Direct observations of microbial community succession on sinking marine particles.

Microbial community dynamics on sinking particles control the amount of carbon that reaches the deep ocean and the length of time that carbon is stored, with potentially profound impacts on Earth's climate. A mechanistic understanding of the controls on sinking particle distributions has been hindered by limited depth- and time-resolved sampling and methods that cannot distinguish individual particles. Here, we analyze microbial communities on nearly 400 individual sinking particles in conjunction with more conventional composite particle samples to determine how particle colonization and community assembly might control carbon sequestration in the deep ocean. We observed community succession with corresponding changes in microbial metabolic potential on the larger sinking particles transporting a significant fraction of carbon to the deep sea. Microbial community richness decreased as particles aged and sank; however, richness increased with particle size and the attenuation of carbon export. This suggests that the theory of island biogeography applies to sinking marine particles. Changes in POC flux attenuation with time and microbial community composition with depth were reproduced in a mechanistic ecosystem model that reflected a range of POC labilities and microbial growth rates. Our results highlight microbial community dynamics and processes on individual sinking particles, the isolation of which is necessary to improve mechanistic models of ocean carbon uptake.

Flagella are required to coordinately activate competition and host colonization factors in response to a mechanical signal.

Bacteria employ antagonistic strategies to eliminate competitors of an ecological niche. Contact-dependent mechanisms, such as the type VI secretion system (T6SS), are prevalent in host-associated bacteria, yet we know relatively little about how T6SS+ strains make contact with competitors in highly viscous environments, such as host mucus. To better understand how cells respond to and contact one another in such environments, we performed a genome-wide transposon mutant screen of the T6SS-wielding beneficial bacterial symbiont, Vibrio fischeri, and identified two sets of genes that are conditionally required for killing. LPS/capsule and flagellar-associated genes do not affect T6SS directly and are therefore not required for interbacterial killing when cell contact is forced yet are necessary for killing in high-viscosity liquid (hydrogel) where cell-cell contact must be biologically mediated. Quantitative transcriptomics revealed that V. fischeri significantly increases expression of both T6SS genes and cell surface modification factors upon transition from low- to high-viscosity media. Consistent with coincubation and fluorescence microscopy data, flagella are not required for T6SS expression in hydrogel. However, flagella play a key role in responding to the physical environment by promoting expression of the surface modification genes identified in our screen, as well as additional functional pathways important for host colonization including uptake of host-relevant iron and carbon sources, and nitric oxide detoxification enzymes. Our findings suggest that flagella may act as a mechanosensor for V. fischeri to coordinately activate competitive strategies and host colonization factors, underscoring the significance of the physical environment in directing complex bacterial behaviors.

Synechococcus nitrogen gene loss in iron-limited ocean regions.

Synechococcus are the most abundant cyanobacteria in high latitude regions and are responsible for an estimated 17% of annual marine net primary productivity. Despite their biogeochemical importance, Synechococcus populations have been unevenly sampled across the ocean, with most studies focused on low-latitude strains. In particular, the near absence of Synechococcus genomes from high-latitude, High Nutrient Low Chlorophyll (HNLC) regions leaves a gap in our knowledge of picocyanobacterial adaptations to iron limitation and their influence on carbon, nitrogen, and iron cycles. We examined Synechococcus populations from the subarctic North Pacific, a well-characterized HNLC region, with quantitative metagenomics. Assembly with short and long reads produced two near complete Synechococcus metagenome-assembled genomes (MAGs). Quantitative metagenome-derived abundances of these populations matched well with flow cytometry counts, and the Synechococcus MAGs were estimated to comprise >99% of the Synechococcus at Station P. Whereas the Station P Synechococcus MAGs contained multiple genes for adaptation to iron limitation, both genomes lacked genes for uptake and assimilation of nitrate and nitrite, suggesting a dependence on ammonium, urea, and other forms of recycled nitrogen leading to reduced iron requirements. A global analysis of Synechococcus nitrate reductase abundance in the TARA Oceans dataset found nitrate assimilation genes are also lower in other HNLC regions. We propose that nitrate and nitrite assimilation gene loss in Synechococcus may represent an adaptation to severe iron limitation in high-latitude regions where ammonium availability is higher. Our findings have implications for models that quantify the contribution of cyanobacteria to primary production and subsequent carbon export.

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.

Protistan plankton communities in the Galápagos Archipelago respond to changes in deep water masses resulting from the 2015/16 El Niño.

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.

Para-Aminobenzoic Acid, Calcium, and c-di-GMP Induce Formation of Cohesive, Syp-Polysaccharide-Dependent Biofilms in Vibrio fischeri.

