethyl-2-methylpyrimidine (FAMP), can be used by diverse marine microbes (bacteria and phytoplankton) to meet their vitamin B1 demands instead of B1 and that FAMP occurs in the surface ocean. FAMP has not yet been accounted for in the ocean and its use likely enables cells to avoid B1 growth deficiency. Additionally, we show FAMP is formed in and out of cells without solar irradiance-a commonly considered route of vitamin degradation in the sea and nature. Altogether, the results expand thinking about oceanic vitamin degradation, but also the marine B1 cycle where it is now crucial to consider a new B1-related compound pool (FAMP), as well as its generation (dark degradation-likely via oxidation), turnover (plankton uptake), and exchange within networks of plankton.

Cobalamin producers and prokaryotic consumers in the Northwest Atlantic.

Cobalamin availability can influence primary productivity and ecological interactions in marine microbial communities. The characterization of cobalamin sources and sinks is a first step in investigating cobalamin dynamics and its impact on productivity. Here, we identify potential cobalamin sources and sinks on the Scotian Shelf and Slope in the Northwest Atlantic Ocean. Functional and taxonomic annotation of bulk metagenomic reads, combined with analysis of genome bins, were used to identify potential cobalamin sources and sinks. Cobalamin synthesis potential was mainly attributed to Rhodobacteraceae, Thaumarchaeota, and cyanobacteria (Synechococcus and Prochlorococcus). Cobalamin remodelling potential was mainly attributed to Alteromonadales, Pseudomonadales, Rhizobiales, Oceanospirilalles, Rhodobacteraceae, and Verrucomicrobia, while potential cobalamin consumers include Flavobacteriaceae, Actinobacteria, Porticoccaceae, Methylophiliaceae, and Thermoplasmatota. These complementary approaches identified taxa with the potential to be involved in cobalamin cycling on the Scotian Shelf and revealed genomic information required for further characterization. The Cob operon of Rhodobacterales bacterium HTCC2255, a strain with known importance in cobalamin cycling, was similar to a major cobalamin producer bin, suggesting that a related strain may represent a critical cobalamin source in this region. These results enable future inquiries that will enhance our understanding of how cobalamin shapes microbial interdependencies and productivity in this region.

Community Interaction Co-limitation: Nutrient Limitation in a Marine Microbial Community Context.

The simultaneous limitation of productivity by two or more nutrients, commonly referred to as nutrient co-limitation, affects microbial communities throughout the marine environment and is of profound importance because of its impacts on various biogeochemical cycles. Multiple types of co-limitation have been described, enabling distinctions based on the hypothesized mechanisms of co-limitation at a biochemical level. These definitions usually pertain to individuals and do not explicitly, or even implicitly, consider complex ecological dynamics found within a microbial community. However, limiting and co-limiting nutrients can be produced in situ by a subset of microbial community members, suggesting that interactions within communities can underpin co-limitation. To address this, we propose a new category of nutrient co-limitation, community interaction co-limitation (CIC). During CIC, one part of the community is limited by one nutrient, which results in the insufficient production or transformation of a biologically produced nutrient that is required by another part of the community, often primary producers. Using cobalamin (vitamin B12) and nitrogen fixation as our models, we outline three different ways CIC can arise based on current literature and discuss CIC's role in biogeochemical cycles. Accounting for the inherent and complex roles microbial community interactions play in generating this type of co-limitation requires an expanded toolset - beyond the traditional approaches used to identify and study other types of co-limitation. We propose incorporating processes and theories well-known in microbial ecology and evolution to provide meaningful insight into the controls of community-based feedback loops and mechanisms that give rise to CIC in the environment. Finally, we highlight the data gaps that limit our understanding of CIC mechanisms and suggest methods to overcome these and further identify causes and consequences of CIC. By providing this framework for understanding and identifying CIC, we enable systematic examination of the impacts this co-limitation can have on current and future marine biogeochemical processes.

Microbial metabolites in the marine carbon cycle.

