Angelicque E. White

Unicellular Cyanobacterial Distributions Broaden the Oceanic N2 Fixation Domain

Oceanic Nitrogen Fixation Nitrogen fixation in the oceans is important in sustaining global marine productivity and balances carbon dioxide export to the deep ocean. It was previously believed that marine nitrogen fixation was due to a single genus of filamentous cyanobacteria, Trichodesmium. The recent discovery of unicellular open-ocean cyanobacteria has raised the question of how they contribute to global ocean nitrogen fixation and how they compare in distribution and activity to Trichodesmium. Using data collected from the southwest Pacific Ocean, Moisander et al. (p. 1512, published online 25 February) show that the unicellular nitrogen-fixing cyanobacteria (UCYN-A and Crocosphaera watsonii) have distinct ecophysiologies and distinct oceanic distributions from each other, and from Trichodesmium. These data can be incorporated into models to retune estimates of the global rates of oceanic nitrogen fixation and carbon sequestration. Nitrogen fixation in the South Pacific Ocean is partitioned among several microbe species with distinct ecophysiologies. Nitrogen (N2)–fixing microorganisms (diazotrophs) are an important source of biologically available fixed N in terrestrial and aquatic ecosystems and control the productivity of oligotrophic ocean ecosystems. We found that two major groups of unicellular N2-fixing cyanobacteria (UCYN) have distinct spatial distributions that differ from those of Trichodesmium, the N2-fixing cyanobacterium previously considered to be the most important contributor to open-ocean N2 fixation. The distributions and activity of the two UCYN groups were separated as a function of depth, temperature, and water column density structure along an 8000-kilometer transect in the South Pacific Ocean. UCYN group A can be found at high abundances at substantially higher latitudes and deeper in subsurface ocean waters than Trichodesmium. These findings have implications for the geographic extent and magnitude of basin-scale oceanic N2 fixation rates.

Microbial community gene expression within colonies of the diazotroph, Trichodesmium, from the Southwest Pacific Ocean.

Trichodesmium are responsible for a large fraction of open ocean nitrogen fixation, and are often found in complex consortia of other microorganisms, including viruses, prokaryotes, microbial eukaryotes and metazoa. We applied a community gene expression (metatranscriptomic) approach to study the patterns of microbial gene utilization within colonies of Trichodesmium collected during a bloom in the Southwest Pacific Ocean in April 2007. The survey generated 5711-day and 5385-night putative mRNA reads. The majority of mRNAs were from the co-occurring microorganisms and not Trichodesmium, including other cyanobacteria, heterotrophic bacteria, eukaryotes and phage. Most transcripts did not share homology with proteins from cultivated microorganisms, but were similar to shotgun sequences and unannotated proteins from open ocean metagenomic surveys. Trichodesmium transcripts were mostly expressed photosynthesis, N2 fixation and S-metabolism genes, whereas those in the co-occurring microorganisms were mostly involved in genetic information storage and processing. Detection of Trichodesmium genes involved in P uptake and As detoxification suggest that local enrichment of N through N2 fixation may lead to a P-stress response. Although containing similar dominant transcripts to open ocean metatranscriptomes, the overall pattern of gene expression in Trichodesmium colonies was distinct from free-living pelagic assemblages. The identifiable genes expressed by Trichodesmium and closely associated microorganisms reflect the constraints of life in well-lit and nutrient-poor waters, with biosynthetic investment in nutrient acquisition and cell maintenance, which is in contrast to gene transcription by soil and coastal seawater microbial assemblages. The results provide insight into aggregate microbial communities in contrast to planktonic free-living assemblages that are the focus of other studies.

In situ transcriptomic analysis of the globally important keystone N 2 -fixing taxon Crocosphaera watsonii

The diazotrophic cyanobacterium Crocosphaera watsonii supplies fixed nitrogen (N) to N-depleted surface waters of the tropical oceans, but the factors that determine its distribution and contribution to global N2 fixation are not well constrained for natural populations. Despite the heterogeneity of the marine environment, the genome of C. watsonii is highly conserved in nucleotide sequence in contrast to sympatric planktonic cyanobacteria. We applied a whole assemblage shotgun transcript sequencing approach to samples collected from a bloom of C. watsonii observed in the South Pacific to understand the genomic mechanisms that may lead to high population densities. We obtained 999 C. watsonii transcript reads from two metatranscriptomes prepared from mixed assemblage RNA collected in the day and at night. The C. watsonii population had unexpectedly high transcription of hypothetical protein genes (31% of protein-encoding genes) and transposases (12%). Furthermore, genes were expressed that are necessary for living in the oligotrophic ocean, including the nitrogenase cluster and the iron-stress-induced protein A (isiA) that functions to protect photosystem I from high-light-induced damage. C. watsonii transcripts retrieved from metatranscriptomes at other locations in the southwest Pacific Ocean, station ALOHA and the equatorial Atlantic Ocean were similar in composition to those recovered in the enriched population. Quantitative PCR and quantitative reverse transcriptase PCR were used to confirm the high expression of these genes within the bloom, but transcription patterns varied at shallower and deeper horizons. These data represent the first transcript study of a rare individual microorganism in situ and provide insight into the mechanisms of genome diversification and the ecophysiology of natural populations of keystone organisms that are important in global nitrogen cycling.

