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Abstract 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. 

Abstract The hundreds of tidewater glaciers found in the Canadian Arctic Archipelago have the potential to enhance delivery of nutrients and other material to the surface ocean. Despite this, their influence on marine ecosystems, specifically phytoplankton, is poorly characterized. Here we developed and applied a quantitative mass spectrometry‐based approach to measure phytoplankton ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) concentrations to examine differences in productivity in glacierized and non‐glacierized marine systems in Jones Sound, Nunavut, within Inuit Nunangat. Comparisons to chloroplast 16S rRNA gene amplicon sequencing data suggested that these measurements detect the majority of Rubisco produced in Jones Sound. Because Rubisco catalyzes carbon fixation, we used these measurements to estimate total and group‐specific primary production potential, which were within the range of historical primary production measurements made using classical methods in this region. Our measurements also revealed that up to 2% of total protein in the water column is Rubisco, and that Rubisco concentrations are correlated with chlorophyll fluorescence, with maxima near the nitracline. Rubisco produced by diatom generaChaetocerosandThalassiosirawere higher in marine regions influenced by glaciers, while Rubisco fromMicromonas(Chlorophyta) was greater in non‐glacierized regions. This suggests that future climate scenarios may favor smaller phytoplankton groups, likeMicromonas, with consequences for food webs and carbon cycling. This study broadens our understanding of how tidewater glaciers will impact phytoplankton communities, now and in a warmer future, and lays the foundation for using this mass spectrometry‐based approach to quantify phytoplankton group‐specific carbon fixation potential in other marine regions. 

ABSTRACT 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. IMPORTANCEResults 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. 

Abstract High‐latitude oceans experience strong seasonality where low light limits photosynthetic activity most of the year. This limitation is pronounced for algae within and underlying sea ice, and these algae are uniquely acclimated to low light levels. During spring melt, however, light intensity and daylength increase drastically, triggering blooms of ice algae that play important roles in carbon cycling and ecosystem productivity. How the algae acclimate to this dynamic and heterogeneous environment is poorly understood. Here, we measured¹⁴C‐carbon fixation rates, photophysiology, and ribulose 1,5‐bisphosphate carboxylase oxygenase (Rubisco) content of sea‐ice algae in coastal waters near the western Antarctic Peninsula during spring, ranging from a low‐light‐acclimated, bottom community to a light‐saturated bloom. Carbon fixation rates by sea‐ice algae were similar to other Antarctic sea‐ice measurements (2–49 mg C m⁻²d⁻¹), and there was little phytoplankton biomass in the underlying water at the time of sampling. Net‐to‐gross ratios of carbon fixation were generally high and showed no relationship with ice type. We found algal photophysiology and Rubisco concentrations varied in relation to the different types of ice, altering the balance between the photochemical and biochemical processes that constrain carbon fixation rates. For algae inhabiting the bottom layers of sea ice, rates of carbon fixation were largely constrained by light availability whereas in surface seawater, interior and rotten/brash ice, carbon fixation rates could be calculated with reasonable accuracy from measurements of Rubisco concentrations. This work provides additional insight and means to evaluate carbon fixation rates as sea ice continues to change in future. 

Abstract. Metaproteomics is an increasingly popular methodology that provides information regarding the metabolic functions of specific microbial taxa and has potential for contributing to ocean ecology and biogeochemical studies. A blinded multi-laboratory intercomparison was conducted to assess comparability and reproducibility of taxonomic and functional results and their sensitivity to methodological variables. Euphotic zone samples from the Bermuda Atlantic Time-Series Study in the North Atlantic Ocean collected by in situ pumps and the AUV Clio were distributed with a paired metagenome, and one-dimensional liquid chromatographic data dependent acquisition mass spectrometry analyses was stipulated. Analysis of mass spectra from seven laboratories through a common informatic pipeline identified a shared set of 1056 proteins from 1395 shared peptides constituents. Quantitative analyses showed good reproducibility: pairwise regressions of spectral counts between laboratories yielded R2 values ranging from 0.43 to 0.83, and a Sørensen similarity analysis of the top 1,000 proteins revealed 70–80 % similarity between laboratory groups. Taxonomic and functional assignments showed good coherence between technical replicates and different laboratories. An informatic intercomparison study, involving 10 laboratories using 8 software packages successfully identified thousands of peptides within the complex metaproteomic datasets, demonstrating the utility of these software tools for ocean metaproteomic research. Future efforts could examine reproducibility in deeper metaproteomes, examine accuracy in targeted absolute quantitation analyses, and develop standards for data output formats to improve data interoperability. Together, these results demonstrate the reproducibility of metaproteomic analyses and their suitability for microbial oceanography research including integration into global scale ocean surveys and ocean biogeochemical models. 

