Aerobic degradation of methylphosphonate (MPn) by marine bacterioplankton has been hypothesized to contribute significantly to the ocean's methane supersaturation, yet little is known about MPn utilization by marine microbes. To identify the microbial taxa and metabolic functions associated with MPn-driven methane production we performed parallel metagenomic, metatranscriptomic, and functional screening of microcosm perturbation experiments using surface water collected in the North Pacific Subtropical Gyre. In nutrient amended microcosms containing MPn, a substrate-driven microbial succession occurred. Initially, the addition of glucose and nitrate resulted in a bloom of Vibrionales and a transcriptional profile dominated by glucose-specific PTS transport and polyhydroxyalkanoate biosynthesis. Transcripts associated with phosphorus (P) acquisition were also overrepresented and suggested that the addition of glucose and nitrate had driven the community to P depletion. At this point, a second community shift occurred characterized by the increase in C-P lyase containing microbes of the Vibrionales and Rhodobacterales orders. Transcripts associated with C-P lyase components were among the most highly expressed at the community level, and only C-P lyase clusters were recovered in a functional screen for MPn utilization, consistent with this pathway being responsible for the majority, if not all, of the methane accumulation we observed. Our results identify specific bacterioplankton taxa that can utilize MPn aerobically under conditions of P limitation using the C-P lyase pathway, and thereby elicit a significant increase in the dissolved methane concentration.
methylphosphonate; phosphonates; genomics; transcriptomics; functional screens
Marine oxygen minimum zones (OMZs) support diverse microbial communities with roles in major elemental cycles. It is unclear how the taxonomic composition and metabolism of OMZ microorganisms vary between particle-associated and free-living size fractions. We used amplicon (16S rRNA gene) and shotgun metagenome sequencing to compare microbial communities from large (>1.6 μm) and small (0.2–1.6 μm) filter size fractions along a depth gradient in the OMZ off Chile. Despite steep vertical redox gradients, size fraction was a significantly stronger predictor of community composition compared to depth. Phylogenetic diversity showed contrasting patterns, decreasing towards the anoxic OMZ core in the small size fraction, but exhibiting maximal values at these depths within the larger size fraction. Fraction-specific distributions were evident for key OMZ taxa, including anammox planctomycetes, whose coding sequences were enriched up to threefold in the 0.2–1.6 μm community. Functional gene composition also differed between fractions, with the >1.6 μm community significantly enriched in genes mediating social interactions, including motility, adhesion, cell-to-cell transfer, antibiotic resistance and mobile element activity. Prokaryotic transposase genes were three to six fold more abundant in this fraction, comprising up to 2% of protein-coding sequences, suggesting that particle surfaces may act as hotbeds for transposition-based genome changes in marine microbes. Genes for nitric and nitrous oxide reduction were also more abundant (three to seven fold) in the larger size fraction, suggesting microniche partitioning of key denitrification steps. These results highlight an important role for surface attachment in shaping community metabolic potential and genome content in OMZ microorganisms.
Archaea; bacteria; microbial diversity; OMZ; oxycline; particle
Molecular surveys are revealing diverse eukaryotic assemblages in oxygen-limited ocean waters. These communities may play pivotal ecological roles through autotrophy, feeding, and a wide range of symbiotic associations with prokaryotes. We used 18S rRNA gene sequencing to provide the first snapshot of pelagic microeukaryotic community structure in two cellular size fractions (0.2–1.6 μm, >1.6 μm) from seven depths through the anoxic oxygen minimum zone (OMZ) off northern Chile. Sequencing of >154,000 amplicons revealed contrasting patterns of phylogenetic diversity across size fractions and depths. Protist and total eukaryote diversity in the >1.6 μm fraction peaked at the chlorophyll maximum in the upper photic zone before declining by ~50% in the OMZ. In contrast, diversity in the 0.2–1.6 μm fraction, though also elevated in the upper photic zone, increased four-fold from the lower oxycline to a maximum at the anoxic OMZ core. Dinoflagellates of the Dinophyceae and endosymbiotic Syndiniales clades dominated the protist assemblage at all depths (~40–70% of sequences). Other protist groups varied with depth, with the anoxic zone community of the larger size fraction enriched in euglenozoan flagellates and acantharean radiolarians (up to 18 and 40% of all sequences, respectively). The OMZ 0.2–1.6 μm fraction was dominated (11–99%) by Syndiniales, which exhibited depth-specific variation in composition and total richness despite uniform oxygen conditions. Metazoan sequences, though confined primarily to the 1.6 μm fraction above the OMZ, were also detected within the anoxic zone where groups such as copepods increased in abundance relative to the oxycline and upper OMZ. These data, compared to those from other low-oxygen sites, reveal variation in OMZ microeukaryote composition, helping to identify clades with potential adaptations to oxygen-depletion.