Summer surface waters in the Gulf of California: Prime habitat for biological N2 fixation

[1] We report significant rates of dinitrogen (N 2 ) fixation in the central basins of the Gulf of California (GC) during July-August 2005. Mixing model estimates based upon δ 15 N values of particulate matter in the surface mixed layer indicate that N 2 fixation provides as much as 35% to 48% of the phytoplankton-based nitrogen demand in the central Guaymas and Carmen basins. Microscopic analyses identify the responsible genera as the N 2 -fixing endosymbiont, Richelia intracellularis, with lesser contributions from the large nonheterocystous diazotroph Trichodesmium. Analyses of remotely sensed chlorophyll a and sea surface temperature indicate that primary production levels are elevated in regions of the GC where oceanographic conditions are ideal in summertime for the growth of N 2 -fixing organisms. These findings suggest that biological N 2 fixation must be taken into account when assessing past and present nitrogen dynamics in this environmentally important region.

SAR11 lipid renovation in response to phosphate starvation

Significance Nonphosphorus lipids produced by heterotrophic bacteria have been measured in marine ecosystems without an understanding of their origins or role. This work shows SAR11 chemoheterotrophic bacteria synthesize multiple nonphosphorus lipids in response to phosphate depletion. Because this process results in a reduced cellular P:C ratio, it impacts our understanding of ocean processes related to cellular elemental stoichiometry by showing how different environmental parameters alter P:C ratios in heterotrophs. Also, SAR11 grown with excess organophosphonate synthesized phosphorus-free lipids. This finding contrasts the contemporary view of organophosphorus utilization because organophosphate-derived phosphorus did not equally substitute for inorganic phosphate in lipids. Considering lipid phosphorus content was lower in cells using organophosphonate, phosphorus-based productivity estimates may vary as a function of phosphorus source. Phytoplankton inhabiting oligotrophic ocean gyres actively reduce their phosphorus demand by replacing polar membrane phospholipids with those lacking phosphorus. Although the synthesis of nonphosphorus lipids is well documented in some heterotrophic bacterial lineages, phosphorus-free lipid synthesis in oligotrophic marine chemoheterotrophs has not been directly demonstrated, implying they are disadvantaged in phosphate-deplete ecosystems, relative to phytoplankton. Here, we show the SAR11 clade chemoheterotroph Pelagibacter sp. str. HTCC7211 renovates membrane lipids when phosphate starved by replacing a portion of its phospholipids with monoglucosyl- and glucuronosyl-diacylglycerols and by synthesizing new ornithine lipids. Lipid profiles of cells grown with excess phosphate consisted entirely of phospholipids. Conversely, up to 40% of the total lipids were converted to nonphosphorus lipids when cells were starved for phosphate, or when growing on methylphosphonate. Cells sequentially limited by phosphate and methylphosphonate transformed >75% of their lipids to phosphorus-free analogs. During phosphate starvation, a four-gene cluster was significantly up-regulated that likely encodes the enzymes responsible for lipid renovation. These genes were found in Pelagibacterales strains isolated from a phosphate-deficient ocean gyre, but not in other strains from coastal environments, suggesting alternate lipid synthesis is a specific adaptation to phosphate scarcity. Similar gene clusters are found in the genomes of other marine α-proteobacteria, implying lipid renovation is a common strategy used by heterotrophic cells to reduce their requirement for phosphorus in oligotrophic habitats.