The marine bacterium Vibrio fischeri efficiently colonizes its symbiotic squid host, Euprymna scolopes, by producing a transient biofilm dependent on the symbiosis polysaccharide (SYP). In vitro, however, wild-type strain ES114 fails to form SYP-dependent biofilms. Instead, genetically engineered strains, such as those lacking the negative regulator BinK, have been developed to study this phenomenon. Historically, V. fischeri has been grown using LBS, a complex medium containing tryptone and yeast extract; supplementation with calcium is required to induce biofilm formation by a binK mutant. Here, through our discovery that yeast extract inhibits biofilm formation, we uncover signals and underlying mechanisms that control V. fischeri biofilm formation. In contrast to its inability to form a biofilm on unsupplemented LBS, a binK mutant formed cohesive, SYP-dependent colony biofilms on tTBS, modified LBS that lacks yeast extract. Moreover, wild-type strain ES114 became proficient to form cohesive, SYP-dependent biofilms when grown in tTBS supplemented with both calcium and the vitamin para-aminobenzoic acid (pABA); neither molecule alone was sufficient, indicating that this phenotype relies on coordinating two cues. pABA/calcium supplementation also inhibited bacterial motility. Consistent with these phenotypes, cells grown in tTBS with pABA/calcium were enriched in transcripts for biofilm-related genes and predicted diguanylate cyclases, which produce the second messenger cyclic-di-GMP (c-di-GMP). They also exhibited elevated levels of c-di-GMP, which was required for the observed phenotypes, as phosphodiesterase overproduction abrogated biofilm formation and partially rescued motility. This work thus provides insight into conditions, signals, and processes that promote biofilm formation by V. fischeri. IMPORTANCE Bacteria integrate environmental signals to regulate gene expression and protein production to adapt to their surroundings. One such behavioral adaptation is the formation of a biofilm, which can promote adherence and colonization and provide protection against antimicrobials. Identifying signals that trigger biofilm formation and the underlying mechanism(s) of action remain important and challenging areas of investigation. Here, we determined that yeast extract, commonly used for growth of bacteria in laboratory culture, inhibits biofilm formation by Vibrio fischeri, a model bacterium used for investigating host-relevant biofilm formation. Omitting yeast extract from the growth medium led to the identification of an unusual signal, the vitamin para-aminobenzoic acid (pABA), that when added together with calcium could induce biofilm formation. pABA increased the concentrations of the second messenger, c-di-GMP, which was necessary but not sufficient to induce biofilm formation. This work thus advances our understanding of signals and signal integration controlling bacterial biofilm formation.

Investigating the microbial ecology of coastal hotspots of marine nitrogen fixation in the western North Atlantic.

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.

Transcriptional activity differentiates families of Marine Group II Euryarchaeota in the coastal ocean.

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.

Microbial Niche Diversification in the Galápagos Archipelago and Its Response to El Niño.

The Galápagos 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 Niño. 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 Niño 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 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.

A Model Roseobacter, Ruegeria pomeroyi DSS-3, Employs a Diffusible Killing Mechanism To Eliminate Competitors.

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.

Draft Genome Sequence of a Harveyi Clade Bacterium Isolated from Lolliguncula brevis Squid.

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.

Coastal Ocean Metagenomes and Curated Metagenome-Assembled Genomes from Marsh Landing, Sapelo Island (Georgia, USA).

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.

Revisiting the distribution of oceanic N2 fixation and estimating diazotrophic contribution to marine production.

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.

Towards Quantitative Microbiome Community Profiling Using Internal Standards.

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.

Linking patterns of net community production and marine microbial community structure in the western North Atlantic.

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 O2/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 Aureococcus anophagefferens, 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.

Mosaic patterns of B-vitamin synthesis and utilization in a natural marine microbial community.

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.

Quantitative Transcriptomics Reveals the Growth- and Nutrient-Dependent Response of a Streamlined Marine Methylotroph to Methanol and Naturally Occurring Dissolved Organic Matter.

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-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-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 IMPORTANCE: Bacteria exert a substantial influence on marine organic matter flux, yet the carbon components targeted by specific bacterial groups, as well as how those groups' metabolic activities change under different conditions, are not well understood. Gene expression studies of model organisms can identify these responses under defined conditions, which can then be compared to environmental transcriptomes to elucidate in situ activities. This integration, however, is limited by the data's relative nature. Here, we report the fully quantitative transcriptome of a marine bacterium, providing a genome-wide survey of cellular transcript abundances and how they change with different states of growth, nutrient conditions, and carbon substrates. The results revealed the dynamic metabolic strategies this methylotroph has for processing both simple one-carbon compounds and the complex multicarbon substrates of naturally derived marine organic matter and provide baseline quantitative data for identifying their in situ activities and impact on the marine carbon cycle.

High molecular weight dissolved organic matter enrichment selects for methylotrophs in dilution to extinction cultures.

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 <1% scored positive for growth in non-DOM-amended controls. The majority (71%) of isolates displayed a distinct increase in cell yields when grown in increasing concentrations of HMW DOM. Whole-genome sequencing was used to screen the culture collection for purity and to determine the phylogenetic identity of the isolates. Eleven percent of the isolates belonged to the gammaproteobacteria including Alteromonadales (the SAR92 clade) and Vibrio. Surprisingly, 85% of isolates belonged to the methylotrophic OM43 clade of betaproteobacteria, bacteria thought to metabolically specialize in degrading C1 compounds. Growth of these isolates on methanol confirmed their methylotrophic phenotype. Our results indicate that dilution to extinction cultivation enriched with natural sources of organic substrates has a potential to reveal the previously unsuspected relationships between naturally occurring organic nutrients and the microorganisms that consume them.

Single-taxon field measurements of bacterial gene regulation controlling DMSP fate.

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 demethylation pathway was relatively greater during a high-DMSP-producing dinoflagellate bloom, and expression of the cleavage pathway was greater in the presence of a mixed diatom and dinoflagellate community [corrected].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.