One-quarter of photosynthesis-derived carbon on Earth rapidly cycles through a set of short-lived seawater metabolites that are generated from the activities of marine phytoplankton, bacteria, grazers and viruses. Here we discuss the sources of microbial metabolites in the surface ocean, their roles in ecology and biogeochemistry, and approaches that can be used to analyse them from chemistry, biology, modelling and data science. Although microbial-derived metabolites account for only a minor fraction of the total reservoir of marine dissolved organic carbon, their flux and fate underpins the central role of the ocean in sustaining life on Earth.

Highly-resolved interannual phytoplankton community dynamics of the coastal Northwest Atlantic.

Microbial observatories can track phytoplankton at frequencies that resolve monthly, seasonal, and multiyear trends in environmental change from short-lived events. Using 4-years of weekly flow cytometry along with chloroplast and cyanobacterial 16S rRNA gene sequence data from a time-series station in the coastal Northwest Atlantic (Bedford Basin, Nova Scotia, Canada), we analyzed temporal observations for globally-relevant genera (e.g., Bolidomonas, Teleaulax, Minidiscus, Chaetoceros, Synechococcus, and Phaeocystis) in an oceanic region that has been recognized as a likely hotspot for phytoplankton diversity. Contemporaneous Scotian Shelf data also collected during our study established that the major phytoplankton within the Bedford Basin were important in the Scotian Shelf during spring and fall, therefore pointing to their broader significance within the coastal Northwest Atlantic (NWA). Temporal trends revealed a subset of indicator taxa along with their DNA signatures (e.g., Eutreptiella and Synechococcus), whose distribution patterns make them essential for timely detection of environmentally-driven shifts in the NWA. High-resolution sampling was key to identifying important community shifts towards smaller phytoplankton under anomalous environmental conditions, while further providing a detailed molecular view of community compositions underpinning general phytoplankton succession within the coastal NWA. Our study demonstrates the importance of accessible coastal time-series sites where high-frequency DNA sampling allows for the detection of shifting baselines in phytoplankton communities.

Proteomic traits vary across taxa in a coastal Antarctic phytoplankton bloom.

Production and use of proteins is under strong selection in microbes, but it is unclear how proteome-level traits relate to ecological strategies. We identified and quantified proteomic traits of eukaryotic microbes and bacteria through an Antarctic phytoplankton bloom using in situ metaproteomics. Different taxa, rather than different environmental conditions, formed distinct clusters based on their ribosomal and photosynthetic proteomic proportions, and we propose that these characteristics relate to ecological differences. We defined and used a proteomic proxy for regulatory cost, which showed that SAR11 had the lowest regulatory cost of any taxa we observed at our summertime Southern Ocean study site. Haptophytes had lower regulatory cost than diatoms, which may underpin haptophyte-to-diatom bloom progression in the Ross Sea. We were able to make these proteomic trait inferences by assessing various sources of bias in metaproteomics, providing practical recommendations for researchers in the field. We have quantified several proteomic traits (ribosomal and photosynthetic proteomic proportions, regulatory cost) in eukaryotic and bacterial taxa, which can then be incorporated into trait-based models of microbial communities that reflect resource allocation strategies.

Cellular costs underpin micronutrient limitation in phytoplankton.

Micronutrients control phytoplankton growth in the ocean, influencing carbon export and fisheries. It is currently unclear how micronutrient scarcity affects cellular processes and how interdependence across micronutrients arises. We show that proximate causes of micronutrient growth limitation and interdependence are governed by cumulative cellular costs of acquiring and using micronutrients. Using a mechanistic proteomic allocation model of a polar diatom focused on iron and manganese, we demonstrate how cellular processes fundamentally underpin micronutrient limitation, and how they interact and compensate for each other to shape cellular elemental stoichiometry and resource interdependence. We coupled our model with metaproteomic and environmental data, yielding an approach for estimating biogeochemical metrics, including taxon-specific growth rates. Our results show that cumulative cellular costs govern how environmental conditions modify phytoplankton growth.

