THE GLOBAL GENOME QUESTION: Microbes

as the Key to Understanding

Evolutionand Ecology

A Report from the American Academy of Microbiology

Copyright © 2004American Academy of Microbiology

1752 N Street, NWWashington, DC 20036http://www.asm.org

This report is based on a colloquium, “The GlobalGenome Question: Microbes as the Key to Understand-ing Evolution and Ecology,” sponsored by the AmericanAcademy of Microbiology and held October 11-13,2002, in Longboat Key, Florida.

The American Academy of Microbiology is the honorificleadership group of the American Society for Micro-biology. The mission of the American Academy ofMicrobiology is to recognize scientific excellence andfoster knowledge and understanding in the microbiolog-ical sciences.

The American Academy of Microbiology is grateful forthe generous support of the following organizations:

National Science FoundationU.S. Department of EnergyNIDCR, National Institutes of HealthThe Ellison Medical Foundation

The opinions expressed in this report are those solely ofthe colloquium participants and may not necessarilyreflect the official positions of our sponsors or theAmerican Society for Microbiology.

THE GLOBAL GENOME QUESTION: Microbes

as the Key to Understanding

Evolutionand Ecology

A Report from the American Academy of Microbiology

Merry R. Buckley

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Board of Governors, American Academy of Microbiology

Eugene W. Nester, Ph.D. (Chair)University of Washington

Kenneth I. Berns, M.D., Ph.D.University of Florida

James E. Dahlberg, Ph.D.University of Wisconsin, Madison

Arnold L. Demain, Ph.D.Drew University

E. Peter Greenberg, Ph.D.University of Iowa

J. Michael Miller, Ph.D.Centers for Disease Control and Prevention

Stephen A. Morse, Ph.D.Centers for Disease Control and Prevention

Harriet L. Robinson, Ph.D.Emory University

Abraham L. Sonenshein, Ph.D.Tufts University Medical Center

David A. Stahl, Ph.D.University of Washington

Judy A. Wall, Ph.D.University of Missouri

Colloquium Steering Committee

Edward F. Delong, Ph.D. (Co- Chair)Monterey Bay Aquarium Research Institute

David A. Relman, M.D. (Co-Chair)Stanford University School of Medicine

W. Ford Doolittle, Ph.D.Dalhousie University, Halifax, Nova Scotia, Canada

Karen E. Nelson, Ph.D.The Institute for Genomic Research

Richard J. Roberts, Ph.D.New England Biolabs

Carol A. ColganAmerican Academy of Microbiology

Colloquium Participants

Siv G.E. Andersson, Ph.D.Uppsala University, Sweden

Martin Blaser, M.D.New York University School of Medicine

Brendan Bohannan, Ph.D.Stanford University

Sallie (Penny) Chisholm, Ph.D.Massachusetts Institute of Technology

Frederick M. Cohan, Ph.D.Wesleyan University

Thomas P. Curtis, Ph.D.University of Newcastle, United Kingdom

Edward F. Delong, Ph.D.Monterey Bay Aquarium Research Institute

W. Ford Doolittle, Ph.D.Dalhousie University, Halifax, Nova Scotia, Canada

Katrina J. Edwards, Ph.D.Woods Hole Oceanographic Institute

T. Martin Embley, Ph.D.Natural History Museum, London, United Kingdom

Theresa Gaasterland, Ph.D.The Rockefeller University

John F. Heidelberg, Ph.D.The Institute for Genomic Research

Matthew D. Kane, Ph.D.National Science Foundation

Eugene Koonin, Ph.D.National Library of Medicine, National Institutes of Health

William F. Martin, Ph.D.Heinrich-Heine University, Dusseldorf, Germany

DeEtta K. Mills, Ph.D.Florida International University

Karen E. Nelson, Ph.D.The Institute for Genomic Research

Howard Ochman, Ph.D.University of Arizona

David A. Relman, M.D.Stanford University School of Medicine

Richard J. Roberts, Ph.D.New England Biolabs

Gabrielle Rocap, Ph.D.University of Washington School of Oceanography

Francisco Rodriguez-Valera, Ph.D.University Miguel Hernandez, San Juan de Alicante, Spain

Thomas M. Schmidt, Ph.D.Michigan State University

Melvin I. Simon, Ph.D.California Institute of Technology

David M. Ward, Ph.D.Montana State University

Ronald Weiner, Ph.D.University of Maryland

Stephen Zinder, Ph.D.Cornell University

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Executive Summary

A colloquium was convened in Longboat Key, Florida, inOctober 2002, by the American Academy of Microbiol-ogy to discuss the role of genomic techniques inmicrobiology research. Research professionals fromboth academia and industry met to discuss the currentstate of knowledge in microbial genomics. Unansweredquestions that should drive future studies, technicalchallenges for applying genomics in microbial systems,and infrastructure and educational needs were dis-cussed. Particular attention was focused on the greatpotential of genomic approaches to advance our under-standing of microbial communities and ecosystems.Recommendations for activities that might promote andaccelerate microbial genome science were identifiedand discussed.

