Brian Palenik - US grants

DNA-dependent RNA polymerase is a multi-subunit enzyme critical to the existence of most organisms and of ancient origin. These properties make it an ideal candidate for a molecular chronometer, both to corroborate 16S rRNA data, and to extend the analysis of evolutionary diversification. n this fellowship research, Dr. Palenik will use antibodies to and oligonucleotide primers developed from Anabaena and E. coli polymerases to study the polymerase subunit structure and sequence of "older" eubacteria (16S rRNA estimate) such as Chloroflexus, and various cyanobacteria, prochlorophytes, and chloroplasts. Analysis of the sequence data obtained will provide new insights into the evolutionary development of photosynthetic organisms. The work will also likely result in the use of RNA polymerase sequences to analyze phytoplankton species diversity in aquatic environments.

9317958 Palenik The flow cytometer provides the unique capability of rapid analyzing the fluorescence, scattering, and several chemical and biochemical parameters of individual cells. This proposal requests funds to acquire a flow cytometer for the Center for Marine Biotechnology and Biomedicine, Scripps Institution of Oceanography (SIO) to be jointly used by several investigators working in diverse areas. The instrument will be used to provide samples. This information will be used to understand the morphological and genetic diversity of these organisms, their effects on the biooptical properties of the water column, and their responses to nutrient and other environmental stresses. In a novel application, the instrument will be used to characterize the bioactive secondary metabolites produced by currently unculturable organisms isolated from field samples by flow cytometric sorting. The flow cytometer will also be used on laboratory cultures to study physiological responses of particular species to various environmental stresses. This research is part of efforts to develop molecular probes as indicators for particular stresses and to parameterize optical models of oceanic photosynthesis as a function of particular stresses.

Palenik 9818543

Molecular Probes for Nutrient Stress in Phytoplankton: Emiliania huxleyi as a model.


Numerous examples exist where both the type and physiological status of the phytoplankton influence major processes in the oceans such as carbon fixation, calcification, sulfur gas emission, and toxic bloom formation. Our ability to understand the physiological status in the field of individual phytoplankton species or individual cells is limited, however. Most biological field measurements (carbon fixation, C:N ratio, Fv/Fm, for example) currently integrate over the whole phytoplankton community. In the long term, an understanding of the physiological ecology of individual "keystone" phytoplankton species will provide a more mechanistic, predictive understanding of ecosystem processes.

Emiliania huxleyi is an abundant, cosmopolitan phytoplankton species which is important in the global biogeochemical cycles of carbon and sulfur because of its production in the water column of calcite (coccoliths) and the sulfur gas DMS. It is present in both oligotrophic and neritic environments and in some locations is known to form massive blooms visible in satellite images. To date our understanding of the ecology of this organism has focused on these bloom phenomena.

One of the research goals of the field of phytoplankton ecology is the development of incubation-independent probes of phytoplankton nutrient status. From previous work an antibody to a cell surface protein, NRP1, that is present in nitrogen-stressed, but not nitrate--replete or ammonia-replete E. huxley has been developed. In this study the antibody will be used to develop a field method for characterizing the physiological state of natural E. huxleyi populations. A whole cell assay will be optimized and its efficiency and sensitivity characterized for examining cell numbers down to those present in oligotrophic environments. Fluorescence microscopy and image analysis will be used to quantify the number of cells expressing NRP1 and ideally the relative level of expression.

An antibody to intact whole cells will be developed to count the total and percent nitrogen-stressed cells in samples. A few previously obtained samples from the California current and the coast of Norway will be tested initially for the presence of nitrogen-stressed cells in the field. The demonstration of the physiological state of a specific phytoplankton group would be novel. This work will involve the training of one graduate student and one or more undergraduates in emerging techniques for applying immunological probes to marine ecological questions.