microeukaryote; ETSP OMZ; diversity; 18S; low oxygen
A major percentage (20 to 40%) of global marine fixed-nitrogen loss occurs in oxygen minimum zones (OMZs). Concentrations of O2 and the sensitivity of the anaerobic N2-producing processes of anammox and denitrification determine where this loss occurs. We studied experimentally how O2 at nanomolar levels affects anammox and denitrification rates and the transcription of nitrogen cycle genes in the anoxic OMZ off Chile. Rates of anammox and denitrification were reversibly suppressed, most likely at the enzyme level. Fifty percent inhibition of N2 and N2O production by denitrification was achieved at 205 and 297 nM O2, respectively, whereas anammox was 50% inhibited at 886 nM O2. Coupled metatranscriptomic analysis revealed that transcripts encoding nitrous oxide reductase (nosZ), nitrite reductase (nirS), and nitric oxide reductase (norB) decreased in relative abundance above 200 nM O2. This O2 concentration did not suppress the transcription of other dissimilatory nitrogen cycle genes, including nitrate reductase (narG), hydrazine oxidoreductase (hzo), and nitrite reductase (nirK). However, taxonomic characterization of transcripts suggested inhibition of narG transcription in gammaproteobacteria, whereas the transcription of anammox narG, whose gene product is likely used to oxidatively replenish electrons for carbon fixation, was not inhibited. The taxonomic composition of transcripts differed among denitrification enzymes, suggesting that distinct groups of microorganisms mediate different steps of denitrification. Sulfide addition (1 µM) did not affect anammox or O2 inhibition kinetics but strongly stimulated N2O production by denitrification. These results identify new O2 thresholds for delimiting marine nitrogen loss and highlight the utility of integrating biogeochemical and metatranscriptomic analyses.
The removal of fixed nitrogen via anammox and denitrification associated with low O2 concentrations in oceanic oxygen minimum zones (OMZ) is a major sink in oceanic N budgets, yet the sensitivity and dynamics of these processes with respect to O2 are poorly known. The present study elucidated how nanomolar O2 concentrations affected nitrogen removal rates and expression of key nitrogen cycle genes in water from the eastern South Pacific OMZ, applying state-of-the-art 15N techniques and metatranscriptomics. Rates of both denitrification and anammox responded rapidly and reversibly to changes in O2, but denitrification was more O2 sensitive than anammox. The transcription of key nitrogen cycle genes did not respond as clearly to O2, although expression of some of these genes decreased. Quantifying O2 sensitivity of these processes is essential for predicting through which pathways and in which environments, from wastewater treatment to the open oceans, nitrogen removal may occur.
This manuscript calls for an international effort to generate a comprehensive catalog from genome sequences of all the archaeal and bacterial type strains.
Microbes hold the key to life. They hold the secrets to our past (as the descendants of the earliest forms of life) and the prospects for our future (as we mine their genes for solutions to some of the planet's most pressing problems, from global warming to antibiotic resistance). However, the piecemeal approach that has defined efforts to study microbial genetic diversity for over 20 years and in over 30,000 genome projects risks squandering that promise. These efforts have covered less than 20% of the diversity of the cultured archaeal and bacterial species, which represent just 15% of the overall known prokaryotic diversity. Here we call for the funding of a systematic effort to produce a comprehensive genomic catalog of all cultured Bacteria and Archaea by sequencing, where available, the type strain of each species with a validly published name (currently∼11,000). This effort will provide an unprecedented level of coverage of our planet's genetic diversity, allow for the large-scale discovery of novel genes and functions, and lead to an improved understanding of microbial evolution and function in the environment.
