Jonathan P. Zehr - US grants

The Microbial Observatory focuses on the microbes found in Mono Lake, an alkaline, hypersaline, currently meromictic (lighter, less saline water overlies heavier, more saline water throughout the year) lake located east of the Sierra Nevada in California. There are a number of reasons why Mono Lake is an ideal site for a Microbial Observatory. It is a well-defined, ecologically simple, microbially dominated ecosystem for which long-term ecological and limnological data exist. Mono Lake is a hydrologically simple system, which makes modeling tractable, yet it contains complex gradients of chemical and physical variables as a result of meromixis. The lake is located close to a major field station (the Sierra Nevada Aquatic Research Laboratory, administered by the University of California, Santa Barbara). There are ongoing studies of the lake's physics, plankton ecology and biogeochemistry that provide a comprehensive framework for the microbial studies. Mono Lake is currently undergoing a human-induced (and thus predictable) limnological transition which imparts a predictable temporal trajectory to physical, biogeochemical and ecological processes. This change mirrors past, natural events in Great Basin lakes resulting from climate oscillations and similar, if less extreme, changes in physical limnology might be expected in other lakes. Mono Lake represents an extreme environment that is likely to harbor unique microbes. However, relatively little is known about the types of microorganisms dwelling in Mono Lake, their phylogenetic diversity, taxonomy, ecology or ecophysiology. For example, recent phylogenetic analysis of an important phytoplankton demonstrates it to be a new class of algae with unusual physiological properties and biochemical composition. While abundant bacterial populations and the existence of pronounced bacterial plates have been noted before in Mono Lake, the temporal and spatial variation in diversity of the bacterial community has only recently begun to be investigated. The primary goal of this research is to examine the distributions of Mono Lake microbes and to understand the response of microbial assemblages to the gradients of physical and chemical variables in relation to temporal changes driven by hydrodynamics. The specific objectives of the project are to: 1) Identify and characterize the microbial assemblages in the unique Mono Lake ecosystem. 2) Determine the spatial and temporal variation of the Mono Lake microbial assemblage, particularly in reference to evolving meromixis. 3) Determine the response of the microbial community to physical processes, especially short-term and small-scale variation in mixing (for example, enhanced vertical diffusion as a result of boundary mixing or localized gravitational circulation). 4) Provide a mechanistic understanding of the interactions between the physical/chemical structure and microbial assemblages as the basis for predictive (long-term) modeling of the relationship between microbial processes, lake biogeochemistry
and primary production. This project provides a unique opportunity to identify novel microorganisms and define interactions among microorganisms in complex gradients of physical/chemical conditions usually only encountered at sediment-water interfaces. This is a collaborative project involving Drs. James Hollibaugh and Samantha Joye, University of Georgia, award #9977886, Dr. Robert Jellison, University of California, Santa Barbara (#9977901) and Dr. Jonathan Zehr, University of California, Santa Cruz (#9977892).

BIOCOMPLEXITY: Collaborative Research: Biocomplexity of aquatic microbial systems -- relating diversity of microorganisms to ecosystem function

Microbial biogeochemical cycling of the elements regulates a dynamic environment in which the cycles of different elements are linked through the physiology of microorganisms. While a certain degree of understanding can be gained through physical/chemical approaches to measurement and modeling of the net transformations, these approaches necessarily rely on gross simplifications about the role and regulation of the various functional groups (guilds) involved. Recent advances in molecular microbial ecology have shown the microbial world to contain immense diversity and complexity at every level: redundancy and duplication of functional genes within a single organism; molecular diversity among functional genes that encode the same process in different organisms; large genetic diversity among different organisms apparently engaged in the same biogeochemical function within single communities; great variability in the species composition of different communities that apparently perform equally well. The goal of this project is to investigate the functional relationship between complexity in microbial communities and the physical/chemical environment at a range of biological and ecological scales. Previously, such analysis was technologically limited by the inability to assay large numbers of samples simultaneously for a large number of genes and phylotypes. Using gene array technology, the researchers will be able to detect the distribution and differential expression of functional genes in natural systems. The results of this study will constitute the first step towards application of DNA chip technology for gene expression of "exotic" (i.e., not of biomedical importance) processes and organisms in the environment. The gene arrays, along with a full suite of ecosystem process measurements, will be deployed along a transect that spans the eutrophic - oligotrophic gradient from the inland waters of the Chesapeake Bay out to the Sargasso Sea. Experiments and functional gene studies will focus on key transformations in the carbon and nitrogen cycles (C fixation, N fixation, nitrification, denitrification, urea assimilation). The diversity of guilds will be interpreted in terms of ecosystem function, assessed using geochemical data and tracer experiments. In addition to field studies designed to investigate and dissect the natural system, the group of collaborating scientists will also perform perturbation experiments using mesocosms. The goal of these experiments is to determine how microbial species diversity affects the major energy and nutrient flows within ecosystems, and to assess the degree of stability or instability associated with changes in redundancy within guilds of microorganisms responsible for major nitrogen and carbon pathways.

