Dual thermal ecotypes coexist within a nearly genetically identical population of the unicellular marine cyanobacterium Synechococcus.

The extent and ecological significance of intraspecific functional diversity within marine microbial populations is still poorly understood, and it remains unclear if such strain-level microdiversity will affect fitness and persistence in a rapidly changing ocean environment. In this study, we cultured 11 sympatric strains of the ubiquitous marine picocyanobacterium Synechococcus isolated from a Narragansett Bay (RI) phytoplankton community thermal selection experiment. Thermal performance curves revealed selection at cool and warm temperatures had subdivided the initial population into thermotypes with pronounced differences in maximum growth temperatures. Curiously, the genomes of all 11 isolates were almost identical (average nucleotide identities of >99.99%, with >99% of the genome aligning) and no differences in gene content or single nucleotide variants were associated with either cool or warm temperature phenotypes. Despite a very high level of genomic similarity, sequenced epigenomes for two strains showed differences in methylation on genes associated with photosynthesis. These corresponded to measured differences in photophysiology, suggesting a potential pathway for future mechanistic research into thermal microdiversity. Our study demonstrates that present-day marine microbial populations can harbor cryptic but environmentally relevant thermotypes which may increase their resilience to future rising temperatures.

Metagenome-Assembled Genomes from Photo-Oxidized and Nonoxidized Oil-Degrading Marine Microcosms.

We performed deep metagenomic sequencing on hydrocarbon-degrading marine microcosms designed to experimentally determine the effect of photo-oxidation on oil biodegradation dynamics. Assembly, binning, and dereplication yielded 73 unique metagenome-assembled genomes (MAGs) from 6 phyla, of which 61 are predicted to be over 90% complete.

Importance of mobile genetic element immunity in numerically abundant Trichodesmium clades.

The colony-forming cyanobacteria Trichodesmium spp. are considered one of the most important nitrogen-fixing genera in the warm, low nutrient ocean. Despite this central biogeochemical role, many questions about their evolution, physiology, and trophic interactions remain unanswered. To address these questions, we describe Trichodesmium pangenomic potential via significantly improved genomic assemblies from two isolates and 15 new >50% complete Trichodesmium metagenome-assembled genomes from hand-picked, Trichodesmium colonies spanning the Atlantic Ocean. Phylogenomics identified ~four N2 fixing clades of Trichodesmium across the transect, with T. thiebautii dominating the colony-specific reads. Pangenomic analyses showed that all T. thiebautii MAGs are enriched in COG defense mechanisms and encode a vertically inherited Type III-B Clustered Regularly Interspaced Short Palindromic Repeats and associated protein-based immunity system (CRISPR-Cas). Surprisingly, this CRISPR-Cas system was absent in all T. erythraeum genomes, vertically inherited by T. thiebautii, and correlated with increased signatures of horizontal gene transfer. Additionally, the system was expressed in metaproteomic and transcriptomic datasets and CRISPR spacer sequences with 100% identical hits to field-assembled, putative phage genome fragments were identified. While the currently CO2-limited T. erythraeum is expected to be a 'winner' of anthropogenic climate change, their genomic dearth of known phage resistance mechanisms, compared to T. thiebautii, could put this outcome in question. Thus, the clear demarcation of T. thiebautii maintaining CRISPR-Cas systems, while T. erythraeum does not, identifies Trichodesmium as an ecologically important CRISPR-Cas model system, and highlights the need for more research on phage-Trichodesmium interactions.

Metagenome-Assembled Genome of a Novel Cyclobacteriaceae Epibiont of a Heterotrophic Diazotroph.

Here, we describe the metagenome-assembled genome (MAG) HetDA_MAG_SS2, in the family Cyclobacteriaceae. It was found in association with a HetDA cyanobiont isolated from a Station ALOHA Trichodesmium colony. Annotation suggests that HetDA_MAG_SS2 is a chemoorganoheterotroph with the potential for lithoheterotrophy, containing genes for aerobic respiration, mixed acid fermentation, dissimilatory nitrate reduction to ammonium, and sulfide oxidation.

Metagenome-Assembled Genome of an Alphaproteobacterium Isolated from an HetDA Enrichment from the North Pacific Subtropical Gyre.