This award will enable the purchase of an instrument that combines a rapid thermocycler with a microspectrofluorimeter. This technology allows the quantification of a PCR reaction as it occurs in real time and thus can convert the qualitative results typical of PCR into quantitative ones using the sensitivity and precision of the fluorimeter. Quantitative PCR is emerging as a potentially crucial, but still "risky" technique in the study of marine organisms. The barrier of instrumentation cost has also limited its development as a tool in marine sciences. The researchers will initially use the instrument in the detection of specific microorganisms (free-living photoautotrophs, heterotrophs, symbionts, and pathogens) in the marine environment, with the goal of developing a fundamental understanding of the mechanisms controlling microbial biodiversity. The detection of the genes for various microbial activities such as toxic metal metabolism or nutrient stress responses is a second potential use. Lastly, population genetic studies of eukaryotic marine organisms such as copepods and sea urchins will benefit from the utilization of the proposed instrument. Three laboratories in the Marine Biology Research Division of Scripps Institution of Oceanography, University of California, San Diego, will begin as major users of the instrument (Palenik, Azam and Burton), but use of the instrument will likely extend to several other labs. In all cases the instrument will be valuable in generating results that will open new research directions in marine sciences and provide preliminary results for future NSF proposals.

#0115801, "Acquisition of instrumentation for marine genomics and proteomics." (NSF Major Research Instrumentation Program). PI: R.S. Burton

ABSTRACT:

A grant has been awarded to Dr. Burton at Scripps Institution of Oceanography (SIO) for the acquisition of instrumentation for analyses of genes and their expression in marine organisms. The instrumentation will enable researchers to study genome-wide responses to environmental stress at both the nucleic acid and protein levels and will form a core facility for use in the research projects of ten different laboratories at SIO. Although the specific research goals vary with the laboratory, the primary questions motivating the projects are similar: What are the molecular mechanisms by which organisms adapt to specific marine environments? What impact do these organismal adaptations have on ecosystem function?

Specific research projects will consist of analyses of microbial communities in the ocean, including both quantitative identification of community members and functional composition of the communities. Both free-living microbes and those involved in symbiotic relationships with invertebrates will be analyzed with a focus on adaptations to temperature, pressure and nutrient regimes. Other studies will address interactions between components of the nuclear and mitochondrial genomes of invertebrate species and signal transduction pathways across marine invertebrate cell membranes.

In addition to enabling multiple specific research projects, this grant will facilitate SIO's mission of training the next generation of ocean scientists. SIO students already take a variety of genomics classes and seminars across the entire UCSD campus; yet without easy access and hands-on training using modern instrumentation, students have been unable to take full advantage of existing technologies in designing their own dissertation research programs. This grant will enhance the research and training environment at SIO so that the institution will continue to attract top faculty, postdoctoral researchers and graduate students and provide a state-of-the-art research environment.

A grant has been awarded to Dr. Brian Palenik of the University of California-San Diego, Scripps Institution of Oceanography in collaboration with Dr. Ian Paulsen, The Institute for Genomic Research, to sequence and analyze the complete genome of a coastal marine cyanobacterium, Synechococcus sp. strain CC9311. This photosynthetic microorganism represents a widespread, ecologically relevant group that greatly contributes to the primary productivity of coastal environments. The investigators have hypothesized that this cyanobacterium, and coastal bacteria in general, have characteristic strategies for living in coastal, compared to open ocean, marine environments. For example these bacteria may have different or more protein pumps for excreting pollutants or toxic metals due to the higher level of these compounds in coastal environments. Using the genome sequence of CC9311 and comparing it to available genome sequences of open ocean cyanobacteria will help reveal these strategies.

The information from this research will be available for future use to develop tools for monitoring cyanobacterial populations and their physiological status in coastal environments. Whether or not these microorganisms are being affected by pollutants at a particular coastal site can potentially be determined if genome information is available to develop monitoring techniques. Thus this project will contribute to the larger societal goal of monitoring coastal water quality. This project is also synergistic with costal marine projects funded by the NSF Microbial Observatories program. A broader impact of this project will be to communicate the basic concepts of genomics, their application to marine science, and the results of this research to the general public in the form of an exhibit at the Birch Aquarium, a significant tourist attraction and an important institution for science education in San Diego California. Training of future scientists (one graduate student and one post-doctoral fellow) will also occur in the emerging field of bioinformatics.