Molecular underpinnings and biogeochemical consequences of enhanced diatom growth in a warming Southern Ocean.

The Southern Ocean (SO) harbors some of the most intense phytoplankton blooms on Earth. Changes in temperature and iron availability are expected to alter the intensity of SO phytoplankton blooms, but little is known about how these changes will influence community composition and downstream biogeochemical processes. We performed light-saturated experimental manipulations on surface ocean microbial communities from McMurdo Sound in the Ross Sea to examine the effects of increased iron availability (+2 nM) and warming (+3 and +6 °C) on nutrient uptake, as well as the growth and transcriptional responses of two dominant diatoms, Fragilariopsis and Pseudo-nitzschia We found that community nutrient uptake and primary productivity were elevated under both warming conditions without iron addition (relative to ambient -0.5 °C). This effect was greater than additive under concurrent iron addition and warming. Pseudo-nitzschia became more abundant under warming without added iron (especially at 6 °C), while Fragilariopsis only became more abundant under warming in the iron-added treatments. We attribute the apparent advantage Pseudo-nitzschia shows under warming to up-regulation of iron-conserving photosynthetic processes, utilization of iron-economic nitrogen assimilation mechanisms, and increased iron uptake and storage. These data identify important molecular and physiological differences between dominant diatom groups and add to the growing body of evidence for Pseudo-nitzschia's increasingly important role in warming SO ecosystems. This study also suggests that temperature-driven shifts in SO phytoplankton assemblages may increase utilization of the vast pool of excess nutrients in iron-limited SO surface waters and thereby influence global nutrient distribution and carbon cycling.

Prediction and Consequences of Cofragmentation in Metaproteomics.

Metaproteomics can provide critical information about biological systems, but peptides are found within a complex background of other peptides. This complex background can change across samples, in some cases drastically. Cofragmentation, the coelution of peptides with similar mass to charge ratios, is one factor that influences which peptides are identified in an LC-MS/MS experiment: it is dependent on the nature and complexity of this dynamic background. Metaproteomics applications are particularly susceptible to cofragmentation-induced bias; they have vast protein sequence diversity and the abundance of those proteins can span many orders of magnitude. We have developed a mechanistic model that determines the number of potentially cofragmenting peptides in a given sample (called cobia, https://github.com/bertrand-lab/cobia ). We then used previously published data sets to validate our model, showing that the resulting peptide-specific score reflects the cofragmentation "risk" of peptides. Using an Antarctic sea ice edge metatranscriptome case study, we found that more rare taxonomic and functional groups are associated with higher cofragmentation bias. We also demonstrate how cofragmentation scores can be used to guide the selection of protein- or peptide-based biomarkers. We illustrate potential consequences of cofragmentation for multiple metaproteomic approaches, and suggest practical paths forward to cope with cofragmentation-induced bias.

Manganese and iron deficiency in Southern Ocean Phaeocystis antarctica populations revealed through taxon-specific protein indicators.

Iron and light are recognized as limiting factors controlling Southern Ocean phytoplankton growth. Recent field-based evidence suggests, however, that manganese availability may also play a role. Here we examine the influence of iron and manganese on protein expression and physiology in Phaeocystis antarctica, a key Antarctic primary producer. We provide taxon-specific proteomic evidence to show that in-situ Southern Ocean Phaeocystis populations regularly experience stress due to combined low manganese and iron availability. In culture, combined low iron and manganese induce large-scale changes in the Phaeocystis proteome and result in reorganization of the photosynthetic apparatus. Natural Phaeocystis populations produce protein signatures indicating late-season manganese and iron stress, consistent with concurrently observed stimulation of chlorophyll production upon additions of manganese or iron. These results implicate manganese as an important driver of Southern Ocean productivity and demonstrate the utility of peptide mass spectrometry for identifying drivers of incomplete macronutrient consumption.

Progress and Challenges in Ocean Metaproteomics and Proposed Best Practices for Data Sharing.