Here, we present HetDA_MAG_SS10, a metagenome-assembled genome (MAG) from an enrichment of a heterocystous diazotroph originally living in association with Trichodesmium spp. obtained near Station ALOHA in the North Pacific Ocean. HetDA_MAG_SS10, an alphaproteobacterium in the order Micavibrionales, is proposed to be photoheterotrophic via rhodopsin and has the potential for dimethylsulfoniopropionate (DMSP) demethylation.

Metagenome-Assembled Genome HetDA_MAG_MH13 of the Family Devosiaceae, from a Marine N2-fixing Cyanobacterial Enrichment Culture.

Here, we present a draft genome in the order Rhizobiales and family Devosiaceae. This draft genome comes from an enrichment of a heterocystous, cyanobacterial diazotroph (HetDA) that was originally living in association with Trichodesmium species. This Rhizobiales organism is proposed to be an anoxygenic phototroph capable of dissimilatory nitrate reduction to ammonia (DNRA).

Genome Sequence of a Heterocystous Diazotroph Isolated from a Trichodesmium Consortium from the North Pacific Subtropical Gyre.

Diazotrophic cyanobacteria play a vital role in the nitrogen influx of the global marine ecosystem. In July 2010, colonies of Trichodesmium spp. were picked near Station ALOHA in the oligotrophic North Pacific Subtropical Gyre, and a novel heterocystous diazotroph (strain HetDA_MAG_MS3) belonging to the genus Rivularia was found living in close association; it was cultured and sequenced.

Metagenome-Assembled Genome of a Cyclobacteriaceae Bacterium, HetDA_MAG_MS6, Isolated from a Trichodesmium Consortium from Station ALOHA.

Here, we describe the metagenome-assembled genome (MAG) HetDA_MAG_MS6. HetDA_MAG_MS6 was obtained from an enrichment of the heterocystous diazotroph HetDA, which was isolated near Station ALOHA. The MAG was placed in the Cyclobacteriaceae family and is predicted to be a chemoorganoheterotroph with the potential for ammonia uptake, phosphonate transport, and sulfolipid biosynthesis.

Genome Sequence of the Euphotic Hawaiian Gammaproteobacterium HetDA_MAG_MS8, in the Order Nevskiales, Family Oceanococcaceae, Genus Oceanococcus.

We present a metagenome-assembled genome (MAG), HetDA_MAG_MS8, that was determined to be unique via relative evolutionary divergence (RED) scores and average nucleotide identity (ANI) values. HetDA_MAG_MS8 is in the order Nevskiales, genus Oceanococcus, and was assembled from a heterocytous cyanobiont enrichment from the Hawaii Ocean Time Series. HetDA_MAG_MS8 is predicted to be a facultative, aerobic, anoxygenic photolithoheterotroph that has the potential for sulfide oxidation and dimethylsulfoniopropionate (DMSP) synthesis.

Microbial functional diversity across biogeochemical provinces in the central Pacific Ocean.

Enzymes catalyze key reactions within Earth's life-sustaining biogeochemical cycles. Here, we use metaproteomics to examine the enzymatic capabilities of the microbial community (0.2 to 3 µm) along a 5,000-km-long, 1-km-deep transect in the central Pacific Ocean. Eighty-five percent of total protein abundance was of bacterial origin, with Archaea contributing 1.6%. Over 2,000 functional KEGG Ontology (KO) groups were identified, yet only 25 KO groups contributed over half of the protein abundance, simultaneously indicating abundant key functions and a long tail of diverse functions. Vertical attenuation of individual proteins displayed stratification of nutrient transport, carbon utilization, and environmental stress. The microbial community also varied along horizontal scales, shaped by environmental features specific to the oligotrophic North Pacific Subtropical Gyre, the oxygen-depleted Eastern Tropical North Pacific, and nutrient-rich equatorial upwelling. Some of the most abundant proteins were associated with nitrification and C1 metabolisms, with observed interactions between these pathways. The oxidoreductases nitrite oxidoreductase (NxrAB), nitrite reductase (NirK), ammonia monooxygenase (AmoABC), manganese oxidase (MnxG), formate dehydrogenase (FdoGH and FDH), and carbon monoxide dehydrogenase (CoxLM) displayed distributions indicative of biogeochemical status such as oxidative or nutritional stress, with the potential to be more sensitive than chemical sensors. Enzymes that mediate transformations of atmospheric gases like CO, CO2, NO, methanethiol, and methylamines were most abundant in the upwelling region. We identified hot spots of biochemical transformation in the central Pacific Ocean, highlighted previously understudied metabolic pathways in the environment, and provided rich empirical data for biogeochemical models critical for forecasting ecosystem response to climate change.