Over the past decade, research employing molecular approaches and other new technologies has permitted increased resolution of plankton diversity and led to the discovery of important new taxa throughout the world's oceans. Progress in understanding the significance of this newfound diversity has been hampered by a lack of high throughput technologies for the simultaneous identification and quantification of the many taxa comprising such communities. Bead arrays offer a promising approach to the problem of high throughput analysis of multiple taxa. The goal of this project is to test the applicability of bead array technology to the quantitative analysis of diverse marine microbial communities. As applied here, a bead array consists of a set of taxon-specific oligonucleotides attached to 5 micron beads. Up to 100 different oligonucleotide probes, each attached to beads of a different color, are used to quantitatively probe DNA extracted from planktonic organisms filtered from bulk seawater. Probes with different levels of resolution can be mixed in a single multiplex assay. Two test systems for the development of this technology will be employed. An array will be developed to assay seasonal changes in picophytoplankton diversity in a coastal environment. A second array will be developed to distinguish among the 25 species of the diatom genus Pseudo-nitzschia, ten of which are known to produce the neurotoxin domoic acid. Multiplex bead arrays will provide a new methodological tool for the analysis of marine microbial communities. The planned investigations also will provide excellent training opportunities for undergraduate, graduate and postdoctoral researchers seeking to apply new molecular technologies to important questions in marine ecology.

The University of Georgia is awarded funds to develop a suite of computational tools in support of elucidation of metabolic and regulatory networks in microbial organisms. Using these computational tools in conjunction with experimental investigation, the team will predict and characterize a number of important metabolic networks and their associated regulatory networks in cyanobacteria. The outcome of these predictions will be wiring diagrams of the selected target networks. The computational elucidation of biological networks will rely mainly on information derived through comparative genome analyses and interpretation of microarray gene expression data. Currently over 40 cyanobacterial genomes have been sequenced or are in the process of being sequenced, providing a rich source of information for network elucidation. In addition, substantial microarray data has been or is being generated for various cyanobacterial organisms, which are or will be publicly accessible in the near future. Using such information, a number of computational tools will be developed to derive as much gene function and association information as possible, which will be then used as constraints in the elucidation of biological networks. These tools will include tools for (a) gene function prediction, (b) prediction of operons and regulons; (c) prediction of protein-protein and protein-DNA interactions; (d) mapping pathways/networks (possibly partial) across genomes; (e) prediction of functional modules that are conserved across multiple microbial organisms; and (f) prediction of the wiring diagrams of metabolic and regulatory networks. The target networks in cyanobacteria with sequenced genomes include (a) photosynthesis and its acclimation to different environmental factors, (b) nitrogen assimilation, (c) phosphorus assimilation, (d) carbon fixation, (e) iron assimilation and regulation and (f) osmolarity regulation. The predictions will be based on both public experimental data and data generated in this project. Each of the tools will provide a new and useful addition to the current pool of computational tools for microbial genome analysis and network elucidation. The computational tools can be used for network elucidation for microbial organisms in general as long as a target organism has its genome and some related genomes sequenced and has microarray gene expression data available relevant to the target networks. The comprehensive nature and the systemic approach of the planned prediction capability will provide a highly effective and transferable framework for microbial network elucidation in general. Other researchers can directly use this framework and the tools developed in this project in their own investigation of pathways and networks as all the prediction programs will be provided as open source. This project provides an ideal training ground for both undergraduate and graduate students to learn bioinformatics tool development and application for solving complex biological problems. New bioinformatics and genomics courses will be developed and taught based on the research results of this project. In addition, annual training workshops will teach interested microbiologists the use of the tools developed in this project and other related tools.

