Fuelled by solar energy and performed by microscopic algae, or phytoplankton, photosynthesis is the primary source of productivity in aquatic ecosystems. However, recent studies suggest that microorganisms with previously unknown capabilities for harnessing solar energy, the so-called photoheterotrophs, are abundant in the oceans and are present in freshwater ecosystems as well. In contrast to algae, which completely depend on chlorophyll-based photosynthesis for their growth, photoheterotrophs use pigments called rhodopsins and bacteriochlorophylls to convert solar energy into a food supplement to their predominant diet of ingested organic matter. The aim of this project is to discover, describe, and document the predominant organisms that make up this unusual group of phytoplankton in temperate freshwater lakes. Due to their light-harvesting capacity and amenability to genetic engineering, photoheterotrophs may be useful in future bioenergy production systems. Research results will provide baseline information for freshwater phytoplankton bioprospecting, in a search for potentially useful microorganisms. The broader impacts of this work also include further development of cutting-edge research technologies, such as the study of a complete genome of a single cell, infrared epifluorescence microscopy for visualizing phytoplankton, and fluorescence-activated cell sorting to isolate and study phytoplankton cells for experiments. One postdoctoral associate and up to three undergraduate students will be trained during the course of the project, with particular effort placed on recruiting Native American students. Wide dissemination of research results will be achieved by workshops and advanced courses to the scientific community, as well as through diverse outreach activities to the general public.
Photosynthetic reactions are the primary sources of energy and organic carbon in most ecosystems. However, recent studies show that microorganisms with alternative ways to harness solar radiation are abundant in the ocean and, potentially, in freshwater environments as well. We refer to them as photoheterotrophs. Two predominant types of photoheterotrophs are proteorhodopsin-containing phototrophs (PRPs) and aerobic anoxygenic phototrophic bacteria (AAPs). With the support of this grant, we employed flow cytometric cell sorting, robotic liquid handling, high throughput single cell genomics, metagenomics, and other cutting-edge technologies to perform the first comprehensive survey of photoheterotrophs in temperate freshwater lakes, spanning oligotrophic, mesotrophic, eutrophic, and dystrophic lake types in North America and Europe. Multi-locus sequencing of 712 individual cells of planktonic bacteria revealed that most of the cosmopolitan freshwater lineages contain photoheterotrophs, including taxonomic phyla Actinobacteria, Proteobacteria, Verrucomicrobia, and Bacteroidetes. Our results suggest that Actinobacteria are the predominant freshwater PRPs, while the Polynucleobacter (Betaproteobacteria) are the most abundant AAPs. Interestingly, we found evidence for frequent inter-phyla horizontal gene transfer and recombination of rhodopsin genes, demonstrating the dynamic nature of genomes in freshwater bacteria. Overall, 10-23% of planktonic bacteria in the lakes studied were found to be photoheterotrophs, with PRPs being significantly more abundant than AAPs. This suggests that photoheterotrophy may be an important source of energy to freshwater plankton. By leveraging multiple productive collaborations and genomic sequencing support from the Department of Energy (DOE), we obtained the first genomic blueprints of several predominant, uncultured lineages of freshwater photoheterotrophs. This provided important insights into their metabolic features, evolutionary adaptations, and potential utility in bioenergy production. Furthermore, this project contributed to a large study, led by the DOE Joint Genome Institute, where genomic sequences of 201 uncultivated cells belonging to 29 candidate divisions, the so-called "microbial dark matter", were obtained. This study resulted in a major revision of the Bacteria and Archaea branches of the tree of life, with multiple discoveries of unexpected metabolic features that extend our understanding of biology and challenge established boundaries among the domains of life. This grant contributed significantly toward further improvements of laboratory and computational tools for microbial single cell genomics, a novel approach in genetic research, which enables the analysis of hereditary information at the most basic level of biological organization. We resolved the key issue of DNA contaminant removal from laboratory reagents, and improved algorithms for the assembly of genomes from next-generation genomic sequencing platforms. This project constituted a major educational opportunity to four postdoctoral scientists at Bigelow Laboratory who were involved in diverse aspects of this work. Another ten postdoctoral scientists and students at collaborating institutions were involved in this project. Five technicians at Bigelow Laboratory were involved in development of single cell genomics technology and in the operation of advanced laboratory instruments and computational tools, significantly benefiting their professional development. In September 2010, we hosted a highly successful second workshop on single cell genomics (www.bigelow.org/scgc/workshop2010). Over 80 participants from 15 countries attended the workshop, which featured presentations by the leading scientists in the field and a two-day bioinformatics tutorial. The dissemination of project results to the research community and broader audiences was achieved through peer-reviewed publications (eight published, six in preparation), conference presentations and seminars (50 so far), and various Bigelow Laboratory outreach activities. At least ten commentaries, popular science, and trade magazine articles have highlighted the results of this project, including Nature, Science, The Scientist, Genome Technology, and others.