Molecular mechanisms underlying iron and phosphorus co-limitation responses in the nitrogen-fixing cyanobacterium Crocosphaera.

In the nitrogen-limited subtropical gyres, diazotrophic cyanobacteria, including Crocosphaera, provide an essential ecosystem service by converting dinitrogen (N2) gas into ammonia to support primary production in these oligotrophic regimes. Natural gradients of phosphorus (P) and iron (Fe) availability in the low-latitude oceans constrain the biogeography and activity of diazotrophs with important implications for marine biogeochemical cycling. Much remains unknown regarding Crocosphaera's physiological and molecular responses to multiple nutrient limitations. We cultured C. watsonii under Fe, P, and Fe/P (co)-limiting scenarios to link cellular physiology with diel gene expression and observed unique physiological and transcriptional profiles for each treatment. Counterintuitively, reduced growth and N2 fixation resource use efficiencies (RUEs) for Fe or P under P limitation were alleviated under Fe/P co-limitation. Differential gene expression analyses show that Fe/P co-limited cells employ the same responses as single-nutrient limited cells that reduce cellular nutrient requirements and increase responsiveness to environmental change including smaller cell size, protein turnover (Fe-limited), and upregulation of environmental sense-and-respond systems (P-limited). Combined, these mechanisms enhance growth and RUEs in Fe/P co-limited cells. These findings are important to our understanding of nutrient controls on N2 fixation and the implications for primary productivity and microbial dynamics in a changing ocean.

Genome Sequence of Synechococcus sp. Strain LA31, Isolated from a Temperate Estuary.

Cluster 5 Synechococcus species are widely acknowledged for their broad distribution and biogeochemical importance. In particular, subcluster 5.2 strains inhabit freshwater, estuarine, and marine environments but are understudied, compared to other subclusters. Here, we present the genome for Synechococcus sp. strain LA31, a strain that was recently isolated from Narragansett Bay, Rhode Island, USA.

Dynamic diel proteome and daytime nitrogenase activity supports buoyancy in the cyanobacterium Trichodesmium.

Cyanobacteria of the genus Trichodesmium provide about 80 Tg of fixed nitrogen to the surface ocean per year and contribute to marine biogeochemistry, including the sequestration of carbon dioxide. Trichodesmium fixes nitrogen in the daylight, despite the incompatibility of the nitrogenase enzyme with oxygen produced during photosynthesis. While the mechanisms protecting nitrogenase remain unclear, all proposed strategies require considerable resource investment. Here we identify a crucial benefit of daytime nitrogen fixation in Trichodesmium spp. that may counteract these costs. We analysed diel proteomes of cultured and field populations of Trichodesmium in comparison with the marine diazotroph Crocosphaera watsonii WH8501, which fixes nitrogen at night. Trichodesmium's proteome is extraordinarily dynamic and demonstrates simultaneous photosynthesis and nitrogen fixation, resulting in balanced particulate organic carbon and particulate organic nitrogen production. Unlike Crocosphaera, which produces large quantities of glycogen as an energy store for nitrogenase, proteomic evidence is consistent with the idea that Trichodesmium reduces the need to produce glycogen by supplying energy directly to nitrogenase via soluble ferredoxin charged by the photosynthesis protein PsaC. This minimizes ballast associated with glycogen, reducing cell density and decreasing sinking velocity, thus supporting Trichodesmium's niche as a buoyant, high-light-adapted colony forming cyanobacterium. To occupy its niche of simultaneous nitrogen fixation and photosynthesis, Trichodesmium appears to be a conspicuous consumer of iron, and has therefore developed unique iron-acquisition strategies, including the use of iron-rich dust. Particle capture by buoyant Trichodesmium colonies may increase the residence time and degradation of mineral iron in the euphotic zone. These findings describe how cellular biochemistry defines and reinforces the ecological and biogeochemical function of these keystone marine diazotrophs.