The most common fate of microbes in aquatic environments is to become a meal for a protest grazer. A major flux of carbon in aquatic environments is thus mediated through microbe-protist interactions. The mechanisms by which microbes attempt to avoid grazers likely include morphological, behavioral, and chemical resistance strategies, yet our knowledge of these is primitive and most progress is currently being made on harmful algal bloom species. One of the insights from the availability of multiple cyanobacterial genomes is that different cyanobacteria have very different cell surface structures as well as potentially different modifications of common structures. In addition, potential chemical resistance strategies have been detected in several available genomes. This project will address protest-cyanobacterial interactions with the collaboration of researchers with expertise in protist biology, cyanobacterial ecology, molecular genetics and genomics to examine the range of interactions between protists and cyanobacteria of the genus Synechococcus.

The investigators propose to use the recent availability of complete genomes and molecular genetics tools to determine the key cell structures under selection by diverse protist grazers. They will use "shot-gun" approaches (transposon mutagenesis and selection) to identify these structures and use available mutants to investigate specific known cell surface structures. They will also examine the role of some potential chemical defense enzymes, found through whole genome comparisons, in deterring grazing. The proposed research will provide qualitative and quantitative information on the role of various grazer deterrence strategies and new paradigms for understanding grazer-prey interactions.

Broader Impacts of the Proposed Research

The broader impacts of this work will include the training of undergraduate and graduate students, including those from under-represented groups. Scientific findings will be disseminated broadly though participation in national meetings and publication in peer-review journals. The findings may indicate a mechanistic basis for understanding microbe-grazer interactions, one of the major processes determining carbon cycling in the oceans. The investigators expect to define one or more model but ecologically relevant grazer-cyanobacteria pairs that could be used by the community for future studies. In addition, novel marine natural products that affect eukaryotic cells, with possible biotechnological applications, may be found through these activities.

Marine cyanobacteria contribute perhaps 25% of global primary productivity and are thus of major importance in the global carbon and other element cycles. Multiple whole genomes are now available for model isolated strains of these organisms. These have been invaluable for understanding some of the different ecological strategies of these organisms and their ability to thrive in diverse environments from the coast to the open ocean. An important issue however is to what extent does the whole genome of a model organism isolated as a single cell from an environment really reflect the total repertoire of that species (referred to as its pan-genome). Some microbes seem to be able to access a larger pool of genes through horizontal transfer of DNA from other organisms. The types of genes they are obtaining and the mechanisms by which they are obtaining them is largely unknown, but could be very important to their ability to fix carbon and compete in an ecosystem. On the other hand, some microbes seem less able to gain genes through horizontal gene transfer. This research seeks to address these issues by performing metagenomic sequencing (DNA sequencing on a pool of environmental DNA) using 454 sequencing technology. Coastal marine cyanobacteria are sorted out of the sample before sequencing, greatly increasing the information available to understand the genetic diversity of these microbes. In this way the pan-genome of two major marine cyanobacteria (Synechococcus) species will be investigated and likely lead to the discovery of novel genes in these microorganisms.

Understanding the genetic diversity of these important microbes will help us understand how they fix carbon and compete in the constantly changing marine environment. It will provide a window into how genes can move between microbes in aquatic environments, a topic of great interest because of its similarity to horizontal gene transfer and associated antibiotic resistance in human health and agriculture. The metagenomic analyses used here are in their infancy in the aquatic sciences and training scientists in this cross-disciplinary area will spur its development. The broader impact of this research thus includes the interdisciplinary research training of an undergraduate, a graduate student, and a post-doctoral fellow. Mentoring undergraduates in an environmental microbiology lab course at UCSD will also further broaden the outreach of the work.