New insights into bacterial acquisition of phosphorus in the surface ocean

Since 1958 when Alfred C. Redfield (1) recognized the similarity between the ratios of elements in living biomass and those dissolved in the surrounding seawater, we have understood that microorganisms largely control the concentrations, distribution, and molecular makeup of nutritional resources in the ocean. The primary elemental ingredients for life, carbon (C), nitrogen (N), and phosphorus (P), are assembled, disassembled, transformed, and consumed by marine microorganisms, resulting in a steady cycling of elements between intracellular, inorganic, and organic reservoirs. Of these pools, dissolved organic matter (DOM) represents the largest C, N, and P reservoir in the surface ocean of most marine habitats, greatly exceeding the respective concentrations of inorganic pools or that found in living organisms. DOM is a source of energy and elements, fueling heterotrophic and autotrophic growth alike (2), yet we understand very little about the biomolecular strategies marine microbes employ to use organic substrates in the global ocean. What is the molecular composition of organic matter, where do these compounds originate, and how much of this is bioavailable? How do microbes hydrolyze and transport constituents of DOM into the cell? What are the factors that regulate enzyme expression and control the decomposition of organic matter? These are but a few of the questions that must be addressed to fundamentally and mechanistically understand how microorganisms assimilate, transform, and turn over elemental resources in the ocean. In this issue of PNAS, Luo et al. (3) use a bioinformatics approach to investigate the diversity and localization of bacterial phosphatases, enzymes specialized for the hydrolysis of a reactive fraction of DOM, P-linked esters. Most notably, their study indicates that a significant fraction of bacteria may transport intact organophosphate compounds across the cell membrane for intracellular depolymerization, a finding counter to the prevailing concept of phosphatases as being largely extracellular. These disparate …

Diversity and Activity of Communities Inhabiting Plastic Debris in the North Pacific Gyre

Marine plastic debris is a growing concern that has captured the general public’s attention. While the negative impacts of plastic debris on oceanic macrobiota, including mammals and birds, are well documented, little is known about its influence on smaller marine residents, including microbes that have key roles in ocean biogeochemistry. Our work provides a new perspective on microbial communities inhabiting microplastics that includes its effect on microbial biogeochemical activities and a description of the cross-domain communities inhabiting plastic particles. This study is among the first molecular ecology, plastic debris biota surveys in the North Pacific Subtropical Gyre. It has identified fundamental differences in the functional potential and taxonomic composition of plastic-associated microbes versus planktonic microbes found in the surrounding open-ocean habitat. ABSTRACT Marine plastic debris has become a significant concern in ocean ecosystems worldwide. Little is known, however, about its influence on microbial community structure and function. In 2008, we surveyed microbial communities and metabolic activities in seawater and on plastic on an oceanographic expedition through the “great Pacific garbage patch.” The concentration of plastic particles in surface seawater within different size classes (2 to 5 mm and >5 mm) ranged from 0.35 to 3.7 particles m−3 across sampling stations. These densities and the particle size distribution were consistent with previous values reported in the North Pacific Ocean. Net community oxygen production (NCP = gross primary production − community respiration) on plastic debris was positive and so net autotrophic, whereas NCP in bulk seawater was close to zero. Scanning electron microscopy and metagenomic sequencing of plastic-attached communities revealed the dominance of a few metazoan taxa and a diverse assemblage of photoautotrophic and heterotrophic protists and bacteria. Bryozoa, Cyanobacteria, Alphaproteobacteria, and Bacteroidetes dominated all plastic particles, regardless of particle size. Bacteria inhabiting plastic were taxonomically distinct from the surrounding picoplankton and appeared well adapted to a surface-associated lifestyle. Genes with significantly higher abundances among plastic-attached bacteria included che genes, secretion system genes, and nifH genes, suggesting enrichment for chemotaxis, frequent cell-to-cell interactions, and nitrogen fixation. In aggregate, our findings suggest that plastic debris forms a habitat for complex microbial assemblages that have lifestyles, metabolic pathways, and biogeochemical activities that are distinct from those of free-living planktonic microbial communities. IMPORTANCE Marine plastic debris is a growing concern that has captured the general public’s attention. While the negative impacts of plastic debris on oceanic macrobiota, including mammals and birds, are well documented, little is known about its influence on smaller marine residents, including microbes that have key roles in ocean biogeochemistry. Our work provides a new perspective on microbial communities inhabiting microplastics that includes its effect on microbial biogeochemical activities and a description of the cross-domain communities inhabiting plastic particles. This study is among the first molecular ecology, plastic debris biota surveys in the North Pacific Subtropical Gyre. It has identified fundamental differences in the functional potential and taxonomic composition of plastic-associated microbes versus planktonic microbes found in the surrounding open-ocean habitat. Author Video: An author video summary of this article is available.