Production of dissolved organic matter (DOM) by marine phytoplankton supplies the majority of organic substrate consumed by heterotrophic bacterioplankton in the sea. This production and subsequent consumption converts a vast quantity of carbon, nitrogen, and phosphorus between organic and inorganic forms, directly impacting global cycles of these biologically important elements. Details regarding the chemical composition of DOM produced by marine phytoplankton are sparse, and while often assumed, it is not currently known if phylogenetically distinct groups of marine phytoplankton release characteristic suites of DOM. To investigate the relationship between specific phytoplankton groups and the DOM they release, hydrophobic phytoplankton-derived dissolved organic matter (DOMP) from eight axenic strains was analyzed using high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS). Identification of DOM features derived from Prochlorococcus, Synechococcus, Thalassiosira, and Phaeodactylum revealed DOMP to be complex and highly strain dependent. Connections between DOMP features and the phylogenetic relatedness of these strains were identified on multiple levels of phylogenetic distance, suggesting that marine phytoplankton produce DOM that in part reflects its phylogenetic origin. Chemical information regarding the size and polarity ranges of features from defined biological sources was also obtained. Our findings reveal DOMP composition to be partially conserved among related phytoplankton species, and implicate marine DOM as a potential factor influencing microbial diversity in the sea by acting as a link between autotrophic and heterotrophic microbial community structures.
dissolved organic matter; untargeted metabolomics; marine phytoplankton; exometabolome; Prochlorococcus; Synechococcus; Thalassiosira; Phaeodactylum
Strain HIMB11 is a planktonic marine bacterium isolated from coastal seawater in Kaneohe Bay, Oahu, Hawaii belonging to the ubiquitous and versatile Roseobacter clade of the alphaproteobacterial family Rhodobacteraceae. Here we describe the preliminary characteristics of strain HIMB11, including annotation of the draft genome sequence and comparative genomic analysis with other members of the Roseobacter lineage. The 3,098,747 bp draft genome is arranged in 34 contigs and contains 3,183 protein-coding genes and 54 RNA genes. Phylogenomic and 16S rRNA gene analyses indicate that HIMB11 represents a unique sublineage within the Roseobacter clade. Comparison with other publicly available genome sequences from members of the Roseobacter lineage reveals that strain HIMB11 has the genomic potential to utilize a wide variety of energy sources (e.g. organic matter, reduced inorganic sulfur, light, carbon monoxide), while possessing a reduced number of substrate transporters.
marine bacterioplankton; Roseobacter; aerobic anoxygenic phototroph; dimethylsulfoniopropionate
The National Science Foundation’s EarthCube End User Workshop was held at USC Wrigley Marine Science Center on Catalina Island, California in August 2013. The workshop was designed to explore and characterize the needs and tools available to the community that is focusing on microbial and physical oceanography research with a particular emphasis on ‘omic research. The assembled researchers outlined the existing concerns regarding the vast data resources that are being generated, and how we will deal with these resources as their volume and diversity increases. Particular attention was focused on the tools for handling and analyzing the existing data, on the need for the construction and curation of diverse federated databases, as well as development of shared, interoperable, “big-data capable” analytical tools. The key outputs from this workshop include (i) critical scientific challenges and cyber infrastructure constraints, (ii) the current and future ocean ‘omics science grand challenges and questions, and (iii) data management, analytical and associated and cyber-infrastructure capabilities required to meet critical current and future scientific challenges. The main thrust of the meeting and the outcome of this report is a definition of the ‘omics tools, technologies and infrastructures that facilitate continued advance in ocean science biology, marine biogeochemistry, and biological oceanography.