Trace metal sample handling and analysis have led to the discovery that tiny (nanomolar) levels of metals such as iron are having profound influences on the diversity, abundance, and carbon fixation of primary producers in the oceans. One of these important primary producers, marine cyanobacteria, have similarly been shown to be affected, positively and negatively, by copper (natural and anthropogenic) levels and may also be influenced by cobalt and nickel levels. At the same time, cyanobacteria and other phytoplankton are changing the distributions of metals and their reactivity through uptake, which causes measurable depletion of metals in surface waters, and through the extra-cellular production of metal binding ligands. Because of the importance of copper in aquatic environments, the PIs will characterize copper metabolism in marine cyanobacteria using model Synechococcus strains from oligotrophic environments and from coastal environments that have very different metal physiologies revealed by the whole genome sequencing projects. They will characterize how the strains respond to different copper levels using whole genome microarrays that probe the global response of the cell and will combine these studies with state of the art characterization of intracellular metal levels and other measures of cellular physiology and photosynthetic capacity. Molecular genetics studies of diverse copper associated genes will be undertaken to determine their function in the cell. Preliminary results have found a potential candidate for an intracellular copper binding protein that is conserved in all marine cyanobacteria. At the same time, preliminary results have found that coastal cyanobacteria have greater resistance to copper than open ocean species and this might be due to a novel copper binding or efflux system. The PIs will illustrate the importance of copper in aquatic environments by developing hands-on lab components that will communicate some basic concepts around metal nutrition/pollution in the marine environment to middle school students. This will be undertaken in collaboration with Aquatic Adventures, who provide educational programs that connect underserved youth to science.
Broader Impacts
This research will reveal some of the major mechanisms by which marine cyanobacteria have adapted to metal levels in coastal and oligotrophic environments. Thus these results will help us understand the distribution and diversity of these organisms in relation to global primary productivity. They should lead to more robust biomarkers for metal stress and pollution in coastal environments. The PIs will expand on a previous outreach activity like their museum exhibit on marine genomics at the Birch Aquarium, La Jolla CA, by developing hands-on lab components that will communicate some basic concepts around metal nutrition/pollution in the marine environment to middle school students. This will be undertaken in collaboration with Aquatic Adventures, who provide educational programs that connect underserved youth to science, inspire environmental action, and increase exposure to marine habitats. In addition an undergraduate and graduate student in the interdisciplinary field of the metal physiology of microbes will be trained.

This EAGER project to Brian Palenik, UC San Diego, is for tools development to identify previously unknown, putative genes in metagenomic sequence data. Metagenomic sequences represent the full complement of microbial genomes in a community and are difficult to analyse because of their complexity. Even the genomes of cultured organisms are difficult to annotate as typically 50% of the genome sequence can contain unknown or novel genes. Proteomics uses mass spectrometry to analyze peptides derived from the proteolysis of samples. This analysis allows for the determination of the presence or absence of specific proteins in the original sample. In some forms, proteomics can use genomic databases as a starting point for peptide and protein identification. More recently, a form of "reverse proteomics" is being tested where mass spectrometry data are used to improve whole genome annotation by demonstrating the presence of proteins not predicted in the initial genome annotation. This technology is now beginning to show promise in the analysis of complex environmental samples, such as metaproteomic samples. This type of community-level analysis can lead to new information about not only the biodiversity but the functioning of ecosystems. However, the coupling of mass spectrometry and genome and metagenome data to analyze environmental samples still requires tool development. This development is best done when the metagenome and metaproteome sampling can be coordinated to optimize and test the tools. This PI is developing several tools for analyzing metaproteomes using metagenomics data obtained from the same site. The tools include computational procedures to discover new open reading frames and genes, methods to identify which species are present without DNA sequencing and an approach for analysing post-translational modifications in Synechococcus metagenomes. This aspect of the work is higher risk than the other aims but is important because this will allow them to evaluate which genes in the metagenome are active. This project represents a novel interdisciplinary collaboration between a marine microbiologist and a mass spectroscopist, and it is timely as it takes advantage of ongoing metagenome sequencing funded by another NSF proposal. If successful, these tools will allow for the identification of activated genes within communities. Further, new or novel genes will be identified in the metagenomes and the products of these novel genes will be identified. This research team will focus on marine cyanobacterial communities as the test metagenome. The PI has already sequenced the genomes of two Synechococcus (marine cyanobacteria) and he will use these as reference genomes for the metagenomic/metaproteomic analyses. Synechococcus assemblages in the ocean are not very diverse, therefore Synechococcus metagenomes are some of the best metagenomes for these tests because these populations can be enriched from seawater to produce a nearly pure samples for proteomic analysis.
The PI is overseeing training of two post-docs as a result of this award.