Why Environmental Biomarkers Work: Transcriptome-Proteome Correlations and Modeling of Multistressor Experiments in the Marine Bacterium Trichodesmium.

Ocean microbial communities are important contributors to the global biogeochemical reactions that sustain life on Earth. The factors controlling these communities are being increasingly explored using metatranscriptomic and metaproteomic environmental biomarkers. Using published proteomes and transcriptomes from the abundant colony-forming cyanobacterium Trichodesmium (strain IMS101) grown under varying Fe and/or P limitation in low and high CO2, we observed robust correlations of stress-induced proteins and RNAs (i.e., involved in transport and homeostasis) that yield useful information on the nutrient status under low and/or high CO2. Conversely, transcriptional and translational correlations of many other central metabolism pathways exhibit broad discordance. A cellular RNA and protein production/degradation model demonstrates how biomolecules with small initial inventories, such as environmentally responsive proteins, achieve large increases in fold-change units as opposed to those with a higher basal expression and inventory such as metabolic systems. Microbial cells, due to their immersion in the environment, tend to show large adaptive responses in both RNA and protein that result in transcript-protein correlations. These observations and model results demonstrate multi-omic coherence for environmental biomarkers and provide the underlying mechanism for those observations, supporting the promise for global application in detecting responses to environmental stimuli in a changing ocean.

Alphaproteobacteria facilitate Trichodesmium community trimethylamine utilization.

In the surface waters of the warm oligotrophic ocean, filaments and aggregated colonies of the nitrogen (N)-fixing cyanobacterium Trichodesmium create microscale nutrient-rich oases. These hotspots fuel primary productivity and harbour a diverse consortium of heterotrophs. Interactions with associated microbiota can affect the physiology of Trichodesmium, often in ways that have been predicted to support its growth. Recently, it was found that trimethylamine (TMA), a globally abundant organic N compound, inhibits N2 fixation in cultures of Trichodesmium without impairing growth rate, suggesting that Trichodesmium can use TMA as an alternate N source. In this study, 15 N-TMA DNA stable isotope probing (SIP) of a Trichodesmium enrichment was employed to further investigate TMA metabolism and determine whether TMA-N is incorporated directly or secondarily via cross-feeding facilitated by microbial associates. Herein, we identify two members of the marine Roseobacter clade (MRC) of Alphaproteobacteria as the likely metabolizers of TMA and provide genomic evidence that they converted TMA into a more readily available form of N, e.g., ammonium (NH4 + ), which was subsequently used by Trichodesmium and the rest of the community. The results implicate microbiome-mediated carbon (C) and N transformations in modulating N2 fixation and thus highlight the involvement of host-associated heterotrophs in global biogeochemical cycling.

DMSP synthesis genes distinguish two types of DMSP producer phenotypes.

Dimethylsulfoniopropionate (DMSP) is an important organic carbon and sulfur source in the surface ocean that fuels microbial activity and significantly impacts Earth's climate. After three decades of research, the cellular role(s) of DMSP and environmental drivers of production remain enigmatic. Recent work suggests that cellular DMSP concentrations, and changes in these concentrations in response to environmental stressors, define two major groups of DMSP producers: high DMSP producers that contain ≥ 50 mM intracellular DMSP and low DMSP producers that contain < 50 mM. Here we show that two recently described DMSP synthesis genes (DSYB and TpMT2) may differentiate these two DMSP phenotypes. A survey of prokaryotic and eukaryotic isolates found a significant correlation between the presence of DSYB and TpMT2 genes and previous measurements of high and low DMSP concentrations, respectively. Phylogenetic analysis demonstrated that DSYB and TpMT2 form two distinct clades. DSYB and TpMT2 were also found to be globally abundant in in situ surface communities, and their taxonomic annotations were similar to those observed for isolates. The strong correlation of the DSYB and TpMT2 synthesis genes with high and low producer phenotypes establishes a foundation for direct quantification of DMSP producers, enabling significantly improved predictions of DMSP in situ.