The development of DNA sequencing methods for characterizing microbial communities has evolved rapidly over the past decades. To evaluate more traditional, as well as newer methodologies for DNA library preparation and sequencing, we compared fosmid, short-insert shotgun and 454 pyrosequencing libraries prepared from the same metagenomic DNA samples. GC content was elevated in all fosmid libraries, compared with shotgun and 454 libraries. Taxonomic composition of the different libraries suggested that this was caused by a relative underrepresentation of dominant taxonomic groups with low GC content, notably Prochlorales and the SAR11 cluster, in fosmid libraries. While these abundant taxa had a large impact on library representation, we also observed a positive correlation between taxon GC content and fosmid library representation in other low-GC taxa, suggesting a general trend. Analysis of gene category representation in different libraries indicated that the functional composition of a library was largely a reflection of its taxonomic composition, and no additional systematic biases against particular functional categories were detected at the level of sequencing depth in our samples. Another important but less predictable factor influencing the apparent taxonomic and functional library composition was the read length afforded by the different sequencing technologies. Our comparisons and analyses provide a detailed perspective on the influence of library type on the recovery of microbial taxa in metagenomic libraries and underscore the different uses and utilities of more traditional, as well as contemporary ‘next-generation' DNA library construction and sequencing technologies for exploring the genomics of the natural microbial world.
454 pyrosequencing; DNA sequencing; metagenomics; fosmid; shotgun library
Planktonic microbial activity and community structure is dynamic, and can change dramatically on time scales of hours to days. Yet for logistical reasons, this temporal scale is typically under-sampled in the marine environment. In order to facilitate higher-resolution, long-term observation of microbial diversity and activity, we developed a protocol for automated collection and fixation of marine microbes using the Environmental Sample Processor (ESP) platform. The protocol applies a preservative (RNALater) to cells collected on filters, for long-term storage and preservation of total cellular RNA. Microbial samples preserved using this protocol yielded high-quality RNA after 30 days of storage at room temperature, or onboard the ESP at in situ temperatures. Pyrosequencing of complementary DNA libraries generated from ESP-collected and preserved samples yielded transcript abundance profiles nearly indistinguishable from those derived from conventionally treated replicate samples. To demonstrate the utility of the method, we used a moored ESP to remotely and autonomously collect Monterey Bay seawater for metatranscriptomic analysis. Community RNA was extracted and pyrosequenced from samples collected at four time points over the course of a single day. In all four samples, the oxygenic photoautotrophs were predominantly eukaryotic, while the bacterial community was dominated by Polaribacter-like Flavobacteria and a Rhodobacterales bacterium sharing high similarity with Rhodobacterales sp. HTCC2255. However, each time point was associated with distinct species abundance and gene transcript profiles. These laboratory and field tests confirmed that autonomous collection and preservation is a feasible and useful approach for characterizing the expressed genes and environmental responses of marine microbial communities.
metatranscriptomics; gene expression; automated sampling; marine bacterioplankton; RNA preservation; Monterey Bay
Proteorhodopsin (PR) is a photoprotein that functions as a light-driven proton pump in diverse marine Bacteria and Archaea. Recent studies have suggested that PR may enhance both growth rate and yield in some flavobacteria when grown under nutrient-limiting conditions in the light. The direct involvement of PR, and the metabolic details enabling light-stimulated growth, however, remain uncertain. Here, we surveyed transcriptional and growth responses of a PR-containing marine flavobacterium during carbon-limited growth in the light and the dark. As previously reported (Gómez-Consarnau et al., 2007), Dokdonia strain MED134 exhibited light-enhanced growth rates and cell yields under low carbon growth conditions. Inhibition of retinal biosynthesis abolished the light-stimulated growth response, supporting a direct role for retinal-bound PR in light-enhanced growth. Among protein-coding transcripts, both PR and retinal biosynthetic enzymes showed significant upregulation in the light. Other light-associated proteins, including bacterial cryptochrome and DNA photolyase, were also expressed at significantly higher levels in the light. Membrane transporters for Na+/phosphate and Na+/alanine symporters, and the Na+-translocating NADH-quinone oxidoreductase (NQR) linked electron transport chain, were also significantly upregulated in the light. Culture experiments using a specific inhibitor of Na+-translocating NQR indicated that sodium pumping via NQR is a critical metabolic process in the light-stimulated growth of MED134. In total, the results suggested the importance of both the PR-enabled, light-driven proton gradient, as well as the generation of a Na+ ion gradient, as essential components for light-enhanced growth in these flavobacteria.