Zehr 9977460 and Montoya 9977528

The availability of nutrients, primarily phosphorus, nitrogen and iron, limits the productivity of the oceans. Nitrogen is often believed to be an important, if not limiting, nutrient in oligotrophic oceans. Nitrogen, in the form of dinitrogen gas, is a major component of the Earth's atmosphere and is abundant in seawater as dissolved gas. However, most organisms can use nitrogen in the form of nitrate, ammonium, or organic nitrogen, but cannot directly use dinitrogen. This form of nitrogen can be used only by diazotrophs, which can "fix" nitrogen into ammonium through the action of the enzyme nitrogenase. Diverse prokaryotic taxa have the ability to fix nitrogen, including heterotrophic bacteria, cyanobacteria and Archaea. Despite the diversity of types of microorganisms that can fix nitrogen, very few nitrogen--fixing taxa have previously been reported from the open ocean. Recent studies in the Atlantic and Pacific Oceans indicate that nitrogen fixation is more important in nitrogen dynamics than previously believed, and that there may be a large, unaccounted for flux of nitrogen into the mixed layer, which may be due to nitrogen fixation. Estimates of the nitrogen fixation by known diazotrophs, the cyanobacterium Trichodesmium and endosymbiont Richelia, cannot account for this estimated N flux.

Using a molecular approach, it has very recently been shown that there are diverse microorganisms in the open ocean environment, which have the genetic capacity for nitrogen fixation. In this project, the ecological and biological significance of these microorganisms will be examined using molecular approaches, cultivation efforts, nitrogen fixation rate measurements, and stable isotope measurements. Recent developments in RNA technology (nifH reverse-transcriptase polymerase chain reaction, RT-PCR) make it possible to examine the expression of the unique nitrogenase phylotypes in seasonal studies, and in experimental manipulations. These studies will focus at the HOT site in order to identify and characterize the novel nitrogen-fixing cyanobacterium, to determine the factors controlling its growth and activity, and to quantify its contribution to the local nitrogen budget using stable isotope techniques. In parallel, experiments on nitrogen-fixation activity associated with invertebrates will be investigated by N-15 tracer measurements and RT-PCR to identify the types of nitrogen-fixing microorganisms responsible for observed nitrogen fixation activity. Finally, the novel diazotroph will be characterized by examining the nitrogen fixation apparatus (nitrogenase genes other than nifH) to determine whether the nitrogen fixation apparatus of these organisms is unusual, which may have biotechnological applications as well as evolutionary implications. The identification and characterization of these novel nitrogen-fixing microorganisms in the open ocean is timely and potentially important for evaluating the nitrogen budget of the sea.

The two intertwined goals of this project are to determine the suite of genes expressed by Pseudo-nitzschia under toxin-producing conditions, and to acquire a better understanding of the connections between environmental conditions and physiological responses leading to toxin production. A set of physiological experiments will permit evaluation of molecular probes generated from gene expression studies. In turn, the molecular probes will be used to
interrogate natural populations and help determine the physiological status of Pseudo-nitzschia in the field. The ultimate goal is to find a specific gene transcript or a pattern of gene expression that is correlated with toxin production in the field. The following hypotheses will be tested:
H1: There are genes or a suite of genes whose expression pattern is highly correlated with toxin production in Pseudo-nitzschia.
H2: A primary trigger for toxin production in Monterey Bay is silicate limitation, so that certain oceanographic conditions permit bloom development.
H3: Silicate limitation may sensitize cells to trace-metal (e.g. copper) stress and the toxin (domoic acid) can function as a metal ion buffer.

Batch and continuous cultures will be stressed with silicate, copper, and iron. Growth, substrate utilization, and physiological parameters (variable fluorescence, nutrient quotas, amino acid pools, including domoic acid) will be assessed. Cells will be harvested for development of cDNA subtraction libraries under different stressors. Gene arrays developed from these libraries will provide molecular probes for field testing. Identification of genes related to toxin production, but not general metabolism, will be facilitated by information generated by the physiology experiments. The laboratory work will be combined with a limited field program for assessment of environmental triggers (e.g. copper, silicate, iron stress) and for testing of the molecular probes. Results from the molecular expression and physiological assays will permit an initial description of the cellular pathways mediating environmental triggers (e.g silicate and metals) for production of toxin.