Mechanisms and heterogeneity of in situ mineral processing by the marine nitrogen fixer Trichodesmium revealed by single-colony metaproteomics.

The keystone marine nitrogen fixer Trichodesmium thrives in high-dust environments. While laboratory investigations have observed that Trichodesmium colonies can access the essential nutrient iron from dust particles, less clear are the biochemical strategies underlying particle-colony interactions in nature. Here we demonstrate that Trichodesmium colonies engage with mineral particles in the wild with distinct molecular responses. We encountered particle-laden Trichodesmium colonies at a sampling location in the Southern Caribbean Sea; microscopy and synchrotron-based imaging then demonstrated heterogeneous associations with iron oxide and iron-silicate minerals. Metaproteomic analysis of individual colonies by a new low-biomass approach revealed responses in biogeochemically relevant proteins including photosynthesis proteins and metalloproteins containing iron, nickel, copper, and zinc. The iron-storage protein ferritin was particularly enriched implying accumulation of mineral-derived iron, and multiple iron acquisition pathways including Fe(II), Fe(III), and Fe-siderophore transporters were engaged. While the particles provided key trace metals such as iron and nickel, there was also evidence that Trichodesmium was altering its strategy to confront increased superoxide production and metal exposure. Chemotaxis regulators also responded to mineral presence suggesting involvement in particle entrainment. These molecular responses are fundamental to Trichodesmium's ecological success and global biogeochemical impact, and may contribute to the leaching of particulate trace metals with implications for global iron and carbon cycling.

Long-Term m5C Methylome Dynamics Parallel Phenotypic Adaptation in the Cyanobacterium Trichodesmium.

A major challenge in modern biology is understanding how the effects of short-term biological responses influence long-term evolutionary adaptation, defined as a genetically determined increase in fitness to novel environments. This is particularly important in globally important microbes experiencing rapid global change, due to their influence on food webs, biogeochemical cycles, and climate. Epigenetic modifications like methylation have been demonstrated to influence short-term plastic responses, which ultimately impact long-term adaptive responses to environmental change. However, there remains a paucity of empirical research examining long-term methylation dynamics during environmental adaptation in nonmodel, ecologically important microbes. Here, we show the first empirical evidence in a marine prokaryote for long-term m5C methylome modifications correlated with phenotypic adaptation to CO2, using a 7-year evolution experiment (1,000+ generations) with the biogeochemically important marine cyanobacterium Trichodesmium. We identify m5C methylated sites that rapidly changed in response to high (750 µatm) CO2 exposure and were maintained for at least 4.5 years of CO2 selection. After 7 years of CO2 selection, however, m5C methylation levels that initially responded to high-CO2 returned to ancestral, ambient CO2 levels. Concurrently, high-CO2 adapted growth and N2 fixation rates remained significantly higher than those of ambient CO2 adapted cell lines irrespective of CO2 concentration, a trend consistent with genetic assimilation theory. These data demonstrate the maintenance of CO2-responsive m5C methylation for 4.5 years alongside phenotypic adaptation before returning to ancestral methylation levels. These observations in a globally distributed marine prokaryote provide critical evolutionary insights into biogeochemically important traits under global change.

Metagenome-Assembled Genome Sequence of Kapabacteriales Bacterium Strain Clear-D13, Assembled from a Harmful Algal Bloom Enrichment Culture.

Metagenomic sequencing of a Dolichospermum circinale enrichment culture resulted in the assembly of several cocultured metagenome-assembled genomes (MAGs). One MAG was affiliated with the class Kapabacteriales and included 5,724,991 bp in 127 contigs with a GC content of 48.4%.

Metagenome-Assembled Genome Sequence of Rhodobacteraceae Bacterium Strain Clear-D3, Assembled from a Dolichospermum Consortium from Clear Lake, California.

Here, we report the metagenome-assembled genome sequence of a Rhodobacteraceae bacterium strain, Clear-D3, that was reconstructed from a cyanobacterial enrichment from a eutrophic lake. The draft genome sequence shows evidence of an anoxygenic photoautotrophic lifestyle. Other potential capabilities include aerobic heterotrophy, flagellar motility, chemotaxis, and utilization of complex C-P compounds.