flavobacteria; marine; photoheterotrophy; proteorhodopsin; transcriptomics
As part of an ongoing survey of microbial community gene expression in the ocean, we sequenced and compared ∼38 Mbp of community transcriptomes and ∼157 Mbp of community genomes from four bacterioplankton samples, along a defined depth profile at Station ALOHA in North Pacific subtropical gyre (NPSG). Taxonomic analysis suggested that the samples were dominated by three taxa: Prochlorales, Consistiales and Cenarchaeales, which comprised 36–69% and 29–63% of the annotated sequences in the four DNA and four cDNA libraries, respectively. The relative abundance of these taxonomic groups was sometimes very different in the DNA and cDNA libraries, suggesting differential relative transcriptional activities per cell. For example, the 125 m sample genomic library was dominated by Pelagibacter (∼36% of sequence reads), which contributed fewer sequences to the community transcriptome (∼11%). Functional characterization of highly expressed genes suggested taxon-specific contributions to specific biogeochemical processes. Examples included Roseobacter relatives involved in aerobic anoxygenic phototrophy at 75 m, and an unexpected contribution of low abundance Crenarchaea to ammonia oxidation at 125 m. Read recruitment using reference microbial genomes indicated depth-specific partitioning of coexisting microbial populations, highlighted by a transcriptionally active high-light-like Prochlorococcus population in the bottom of the photic zone. Additionally, nutrient-uptake genes dominated Pelagibacter transcripts, with apparent enrichment for certain transporter types (for example, the C4-dicarboxylate transport system) over others (for example, phosphate transporters). In total, the data support the utility of coupled DNA and cDNA analyses for describing taxonomic and functional attributes of microbial communities in their natural habitats.
metatranscriptomics; metagenomics; bacterioplankton; microbial gene expression/regulation; biogeochemical processes
Sequencing of microbial community RNA (metatranscriptome) is a useful approach for assessing gene expression in microorganisms from the natural environment. This method has revealed transcriptional patterns in situ, but can also be used to detect transcriptional cascades in microcosms following experimental perturbation. Unambiguously identifying differential transcription between control and experimental treatments requires constraining effects that are simply due to sampling and bottle enclosure. These effects remain largely uncharacterized for “challenging” microbial samples, such as those from anoxic regions that require special handling to maintain in situ conditions. Here, we demonstrate substantial changes in microbial transcription induced by sample collection and incubation in experimental bioreactors. Microbial communities were sampled from the water column of a marine oxygen minimum zone by a pump system that introduced minimal oxygen contamination and subsequently incubated in bioreactors under near in situ oxygen and temperature conditions. Relative to the source water, experimental samples became dominated by transcripts suggestive of cell stress, including chaperone, protease, and RNA degradation genes from diverse taxa, with strong representation from SAR11-like alphaproteobacteria. In tandem, transcripts matching facultative anaerobic gammaproteobacteria of the Alteromonadales (e.g., Colwellia) increased 4–13 fold up to 43% of coding transcripts, and encoded a diverse gene set suggestive of protein synthesis and cell growth. We interpret these patterns as taxon-specific responses to combined environmental changes in the bioreactors, including shifts in substrate or oxygen availability, and minor temperature and pressure changes during sampling with the pump system. Whether such changes confound analysis of transcriptional patterns may vary based on the design of the experiment, the taxonomic composition of the source community, and on the metabolic linkages between community members. These data highlight the impressive capacity for transcriptional changes within complex microbial communities, underscoring the need for caution when inferring in situ metabolism based on transcript abundances in experimental incubations.