0116278
Zehr
This Major Research Instrumentation award to University of California Santa Cruz will provide support for acquisition of a genetic analyzer for DNA sequencing, liquid handling robotics, computer workstations and software for DNA analysis, and technical support for the Molecular Ecology and Evolutionary Genetics facility. It is expected that the new instrumentation and the interdepartmental research and training facility will foster interdisciplinary approaches to a broad range of problems in marine and terrestrial ecology and evolutionary biology. UCSC will contribute cost-sharing of more than 30% of the cost of this project from non-federal funds.
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We propose a study of symbioses between cyanobacteria and planktonic diatoms, dinoflagellates, radiolarians, silicoflagellates and other planktonic protozoans in the equatorial oligotrophic Atlantic and Pacific Oceans. We have recently observed that these symbioses are diverse and abundant, and some have not yet been reported in the scientific literature. The biology and the phylogeny of these symbioses are virtually unstudied. The 16S rDNA sequences of cyanobacterial symbionts within a diatom (Climacodium) showed that they are closely related to the N2 fixing genus Cyanothece (which fixes during the night), which suggests that some of these symbioses may involve N2 fixation.

We hypothesize that cyanelle symbionts may benefit host species either via incorporation of fixed N or C (via DON or DOC release or by being phagocyticized). The research approach will use combined microscopy and molecular biology approaches to link observed relationships between cyanobacteria and the eukaryotic microalgae with phylogenetic information and detection of the genes involved in nitrogen fixation. Using samples collected directly by microscopy, and bulk filtered water samples, the identity, nitrogen fixation potential (presence of nitrogenase genes) and expression of nitrogen fixation genes will be related to specific organisms and quantified in the water column. These approaches will be based on amplification and sequencing of 16S rDNA from the symbionts to understand the phylogeny of the cyanobacteria, and detection of the presence of nifH as an indicator of capacity to fix N2, examination of the ultrastructure of host and cyanelle, use of 14 C autoradiography to examine C transfer, measurements of abundance and distribution to quantify ecological importance, along with culture attempts to allow laboratory studies directed at determining the nature of the symbiotic interactions using GC/MS. The proposed research will take advantage of four already- funded (NSF) research cruises scheduled over the next three years in the Atlantic and Pacific Oceans.

The productivity of the oceans is limited by the availability of nutrients, which has implications for the fluxes of carbon between the atmosphere and oceans. In a previous award the PIs found that previously unrecognized N2-fixing unicellular cyanobacteria are active and abundant in oligotrophic oceans. This finding has important implications for nitrogen cycling in the oceans and for the role of "new" nitrogen in carbon fixation. The PIs will address three major issues: First, there are at least two distinct groups of cyanobacteria that appear to be separated in space and time, due to unknown ecological variables. Second, the geographic distribution and factors controlling the distribution are unknown, so it is not clear how these organisms should be included in biogeochemical models. Finally, one of the groups of cyanobacteria appears to fix N2 during the day, which revives the enigma of simultaneous nitrogen fixation and photosynthesis that was previously limited to discussions of Trichodesmium.

Diatoms (i.e., Hemiaulus) with symbiotic heterocystous cyanobacteria (i.e., Richelia) are considered responsible for a significant proportion of di-nitrogen (N2) and carbon (C) fixation in the World's oceans. Although these associations often form large and expansive blooms, measures of their N2 and C fixation are few, and current nitrogen (N) and C budget models do not include their contributions to new and primary production. This is largely due to the difficulty in collection and identification of these unique consortia. The investigators will use newly developed genetic and stable isotope techniques and high resolution nanometer-scale secondary ion mass spectrometry (nanoSIMS) approaches to directly resolve the N2 (and C) fixation contributions of these symbiotic diatoms on a relevant spatial scale.

The project will enable investigators to explore the metabolic interactions of diatom-diazatoph symbioses and identify environmental conditions that promote the activity of one symbiosis over the other. The three general questions driving the research are: 1) How do the individual diatom-cyanobacteria symbioses differ with respect to their cell specific N2 and C fixation rates; 2) What is the time scale at which N is transferred from symbiont to host; 3) What are the physical and chemical conditions that promote the activity of the different symbioses.

The broader impacts of the research involve advancing our knowledge of metabolic interactions between marine microorganisms, and providing a better understanding of marine planktonic symbioses. In addition, the project will contribute to an on-line web library of photographs for educational outreach. Furthermore, it will support training and education to an undergraduate senior thesis project, and will advance our knowledge of the activity of marine microorganisms.

