Microbial Research: Progress and Potential NSF Microbial Observatory/Life in Extreme Environments Principal Investigators’ Workshop Sept. 22-24, 2002, Arlington, VA Editors: Mary Ann Moran Department of Marine Sciences University of Georgia Sherry L. Cady Department of Geology Portland State University Table of Contents Executive Summary . 3 Introduction . 7 Developing Molecular Technologies . 8 Metagenomics . 8 Box: Metagenomics at Microbial Observatories . 9 Environmental Microarrays. 10 Other Molecular Approaches and Issues . 11 Box: Working in the Antarctic . 11 Recommendations for Future Research . 12 Novel Approaches for Culturing and Isolating Microorganisms . 12 Box: Making Culturing Better . 14 Recommendations for Future Research . 14 Environmental Sequence Databases . 14 Box: Sequence Databases at Microbial Observatories . 15 Recommendations for Future Research . 16 Recommendations for Microbial-Life Research Funding at NSF. 16 Box: Recommendations . 17 Conclusion . 18 References . 18 Acknowledgements The MO and LExEN Principal Investigators’ Workshop and production of this report were funded by the National Science Foundation grant MCB-0234272 to the University of Georgia. We thank the workshop participants for sharing their ideas, and especially David Emerson, Jan Amend, Alison Murray, Feng Chen, Mark Schneegurt, Paul Blum, and Steve Zinder for input to this report. Matt Kane, Ronald Weiner, and Gary Toranzos of NSF pro- vided guidance and logistical support for the workshop. We thank Myrna Watanabe for help with report preparation. 2 13,000 new prokaryotes. These technolo- NSF Microbial Observatory/Life in gies have also allowed access to functional Extreme Environments genes, providing a mechanism to assess Principal Investigators’ Workshop both capability for and expression of eco- logically important microbial processes in Sept. 22-24, 2002, Arlington, VA natural environments. The challenges now facing microbial-life researchers are to move Mary Ann Moran beyond the limitations of 16S rRNA-based Department of Marine Sciences, University approaches and characterized functions to of Georgia, Athens, GA 30602-3636 access the unknown organisms, functions, and activities of uncultured members of Sherry L. Cady microbial communities. Department of Geology, Portland State University Portland, OR 97201 Metagenomics Genomic analysis, now used prima- rily to observe the structures of genomes of Executive Summary individual organisms, can also be used to study the genetic reservoir of entire commu- Introduction nities, i.e., the metagenome (Vergin et al., The National Science Foundation 1998; Rondon et al., 2000; Béjà et al., (NSF) Microbial Observatory (MO) and Life 2000b). With this approach, genetic material in Extreme Environments (LExEn) programs from a natural community can be analyzed have fostered significant advances in micro- without first culturing all the organisms. bial ecosystems research in a wide variety Several MO projects are making use of this of natural environments. The investigators new technology to describe and discover funded by these programs have extended microbes and microbial products from envi- the frontiers of microbial diversity and micro- ronments as diverse as soils, the ocean, bial biogeochemistry research, discovering and the guts of insects. Before novel microbial lineages, describing the metagenomics can take a leading position in complexity of natural microbial communities, microbial-life research, however, technologi- and linking microbial taxa to critical ecosys- cal capabilities must be improved for obtain- tem functions. This workshop, the first of its ing sufficient DNA from natural environ- kind, provided a platform for MO and LExEn ments; for cloning, sequencing, and storing researchers to discuss recent accomplish- metagenomic DNA; and for capturing the ments and future directions in microbial dynamics of microbial communities over ecosystem research. time and space through gene sequencing approaches. Developing Molecular Technologies Environmental Microarrays Use of microarray technology repre- LExEn and MO projects have greatly sents another potentially valuable, yet chal- benefited from the use of new molecular lenging, prospect for microbial-life research. technologies to identify organisms and their A reasonably complete set of functional activities in natural environments. Molecular genes collected from an environment or methods have provided microbial ecologists collection of environments provides a win- with invaluable information about the enor- dow into the composition and activity of mous reservoir of uncultured microbes; 16S complex microbial communities. Sensitivity rRNA work alone has identified more than 3 and specificity are chief among the