OLI Grant: Development of Genomic Techniques for Functional Studies of Uncultured Magnetotactic Bacteria: Establishing Linkages between Physiology and Phylogeny
Grant Funded: 2002
Proposed ResearchOn earth microbes are the primary mediators of the major biogeochemical cycles (e.g., C and S). Despite their importance in global biogeochemistry the vast majority of microbes are not amenable to laboratory growth. In recent years many new molecular techniques (ranging from 16S rDNA analyses to environmental genomics) have been developed that can circumvent the requirement for pure cultures to gain insight into the physiology and ecological impact of key microorganisms. Using these techniques we propose to initiate a study designed to establish linkages between the phylogeny and functional activity of coastal marine magnetotactic bacteria (MB) within the context of environmental geochemistry. The MB are a ubiquitous group of prokaryotes that precipitate the intracellular minerals magnetite Fe3O4, greigite Fe3S4, or both, causing the cells to be oriented along the Earth?s geomagnetic field lines as they swim. In oxic/anoxic zones of the coastal and pelagic ocean many investigators have observed that MB appear qualitatively numerous, but are largely uncharacterized due to the difficulties associated with their culturing.
Major specific objectives of this work include:
- Use 16s phylogenetic analyses and fluorescent in situ hybridization to quantitatively correlate the abundances, distributions, and types of MB with environmental geochemistry at Salt Pond;
- Test the hypothesis that some of these organisms are sulfate reducing bacteria (SRB) using a culture-independent method (bacterial artificial chromosomes, BAC) to confirm the physiology of Fe-sulfide MB;
- Use the data acquired and techniques developed in 1 and 2 to submit an environmental genomics biocomplexity proposal in (target deadline Spring 2003).
Progress ReportWe proposed to develop a genomic technique for the purposes of screening magnetotactic bacteria (MB) for functional activities in the environment. To date we have used two approaches. Our first approach involved a genomic plug extraction using methods similar to those discussed by Rondon et al.(2000). While this approach has been successful, our yields have been low and the procedure fairly lengthy and cumbersome. We have also applied a whole-genome rolling-circle amplification method similar to the one discussed by Detter et al. (2002). Using this approach we have successfully amplified 10-100 kb genomic DNA from the MB and confirmed its size using field inversion gel electrophoresis. To preliminarily test the utility of this DNA for PCR-based screens, we designed and applied degenerate primers for key magnetosome genes including two for MamA and one for MamC. We have applied these primers to genomic amplification products; PCR products have been identified for MamA, which have been cloned and are awaiting sequencing. We are currently building a fosmid library for further screens.
Based on these data and other environmental data we have collected, we have submitted a proposal to the Biogeosciences program at NSF for support of further studies. The methodologies developed through this funding have also led to the initiation of a similar genomic study at Loihi seamount on iron-oxidizing bacteria. Our approach is similar and preliminarily appears to be highly successful. This work will likely lead to another grant proposal for in-depth studies.
Detter, J. C., J. M. Jett, S. M. Lucas, E. Dalin, A. R. Arellano, M. Wang, J. R. Nelson, J. Chapman, Y. Lou, and D. Rokhsar. 2002. Isothermal Strand-Displacement Amplification Applications for High-Throughput Genomics. Genomics 80:691-698.
Rondon, M. R., et al. 2000. Cloning the soil metagenome: A strategy for accessing the genetic and functional diversity of uncultured microorganisms. Appl Environ Microbiol 66:2541-2547.