Intellectual merit. Marine phytoplankton are a diverse group of Prokaryotic and Eukaryotic unicellular organisms that account for approximately 50% of global carbon fixation. Nitrogen (N) is an essential element for microbial growth, but concentrations of bioavailable nitrogen in vast regions of subtropical ocean gyres are extremely low (submicromolar to nanomolar concentrations), and generally limit phytoplankton growth. Phytoplankton taxa differ in their genetic capabilities to take up and assimilate nutrients, and thus competition for different chemical forms of N (NH4+, NO3- and urea) and supply of these N-containing compounds are important controls on phytoplankton growth, productivity, and ultimately ecosystem function. The form and supply of N to phytoplankton have already been altered by anthropogenic activities, and with increasing environmental perturbations the effects will accelerate. To date however, there is limited information on how the N forms and fluxes impact the marine phytoplankton community composition and primary production. Similarly, determining the mechanisms of the response are crucial to assessing how ocean ecosystem function will respond to global climate change. This project seeks to determine how taxonomic, genetic and functional dimensions of phytoplankton diversity are linked with community-level responses to the availability of different N substrates (NH4+, NO3-, and urea) in one of Earth's largest aquatic habitats, the North Pacific Subtropical Gyre. The project will characterize phytoplankton community composition change and gene expression, photosynthetic performance, carbon fixation, and single-cell level N and C uptake in different taxa within the phytoplankton assemblage in response to different N compounds. The research project is unique in investigating community-to-single-cell level function and species (strain)-specific gene expression patterns using state-of-the-art methods including fast repetition rate fluorometry, nanoscale secondary ion mass spectrometry and a comprehensive marine microbial community microarray. The results will provide predictive understanding of how changes in the availability of key nitrogen pools (N) may impact phytoplankton dynamics and function in the ocean.

Broader impacts. This project seeks to understand the ecological basis linking the metabolism of N to phytoplankton biodiversity in the open ocean. The underlying concept that links ecological competition for nutrients (in this case N) to phytoplankton diversity will provide a universal framework for understanding how ecosystem functions are linked to biodiversity. By applying state-of-the-art molecular and genetic methods to address ecological questions, the project seeks to develop an innovative workflow to assess eukaryotic and prokaryotic gene functions in the environment, and provides modern analytical and bioinformatic training for graduate students and postdoctoral researchers. The microarray tool has been designed by involving the larger marine microbiology community and is available to the greater scientific community, and this project is one of the first implementations. The fundamental concepts of microbial ecology and genomics will be used in educational activities in undergraduate and graduate-level classes as well as research training for undergraduates and graduates. Students and the postdoctoral researcher supported by this project will be engaged in development of microbiological and molecular biological displays and presentations at the Exploratorium, a science museum in San Francisco, California. Project personnel will collaboratively develop modules for the Exploratorium. The Exploratorium partnership will provide a mechanism for educational outreach for students and post-docs, as well as an efficient means to communicate the importance of ocean microbes and genomics to the public (over 600,000 visitors per year). The PIs will work with the education team in the Center for Microbial Oceanography: Research and Education (C-MORE) scholars program, at the University of Hawaii, to recruit an undergraduate student to participate in this project. The C- MORE scholars program seeks to promote workforce diversity by identifying faculty mentors to work with students of traditionally underrepresented backgrounds in the STEM disciplines.

Integration. This project integrates multiple perspectives on microbial biodiversity. The project seeks to understand how nitrogenous nutrients regulate the taxonomic, genetic, and functional diversity of phytoplankton communities through differential gene expression and functional properties of phytoplankton taxa.

The extent of summer Arctic sea ice loss is increasing and now occurs earlier in the year. As a result, it is predicted that the rate of growth of phytoplankton, the base of the marine food web that sustains subsistence marine harvests by native populations, will increase within the Arctic seas. The limited amount of available nitrogen, a required nutrient for phytoplankton, eventually will restrict the level of growth. Nitrogen gas dissolved in the ocean can be converted to a form readily utilizable by phytoplankton, but this has been considered primarily a warm-water process. The principal investigators of this project recently have observed this process in the Arctic Ocean, but there is so little data that its extent remains highly speculative. If it is widespread, it will change the way we think about future scenarios for the changing Arctic marine ecosystem, subsistence fishing, and, potentially, commercial fishing in the Arctic. The project will also contribute to workforce development. The principal investigator is an early-career, female scientist. She will use the project as a mechanism to entrain an undergraduate student into research. She will also use the project to sustain an existing educational collaboration between the Virginia Institute of Marine Sciences and Hampton University, a historically black university.