Abstract. Metaproteomics is an increasingly popular methodology that provides information regarding the metabolic functions of specific microbial taxa and has potential for contributing to ocean ecology and biogeochemical studies. A blinded multi-laboratory intercomparison was conducted to assess comparability and reproducibility of taxonomic and functional results and their sensitivity to methodological variables. Euphotic zone samples from the Bermuda Atlantic Time-series Study (BATS) in the North Atlantic Ocean collected by in situ pumps and the autonomous underwater vehicle (AUV) Clio were distributed with a paired metagenome, and one-dimensional (1D) liquid chromatographic data-dependent acquisition mass spectrometry analysis was stipulated. Analysis of mass spectra from seven laboratories through a common bioinformatic pipeline identified a shared set of 1056 proteins from 1395 shared peptide constituents. Quantitative analyses showed good reproducibility: pairwise regressions of spectral counts between laboratories yielded R2 values averaged 0.62±0.11, and a Sørensen similarity analysis of the top 1000 proteins revealed 70 %–80 % similarity between laboratory groups. Taxonomic and functional assignments showed good coherence between technical replicates and different laboratories. A bioinformatic intercomparison study, involving 10 laboratories using eight software packages, successfully identified thousands of peptides within the complex metaproteomic datasets, demonstrating the utility of these software tools for ocean metaproteomic research. Lessons learned and potential improvements in methods were described. Future efforts could examine reproducibility in deeper metaproteomes, examine accuracy in targeted absolute quantitation analyses, and develop standards for data output formats to improve data interoperability. Together, these results demonstrate the reproducibility of metaproteomic analyses and their suitability for microbial oceanography research, including integration into global-scale ocean surveys and ocean biogeochemical models. 

Summary A network of peptidases governs proteostasis in plant chloroplasts and mitochondria. This study reveals strong genetic and functional interactions in Arabidopsis between the chloroplast stromal CLP chaperone‐protease system and the PREP1,2 peptidases, which are dually localized to chloroplast stroma and the mitochondrial matrix.Higher order mutants defective in CLP or PREP proteins were generated and analyzed by quantitative proteomics and N‐terminal proteomics (terminal amine isotopic labeling of substrates (TAILS)).Strong synergistic interactions were observed between the CLP protease system (clpr1‐2,clpr2‐1,clpc1‐1,clpt1,clpt2)and both PREP homologs (prep1,prep2) resulting in embryo lethality or growth and developmental phenotypes. Synergistic interactions were observed even when only one of the PREP proteins was lacking, suggesting that PREP1 and PREP2 have divergent substrates. Proteome phenotypes were driven by the loss of CLP protease capacity, with little impact from the PREP peptidases. Chloroplast N‐terminal proteomesshowed that many nuclear encoded chloroplast proteins have alternatively processed N‐termini inprep1prep2,clpt1clpt2andprep1prep2clpt1clpt2.Loss of chloroplast protease capacity interferes with stromal processing peptidase (SPP) activity due to folding stress and low levels of accumulated cleaved cTP fragments. PREP1,2 proteolysis of cleaved cTPs is complemented by unknown proteases. A model for CLP and PREP activity within a hierarchical chloroplast proteolysis network is proposed.