Ocean metaproteomics is an emerging field enabling discoveries about marine microbial communities and their impact on global biogeochemical processes. Recent ocean metaproteomic studies have provided insight into microbial nutrient transport, colimitation of carbon fixation, the metabolism of microbial biofilms, and dynamics of carbon flux in marine ecosystems. Future methodological developments could provide new capabilities such as characterizing long-term ecosystem changes, biogeochemical reaction rates, and in situ stoichiometries. Yet challenges remain for ocean metaproteomics due to the great biological diversity that produces highly complex mass spectra, as well as the difficulty in obtaining and working with environmental samples. This review summarizes the progress and challenges facing ocean metaproteomic scientists and proposes best practices for data sharing of ocean metaproteomic data sets, including the data types and metadata needed to enable intercomparisons of protein distributions and annotations that could foster global ocean metaproteomic capabilities.

Hemimastigophora is a novel supra-kingdom-level lineage of eukaryotes.

Almost all eukaryote life forms have now been placed within one of five to eight supra-kingdom-level groups using molecular phylogenetics1-4. The 'phylum' Hemimastigophora is probably the most distinctive morphologically defined lineage that still awaits such a phylogenetic assignment. First observed in the nineteenth century, hemimastigotes are free-living predatory protists with two rows of flagella and a unique cell architecture5-7; to our knowledge, no molecular sequence data or cultures are currently available for this group. Here we report phylogenomic analyses based on high-coverage, cultivation-independent transcriptomics that place Hemimastigophora outside of all established eukaryote supergroups. They instead comprise an independent supra-kingdom-level lineage that most likely forms a sister clade to the 'Diaphoretickes' half of eukaryote diversity (that is, the 'stramenopiles, alveolates and Rhizaria' supergroup (Sar), Archaeplastida and Cryptista, as well as other major groups). The previous ranking of Hemimastigophora as a phylum understates the evolutionary distinctiveness of this group, which has considerable importance for investigations into the deep-level evolutionary history of eukaryotic life-ranging from understanding the origins of fundamental cell systems to placing the root of the tree. We have also established the first culture of a hemimastigote (Hemimastix kukwesjijk sp. nov.), which will facilitate future genomic and cell-biological investigations into eukaryote evolution and the last eukaryotic common ancestor.

Quantification of Vitamin B12-Related Proteins in Marine Microbial Systems Using Selected Reaction Monitoring Mass Spectrometry.

Mass spectrometry-based proteomic approaches to studying microbial systems enable assessment of taxonomically resolved functional capacity. A subset of these proteomic approaches are absolutely quantitative, enabling comparisons of protein expression patterns between different studies and across environments. This chapter outlines a method for applying quantitative assays in marine microbial communities, using proteins involved in vitamin B12 (cobalamin) utilization and production as specific examples. This approach involves identifying important protein targets, determining taxonomic resolution of the required assays, identifying suitable peptides, developing and optimizing liquid chromatography-selected reaction monitoring mass spectrometry assays (LC-SRM-MS), and processing the resulting data. Implementing the method outlined here results in measurements (fmol diagnostic peptide per µg of total bulk protein) that, in this case, define the nutritional status of microbial community members with respect to vitamin B12, and are comparable across and between marine microbial systems.

Biosynthesis of the neurotoxin domoic acid in a bloom-forming diatom.

Oceanic harmful algal blooms of Pseudo-nitzschia diatoms produce the potent mammalian neurotoxin domoic acid (DA). Despite decades of research, the molecular basis for its biosynthesis is not known. By using growth conditions known to induce DA production in Pseudo-nitzschia multiseries, we implemented transcriptome sequencing in order to identify DA biosynthesis genes that colocalize in a genomic four-gene cluster. We biochemically investigated the recombinant DA biosynthetic enzymes and linked their mechanisms to the construction of DA's diagnostic pyrrolidine skeleton, establishing a model for DA biosynthesis. Knowledge of the genetic basis for toxin production provides an orthogonal approach to bloom monitoring and enables study of environmental factors that drive oceanic DA production.