This project utilizes three instruments, 1) a Becton Dickinson FACsAria II sorting flow cytometer, 2) an Amnis ImageStream multispectral cytometer with imaging capability, and 3) a Beckmann Coulter Multisizer 4 Coulter Counter, to provide a state-of-the-art suite of analysis tools for microbial oceanography and biotechnology. These instruments will enable population-scale evaluation of individual microbial cells for applications in 1) population analysis of ocean microbes and their ecophysiology, 2) metagenomics, 3) algal physiology, 4) algal biofuels, and 5) biotechnology. Microbes in the ocean ranging from bacteria to unicellular phytoplankton do not exist in isolation, but rather interact in populations on both the large and small scale. Understanding factors involved in ocean productivity involves monitoring the dynamics of changes in population over time, and in response to environmental changes. These instruments will enable the study of ocean microbe population dynamics in unprecedented detail. Technological applications of microalgae are of great scientific and societal interest, and flow cytometric analyses will aid in the development of renewable fuels, discovery and production of pharmaceuticals, and nanotechnology. These instruments will provide a state-of-the-art foundation for marine microbial and biotechnological research in the ocean and environmental sciences. This will enhance the ability to provide students and researchers with the training and modern tools to do cutting edge research in these areas. Broader impacts will be achieved through the operation of a shared research platform and strategic community-building activities. Perhaps the three most pressing science issues relating to society today are climate change, renewable fuels, and nanotechnology. Research resulting from the use of the proposed instruments will advance knowledge in all three areas.

Terrestrial plants have diverse and sophisticated mechanisms for defending against predation and pathogens, but the defenses of photosynthetic microbes in aquatic ecosystems are much less well understood. Synechococcus are aquatic/marine cyanobacteria that substantially contribute to global primary productivity. This project will employ two main approaches to study Synechococcus defenses against protist predators. Experiments will examine the mechanism(s) by which cell surface proteins provide a constitutive (continuously expressed) defense and assay the generality of this defense among Synechococcus strains and against diverse predator types. Secondly, co-culture techniques, in which Synechococcus are grown in the presence of predators, will be used to characterize protein-based induced defenses that may arise in response to predation. The formation of aggregates as a possible induced defense by Synechococcus will also be examined in field and laboratory settings. Aggregate formation is important for the biogeochemistry and ecological functioning of planktonic communities since aggregates can be the site of unique biogeochemical processes. The research is cross-cutting on a number of levels: it examines constitutive and induced defenses in both controlled laboratory and natural settings; it leverages the suite of available Synechococcus genomes to enable a mechanistic investigation of microbial defense processes; and it represents a synthesis of ecological and genomic approaches to the study of microbial interactions that are fundamental to biogeochemical cycling and the maintenance of biodiversity in aquatic ecosystems.

Broader impacts of this project include support of graduate and undergraduate students, including ethnic minority participants in Shannon Point Marine Center's Multicultural Initiatives in Marine Science Undergraduate Program (MIMSUP). Other impacts include development of instructional modules for elementary and high school students, and maintenance and distribution of culture collections.