An Open Ocean Trial of Controlled Upwelling Using Wave Pump Technology

In 1976, John D. Isaacs proposed to use wave energy to invert the density structure of the ocean and pump deep, nutrient-rich water into the sunlit surface layers. The basic principle is simple: a length of tubing attached to a surface buoy at the top, and a one-way valve at the bottom can be extended below the euphotic zone to act as a conduit for deep water. The vertical motion of the ocean forces the attached valve to open on the downslope of a wave and close on the upslope, thus generating upward movement of deep water to the surface ocean. Although Isaacs’s wave-powered pump has taken many forms, from energy production to aquaculture to the more recent suggestion that artificial upwelling could be used to stimulate primary productivity and carbon sequestration, the simple engineering concept remains the same. In June 2008, the authors tested a commercially available wave pump (Atmocean) north of Oahu, Hawaii, to assess the logistics of at-sea deployment and the durability of the equipment under open ocean conditions. This test was done as part of an experiment designed to evaluate a recently published hypothesis that upwelling of water containing excess phosphate (P) relative to nitrogen (N) compared to the canonical ‘‘Redfield’’ molar ratio of 16N:1P would generate a two-phased phytoplankton bloom. The end result of this field experiment was rapid delivery (,2 h for a 300-m transit) of deep water to the surface ocean followed by catastrophic failure of pump materials under the dynamic stresses of the oceanic environment. Wave-driven upwelling of cold water was documented for a period of ;17 h, with a volumetric upwelling rate of ;45 m 3 h 21 and an estimated total input of 765 m 3 of nutrient-enriched deep water. The authors discuss the deployment of a 300-m wave pump, the strategy to sample a biogeochemical response, the engineering challenges faced, and the implications of these results for future experiments aimed at stimulating the growth of phytoplankton.

Climatic regulation of the neurotoxin domoic acid

Significance We investigate regulation of domoic acid, a potent marine phycotoxin, at the climate scale. Due to the threat domoic acid can pose to public health, marine wildlife, and coastal economies, decades of laboratory experiments have examined controls on domoic acid production without reaching consensus on reliable toxin-producing conditions. Our findings reveal an association between domoic acid in shellfish and climate-scale warm ocean conditions, a unique, large-scale perspective relative to previous work. Domoic acid is a potent neurotoxin produced by certain marine microalgae that can accumulate in the foodweb, posing a health threat to human seafood consumers and wildlife in coastal regions worldwide. Evidence of climatic regulation of domoic acid in shellfish over the past 20 y in the Northern California Current regime is shown. The timing of elevated domoic acid is strongly related to warm phases of the Pacific Decadal Oscillation and the Oceanic Niño Index, an indicator of El Niño events. Ocean conditions in the northeast Pacific that are associated with warm phases of these indices, including changes in prevailing currents and advection of anomalously warm water masses onto the continental shelf, are hypothesized to contribute to increases in this toxin. We present an applied domoic acid risk assessment model for the US West Coast based on combined climatic and local variables. Evidence of regional- to basin-scale controls on domoic acid has not previously been presented. Our findings have implications in coastal zones worldwide that are affected by this toxin and are particularly relevant given the increased frequency of anomalously warm ocean conditions.

Small phytoplankton drive high summertime carbon and nutrient export in the Gulf of California and Eastern Tropical North Pacific

Summertime carbon, nitrogen, and biogenic silica export was examined using 234Th:238U disequilibria combined with free floating sediment traps and fine scale water column sampling with in situ pumps (ISP) within the Eastern Tropical North Pacific and the Gulf of California. Fine scale ISP sampling provides evidence that in this system, particulate carbon (PC) and particulate nitrogen (PN) concentrations were more rapidly attenuated relative to 234Th activities in small particles compared to large particles, converging to 1–5 µmol dpm−1 by 100 m. Comparison of elemental particle composition, coupled with particle size distribution analysis, suggests that small particles are major contributors to particle flux. While absolute PC and PN export rates were dependent on the method used to obtain the element/234Th ratio, regional trends were consistent across measurement techniques. The highest C fixation rates were associated with diatom-dominated surface waters. Yet, the highest export efficiencies occurred in picoplankton-dominated surface waters, where relative concentrations of diazotrophs were also elevated. Our results add to the increasing body of literature that picoplankton- and diazotroph-dominated food webs in subtropical regions can be characterized by enhanced export efficiencies relative to food webs dominated by larger phytoplankton, e.g., diatoms, in low productivity pico/nanoplankton-dominated regions, where small particles are major contributors to particle export. Findings from this region are compared globally and provide insights into the efficiency of downward particle transport of carbon and associated nutrients in a warmer ocean where picoplankton and diazotrophs may dominate. Therefore, we argue the necessity of collecting multiple particle sizes used to convert 234Th fluxes into carbon or other elemental fluxes, including <50 µm, since they can play an important role in vertical fluxes, especially in oligotrophic environments. Our results further underscore the necessity of using multiple techniques to quantify particle flux given the uncertainties associated with each collection method.