Author Correction: Carboxythiazole is a key microbial nutrient currency and critical component of thiamin biosynthesis.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Carboxythiazole is a key microbial nutrient currency and critical component of thiamin biosynthesis.

Almost all cells require thiamin, vitamin B1 (B1), which is synthesized via the coupling of thiazole and pyrimidine precursors. Here we demonstrate that 5-(2-hydroxyethyl)-4-methyl-1,3-thiazole-2-carboxylic acid (cHET) is a useful in vivo B1 precursor for representatives of ubiquitous marine picoeukaryotic phytoplankton and Escherichia coli - drawing attention to cHET as a valuable exogenous micronutrient for microorganisms with ecological, industrial, and biomedical value. Comparative utilization experiments with the terrestrial plant Arabidopsis thaliana revealed that it can also use exogenous cHET, but notably, picoeukaryotic marine phytoplankton and E. coli were adapted to grow on low (picomolar) concentrations of exogenous cHET. Our results call for the modification of the conventional B1 biosynthesis model to incorporate cHET as a key precursor for B1 biosynthesis in two domains of life, and for consideration of cHET as a microbial micronutrient currency modulating marine primary productivity and community interactions in human gut-hosted microbiomes.

Nutrient co-limitation at the boundary of an oceanic gyre.

Nutrient limitation of oceanic primary production exerts a fundamental control on marine food webs and the flux of carbon into the deep ocean. The extensive boundaries of the oligotrophic sub-tropical gyres collectively define the most extreme transition in ocean productivity, but little is known about nutrient limitation in these zones. Here we present the results of full-factorial nutrient amendment experiments conducted at the eastern boundary of the South Atlantic gyre. We find extensive regions in which the addition of nitrogen or iron individually resulted in no significant phytoplankton growth over 48 hours. However, the addition of both nitrogen and iron increased concentrations of chlorophyll a by up to approximately 40-fold, led to diatom proliferation, and reduced community diversity. Once nitrogen-iron co-limitation had been alleviated, the addition of cobalt or cobalt-containing vitamin B12 could further enhance chlorophyll a yields by up to threefold. Our results suggest that nitrogen-iron co-limitation is pervasive in the ocean, with other micronutrients also approaching co-deficiency. Such multi-nutrient limitations potentially increase phytoplankton community diversity.

Phytoplankton-bacterial interactions mediate micronutrient colimitation at the coastal Antarctic sea ice edge.

Southern Ocean primary productivity plays a key role in global ocean biogeochemistry and climate. At the Southern Ocean sea ice edge in coastal McMurdo Sound, we observed simultaneous cobalamin and iron limitation of surface water phytoplankton communities in late Austral summer. Cobalamin is produced only by bacteria and archaea, suggesting phytoplankton-bacterial interactions must play a role in this limitation. To characterize these interactions and investigate the molecular basis of multiple nutrient limitation, we examined transitions in global gene expression over short time scales, induced by shifts in micronutrient availability. Diatoms, the dominant primary producers, exhibited transcriptional patterns indicative of co-occurring iron and cobalamin deprivation. The major contributor to cobalamin biosynthesis gene expression was a gammaproteobacterial population, Oceanospirillaceae ASP10-02a. This group also contributed significantly to metagenomic cobalamin biosynthesis gene abundance throughout Southern Ocean surface waters. Oceanospirillaceae ASP10-02a displayed elevated expression of organic matter acquisition and cell surface attachment-related genes, consistent with a mutualistic relationship in which they are dependent on phytoplankton growth to fuel cobalamin production. Separate bacterial groups, including Methylophaga, appeared to rely on phytoplankton for carbon and energy sources, but displayed gene expression patterns consistent with iron and cobalamin deprivation. This suggests they also compete with phytoplankton and are important cobalamin consumers. Expression patterns of siderophore- related genes offer evidence for bacterial influences on iron availability as well. The nature and degree of this episodic colimitation appear to be mediated by a series of phytoplankton-bacterial interactions in both positive and negative feedback loops.