chal- known only through nucleic acid-based lenges of using environmental microarrays studies. It is generally argued that less than when working with small amounts of highly one percent of all microorganisms is known complex DNA or RNA. and culturable. But are any microorganisms truly “unculturable,” or have our attempts Other Molecular Approaches and Issues simply failed to provide the environmental Proteomics and protein arrays will conditions essential for growth? The chal- play important future roles in understanding lenges now lie in overcoming the limitations microbial activities, as will faster and more of traditional culturing techniques—those of affordable genetic fingerprinting methods. selectivity—and improving culturing tech- The expansion of molecular ecology tool kits niques to isolate novel organisms known to include eukaryotic microbes is also only from 16S rRNA sequences. needed. Long-term storage of environmental samples and culture collections, including Recommendations for Future Research mechanisms for distribution to other re- ● The continued development of culture- searchers, is costly but necessary for en- based technologies that take advantage hancing the value of microbial life data. of recent advances in materials, microfluidics, and other micro- and Recommendations for Future Research nanotechnologies; ● The continued development of environ- ● Development of improved microsensor mental genomics with particular attention techniques to identify and quantitate to capturing ecological heterogeneity; important organic and inorganic metabo- ● The continued development of environ- lites in situ, as well as to follow reactant mental microarray technology with par- sources and products in real time; and ticular emphasis on understanding sensi- ● Building of new data schema for rapidly tivity and complexity; identifying novel microbes that can be ● The development of environmental coupled with advances in molecular proteomics; genotyping and phenotyping methods. ● The expansion of molecular environmental methods to include eukaryotic organisms; Environmental Sequence ● The encouragement of bioinformatics and Databases modeling as research tools for solving Existing public sequence databases critical problems (e.g., reconstructing are insufficient for ecological purposes. The genomic information from mixed samples, development of sequence databases with understanding the ecological significance an environmental slant will build knowledge of genomic complexity and evolution); and of where, when, and under what conditions ● The development of cost-effective mecha- microbial sequences were retrieved. Such nisms for environmental sample and databases can provide mechanisms for data culture collection archiving and for dis- exchange within the research community, semination to microbial-life researchers. enhance the value of sequences obtained in single laboratories, and provide a data Novel Approaches for Isolating and Culturing Microorganisms Interestingly, the recent emphasis on molecular characterization of microbial communities is leading to a renewed interest in cultivating representatives of microbes 4 catalog that can be mined at different times and with different questions by microbial diversity and microbial biogeography re- searchers. Recommendations for Future Research ● Determination of the feasibility and desir- ability of building a centralized, ecological sequence database that will serve as a Final Recommendations community-wide resource; and 1. The MO Program has played a major role ● Consideration of the relationship of an in advancing research on microbial life and environmental database to existing gene should be continued in its current form, archives, including the Ribosomal Data- perhaps broadening its scope to include base Project (RDP; rdp.cme.msu.edu/ extreme environments and cover habitat html) and NCBI/GenBank types, rather than single sites. (www.ncbi.nlm.nih.gov). 2. Nonetheless, significant and critical gaps exist in funding research on microbial life Recommendations for Microbial that can only be filled with an increased Life Research Funding at NSF investment by NSF. Workshop participants judged the MO 3. Consideration should be given to estab- program to be overwhelmingly successful in lishing long-term, renewable MO projects, addressing a critical research need in site- perhaps analogous to the Long-Term Eco- based microbial discovery and activity. Yet, logical Research (LTER) projects that focus despite the success of this and other NSF on diversity and function, and are not neces- environmental microbiology funding opportu- sarily restricted to a single locale. nities, significant funding gaps were identi- 4. Consideration should be given to estab- fied. Critical