It is hypothesized that microorganisms capable of fixing N2 (diazotrophs) are present in the Chukchi and Beaufort Seas, that they produce measurable rates of N2 fixation in near shore and offshore Arctic marine waters and that diazotroph community composition will differ between coastal sites, which are influenced by terrestrial inputs, versus open water sites. The Chukchi and Beaufort Seas will be sampled during a cruise in late summer 2016, when hydrographic and nutrient conditions are likely to favor diazotrophic populations. The impact that N2 fixation will have on Arctic ecosystems is dependent on its rate, spatial extent and the conditions that favor it. As a consequence, on each cruise the PIs propose to determine diazotroph community composition, examine their distributions based on the presence of the nitrogenase gene (nifH), measure rates of primary productivity and uptake of inorganic and organic N and C substrates using 15N and 13C tracer techniques, and to compare these to hydrographic, nutrient, and overall microbial community composition profiles made along cruise transects. The proposed work will determine the extent of active N2 fixation within the region in the context of other key biogeochemical and microbial community parameters.

Nitrogen is a nutrient whose availability limits growth and productivity of ecosystems. Nitrogen is extremely abundant in the atmosphere in the inert form of gaseous N2, but most organisms cannot reduce N2 into a biologically available form. In all environments, including agricultural soils, there are microorganisms that can make available the N from gaseous N2 by reducing it to the biologically available form, ammonium. In the vast expanses of the open ocean, few organisms are known to have this ability, and recently a unique symbiosis between a single-celled cyanobacterium and a single-celled algae was discovered, which appears to be very widely distributed and likely of global biogeochemical significance. The cyanobacterium in this symbiotic partnership has very unusual metabolism and genomic streamlining. Little is known of the symbiosis because it is not detectable except by modern molecular biological techniques. Recent work has shown this symbiosis to be very widely spread through the oceans, and that there is previously unrecognized diversity in both the cyanobacterial and algal hosts. This research will examine the environmental distributions and the biogeochemical significance of this diversity in coastal US waters. The investigators will engage the public in ocean sciences through internship programs at local high schools and for undergraduate students at Stanford, and by documenting their field research in a 'virtual cruise' blog.

In the marine environment, the contribution of N2 fixation to the fixed nitrogen (N) pool is poorly quantified, in part due to an incomplete understanding on the abundance, activity, and physiology of diazotrophs. The symbiotic unicellular cyanobacteria (UCYN-A) is a poorly characterized, yet globally important, group of marine diazotrophs. UCYN-A is widely distributed in the marine environment, and lives symbiotically with a picoeukaryotic prymnesiophyte alga. We now know that there are multiple ecotypes of UCYN-A, which may be adapted to specific locations in the water-column and different oceanic provinces. Typically N2 fixation was considered unimportant in coastally influenced and non-tropical waters, however recent data shows that multiple subclades of UCYN-A are present. The distribution and rate of N2 fixation by UCYN-A subclades in coastal/nearshore environments is a major unknown in the oceanic N cycle. Its presence in nearshore waters may change the paradigm of the balance between basin N sources (N2 fixation) and sinks (denitrification). Likewise, significant N2 fixation by UCYN-A will need to be considered when determining estimates of new production in coastally influenced waters. This project aims to quantify the significance of different UCYN-A subclades to coastal/nearshore N budgets. It tackles the issue of determining N2 fixation rates by different UCYN-A subclades in coastal waters through rigorous fieldwork off the west coast of North America. The temporal and spatial distribution of UCYN-A subclades, as well as the rates of N2 fixation, will be determined by coupling N2 fixation measurements of bulk communities and individual cells (nanoSIMS) with molecular assays to study these widespread, but dilute, diazotrophic symbionts and their hosts. Additionally the investigators will conduct experiments aimed at constraining the effects of light and nutrient ratios (N/P) on UCYN-A N2 fixation rates, and the prymnesiophyte host's rate of carbon fixation. They will conduct this work through seasonal sampling of a coastal site in the Southern California Bight (Scripps Pier) and on two process cruises in the coastal waters between central California and the Baja Peninsula. The cruise work will provide an opportunity to understand the temporal dynamics of the UCYN-A/prymnesiophyte associations over larger spatial scales. Finally, evidence suggests that unidentified UCYN-A subclades and hosts exist and the investigators have developed a strategy to identify and quantify their temporal and spatial distributions as well as their N2 fixation activities. Data on the coastal distribution, ecology and activity of UCYN-A is critical for obtaining a better understanding of their contribution to fixed N to the marine environment. The group-specific and bulk rates of N2 fixation measured in this study of coastally influenced waters, will provide data for future modeling efforts, which will make an important contribution to constraining oceanic N2 fixation inputs.