Session 2: Genomics and Proteomics

HIGH-THROUGHPUT CULTURING FOR MICROBIAL DISCOVERY

Stephen J. Giovannoni, Ph.D.

Director, Molecular and Cellular Biology Program

Oregon State University

It is estimated that less than 1 percent of the earth’s microbial life can be grown using standard agar plating techniques; of over 40 known prokaryotic phyla, only 23 have cultured representatives. Uncultured microorganisms are a vast reservoir of biodiversity from which new small molecules and enzymes can be recruited for applications in medicine, industry, and agriculture. To culture novel microorganisms, we developed high-throughput-culturing (HTC) procedures to isolate cells in very low nutrient media. This approach was designed to address microbial metabolic processes that occur at natural substrate concentrations and cell densities, which are typically about three orders of magnitude less than those in common laboratory media. The approach makes use of microtiter dishes and a newly developed procedure in which cell arrays are made on microscope slides and screened by fluorescence in situ hybridization (FISH). Approximately 2,500 cultures of pelagic marine bacteria were examined over the course of 3 years and 14 separate samplings. Up to 14 percent of cells from coastal seawater were cultured using this method— a number that is 1,400 to 140-



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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products Session 2: Genomics and Proteomics HIGH-THROUGHPUT CULTURING FOR MICROBIAL DISCOVERY Stephen J. Giovannoni, Ph.D. Director, Molecular and Cellular Biology Program Oregon State University It is estimated that less than 1 percent of the earth’s microbial life can be grown using standard agar plating techniques; of over 40 known prokaryotic phyla, only 23 have cultured representatives. Uncultured microorganisms are a vast reservoir of biodiversity from which new small molecules and enzymes can be recruited for applications in medicine, industry, and agriculture. To culture novel microorganisms, we developed high-throughput-culturing (HTC) procedures to isolate cells in very low nutrient media. This approach was designed to address microbial metabolic processes that occur at natural substrate concentrations and cell densities, which are typically about three orders of magnitude less than those in common laboratory media. The approach makes use of microtiter dishes and a newly developed procedure in which cell arrays are made on microscope slides and screened by fluorescence in situ hybridization (FISH). Approximately 2,500 cultures of pelagic marine bacteria were examined over the course of 3 years and 14 separate samplings. Up to 14 percent of cells from coastal seawater were cultured using this method— a number that is 1,400 to 140-

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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products fold higher than that obtained by traditional microbiological culturing techniques. Among the cultured organisms are many unique cell lineages that will be named as new species and genera by microbial systematists. Ninety percent of the cells recovered by the project in these early experiments do not replicate in Petri dishes of agar media, the most common method of microbial cell cultivation. THE GENOMICS REVOLUTION: CHALLENGES AND OPPORTUNITIES Claire M. Fraser, Ph.D. President, The Institute for Genomic Research The genomics revolution has provided the DNA sequence for nearly 60 microbial species and a number of important animal and plant species, including human. This information provides the foundation from which a new, comprehensive, and profound understanding of the biology of living systems, the history of life on earth, and the role of genes in disease and disease susceptibility can emerge. Genome-enabled studies on microbial species have revealed new insights about mechanisms of microbial evolution, novel metabolic capabilities, novel approaches to the diagnosis and treatment of infectious disease, and potential biological solutions to environmental remediation and alternative energy sources. Genome sequencing still remains the most robust method for assessing the overall gene complement of any organism, and as costs for DNA sequencing have dramatically decreased, the possibility of using this approach to study new species and microbial populations has become more realistic. It is important to keep in mind that all the work done to date in microbial genomics has focused on species that can be cultured in the laboratory or grown in animal cells. However, uncultured species, particularly from the marine environment, should be a priority for future genomic studies, and the technology now exists to allow us to think about microbial-community genomic projects. One of the most profound lessons that we have learned from the genomics revolution is how little we actually understand about the biology of life on earth. With these immense data sets in hand, we will now be able to pursue avenues of research that were impossible just a few years ago. Although the benefits of this new understanding are apparent, the path forward is formidable. This goal will require the marriage of pow-

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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products erful new technologies from the fields of biology, mathematics, computational biology, engineering, and physics to achieve an understanding of systems biology. Moreover, it will require that investigators entering the life sciences as well as established investigators be provided opportunities to receive training in genomic analysis and bioinformatics to fully exploit the information that is being compiled in numerous databases around the world. MICROBIAL GENOMICS: WHERE DO WE GO NOW? Daniel Drell, Ph.D. Program Manager, Microbial Genome and Cell Projects U.S. Department of Energy Since its beginning in 1994, the U.S. Department of Energy (DOE) Microbial Genome Program has sparked a revolution in microbiology. To date, complete genome sequences of approximately 52 microbes have been published; sequencing of at least a dozen more is known to be complete but not yet published, and sequencing projects of approximately 140 additional microbes are known to be in varying stages of progress. Activity in the private sector has also been intense. Sequencing technologies have progressed to the point where a high-throughput facility, such as the DOE Joint Genome Institute, can draft the sequence of a 2.5 Mb microbe in 1 day and about 65 Mb of sequence in 1 month, and this productivity is rising. This torrent of sequences is enabling a variety of new discoveries. These include new genes and pathways, and the insights that horizontal transfer of genetic information might have been frequent in microbial evolution. Genes of unknown function are astonishingly frequent. Microbes have been isolated (and their genomes sequenced) from extreme environments characterized by low pH, temperatures above boiling water, pressures greater than 200 atmospheres, highly toxic metal concentrations, high radiation fluxes, high salinity, and just about every other inhospitable condition imaginable. Most microbes do not cause diseases and, in fact, their important roles in maintaining the ecology of the earth are becoming clearer. Their sequences will contribute to a deeper and richer understanding of microbial life on the earth. DOE is planning to take the next steps with its ambitious Genome to Life initiative. Its thrusts are four: (1) Understanding protein complexes

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Marine Biotechnology in the Twenty-First Century: Problems, Promise, and Products will increase the number of targets for pharmaceuticals, and will increase the numbers of points of intervention in cell functioning. (2) Better understanding of regulatory networks will also help in modifying cell function in ways that potentially can be useful in addressing mission needs. (3) Exploring functional diversity will add to the limited repertoire of microbial mechanisms for adapting to a variety of environments and for interfacing with the spectrum of substances, both organic and inorganic, that microbes encounter. After all, microbes have been on earth for more than 3.5 billion years and have learned to thrive in many niches exploiting available energy sources and nutrients. (4) The ability to model cell behavior in silico will generate many testable hypotheses (much as gene sequences do today) for a much deeper understanding of cell structure and function. Although enormous value is still to be gained from continued sequencing, we now need to learn how to put the biological “parts” together into understandings of cell processes and functions. The familiar (and very successful) reductionist approach needs to be supplemented by a new “reconstructionist” approach that recognizes that complex biological systems are more than the simple sum of their parts. Starting with “simple” microbial cells and being aware that multicellular life evolved from unicellular forms, we can expect this to be a massive challenge for all of biology.