Cyanobacteria in the genus Synechococcus are minute, but ubiquitous phytoplankton of temperate oceans that contribute a large amount of the biomass produced at the base of marine food webs. Seasonal blooms of Synechococcus are a remarkably consistent feature of coastal water of both the Atlantic and Pacific oceans. However, new molecular techniques have revealed that underlying the consistency of these blooms is a wealth of genetic variation that exhibits rapid dynamical turnover. This has led to a hypothesis that genetic diversity within Synechococcus is ecologically important to sustaining seasonal blooms, and to predicting marine ecosystem response to longer term environmental change. This project will combine ecological and genomic approaches to understand how genetic diversity interacts with the physical and food web processes that drive seasonal cycles of Synechococcus blooms. The genetic diversity of Synechococcus and co-occurring microbes will be quantified on a weekly basis during bloom periods in Booth Bay, Gulf of Maine and Scripps Pier, Southern California. Flow cytometric cell sorting in combination with molecular approaches will be utilized to investigate the diversity of bloom assemblages using a three-tiered approach including: DNA and RNA analysis of the whole unsorted community, DNA analysis of flow-sorted communities, and single cell genomics of Synechococcus and their protist grazers. Ecological processes that cause Synechococcus blooms to end will be investigated through experiments designed to measure the relative importance of mortality from protist grazers versus from viruses on various Synechococcus ecotypes. These results will yield insights into bloom dynamics and how genetic diversity interacts with ecosystem processes.

This project will develop methods and resources that will be generally useful for quantifying variability of the seasonal bloom of phytoplankton, which is likely to be an early indicator of ecosystem responses to longer term climate change. Quantitative PCR methods will be designed for rapid quantification of diversity in Synechococcus and in Synechococcus grazers in marine waters. Numerous protist and Synechococcus single amplified genomes will be produced and cryo-preserved to enable future studies. This project will also support the research training of a graduate student and several undergraduate students, who will be intimately involved with individual projects. Results will be translated to formal courses held at Bigelow Laboratory and to the public through presentations at a successful weekly Café Scientifique series held in Boothbay Harbor each summer.

Cyanobacteria are photosynthetic microorganisms at the base of aquatic food webs. They occupy different types of environments from pristine ocean waters to high nutrient, sometimes metal-polluted bays. The investigators have found that one cyanobacterial species has transitioned from a marine environment into the San Diego Bay, which would require it to adapt to different conditions such as light quality and polluted waters. They will examine if specific genes in this cyanobacterium changed in response to the new environment or if the cyanobacterium picked up new genes from DNA in its environment to help it adapt to the San Diego Bay environment. Since the earth?s environment is changing rapidly, it is important to understand the mechanisms of how organisms such as cyanobacteria will change in response, and what might be the limits on their ability to adapt. The main goal of this research is to determine how cyanobacteria are able to adapt to different environments. In addition, the project will train a post-doctoral fellow and undergraduates in techniques in molecular ecology. The PIs also propose to develop, in collaboration with the Living Coast Discovery Center in south San Diego Bay in Chula Vista, a program for a Wildlife Day Camp focused on microbiology. Campers will discover how the San Diego Bay is filled with, and dependent on, the world of plankton, microbes, and other small species. This program will be unique in that students will have an opportunity to participate in sampling on the Bay and use microscopes to view photosynthetic microbes.

Aquatic microbes can adapt and diversify to occupy new ecological niches. For cyanobacteria, the complex traits of light-harvesting and metal homeostasis are examples of traits that adapt to new environments with differences in light quality (color) or amounts of trace metals. Environmental sequence data show that a normally oligotrophic ?species? of marine cyanobacteria, the Synechococcus clade II, has crossed over the coastal zone (dominated by other clades) and adapted to the conditions inside eutrophic San Diego Bay. This phenomenon is an ideal case study for understanding how microbial adaptation of complex traits occurs at the molecular level. Using isolates and single cell genomes of flow-sorted cells, the investigators will determine if positive selection has been operating on specific genes in the Synechococcus genome. In addition, Synechococcus clade II strains may also be acquiring novel genes in San Diego Bay through horizontal gene transfer. Gene knockouts/knockins will be used to test the functional advantage of specific genes. The PIs propose to develop, in collaboration with the Living Coast Discovery Center, located on San Diego Bay, a program for a Wildlife Day Camp focused on microbiology. Campers will discover how the San Diego Bay is filled with, and dependent on, microbes. This program will be unique in that students will have an opportunity to meet scientists, visit working labs, participate in sampling on the Bay and utilize light and epifluorescence microscopes to view microbes. The project will also train a post-doctoral fellow and undergraduates in techniques in molecular ecology and evolution.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.