Microbiology has always advanced in tandem with newtechnologies. Beginning with the first observations ofmicroscopic organisms with early microscopes in the17th century, the tools and methods for studyingmicrobes have continually evolved. Slowly at first, andnow with startling speed, scientists have developedincreasingly complex and informative tools for analyzingthe functions, interactions, and diversity of microorgan-isms. Today, genomic technologies are revolutionizingmicrobiology. Genomics employs all or part of thegenome to answer questions about an organism andrepresents a generic tool that can be used to dissectany or all living cells. In this report, the term “genomics”includes structural genomic methods that focus on thedetermination of genomic sequence and higher orderstructural features, as well as functional genomic meth-ods, which focus on the activities and productsencoded by the genome.

To date, microbial genomics has largely been applied toindividual, isolated microbial strains, with the resultsextrapolated to the wider world of microbial diversity.We are now presented with an opportune moment tomove beyond studies of single isolates and to applygenome sciences directly to the study of microbial com-munities. It is now possible to adapt genomic tools andapproaches to more realistic models of genome evolu-tion and ecology involving natural microbialcommunities. Microbial communities are formed byorganized groups of microbial species, each having dif-ferent, often complementary functions or activities. Inaggregate, the microbial community has emergentproperties greater than the sum of its individual mem-bers. Outside the laboratory, virtually all microorganismsexist in complex assemblages, in which they exchangegenetic material, nutrients, and biochemical signals withone another. While analysis of individual strains hasbeen a highly profitable enterprise, greater strides can

now be made by focusing attention on microbial communities. These are the entities that encompassthe bulk of microbial interactions, evolutionaryprocesses, and biogeochemical activities, with resultingimmense impacts on human health and the entire planetary biosphere.

The natural microbial world can be viewed as a land-scape of genes and genome ecology, in whichorganisms exchange genetic information and co-evolvewith one another, shaping themselves and the bios-phere over time. Microbial genomic evolution is craftedin microbial communities through the dynamic interplayof mutation, genetic drift, gene transfer, and naturalselection. As it is currently envisioned, the applicationof genomic approaches to the study of microbial communities, i.e. “community genomics” or “meta-genomics,” entails large-scale sequencing of pooled,community genomic material, with either random or targeted approaches, assembly of sequences intounique genomes or genome clusters, determination ofvariation in community gene and genome content orexpression over space and time, and inference of globalcommunity activities, function, differentiation, and evolution from community genomic data.

With the aid of genomic techniques, scientists arepoised to answer fundamental questions about thenature of microbial communities and the processes thatshape and sustain them. Although there are few limitsas to the phenomena that can be explored using thesetools, certain areas of research deserve particular atten-tion, due to their fundamental importance forunderstanding microbial life and due to their relativeaccessibility, given the current state-of-the-art. Out-standing questions about diversity and its generationand maintenance, ecosystem and community stability,and the relative significance of gene transfer in micro-bial communities need to be addressed.

Genomic techniques are a powerful set of methods, butthere are certain technical hurdles to overcome beforethese techniques can be universally applied. Amongthese hurdles are the challenges of coordinating pro-ductive research programs centered around significantand tractable biological questions and applying appro-priate and cost-effective technologies to answer them.One of the biggest challenges is the difficulty of copingwith the tremendous complexity of microbial communi-ties and their habitats and the difficulties in measuringall relevant biotic and environmental variables. Certaintechnical problems, such as identifying minority popula-tions, deciphering diverse chromosome structures, andde-convoluting complex genome assembly problems,

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all need to be tackled to accelerate progress in micro-bial community genomics. In light of these technicalchallenges, establishing the proper biological and environmental contexts for genomic studies and devel-oping new technology platforms and strategies are high priorities.

Microbial genome science can best be advanced byadopting multiple strategies and by addressing multiplelevels of complexity in study systems. Investigationsthat focus on more simplistic model microbial commu-nities will facilitate methods development. Lesscomplex model systems will also allow multipleresearch groups to coordinate their work. Several recommendations are made with respect to the optimalattributes of model systems, and a few examples ofsuch systems are explored. At the same time, genomictechnologies are mature enough now to decipher thegenomic characteristics of more complex microbialcommunities. Genomic investigations of microbial com-munities of global ecological, medical, or industrialimportance should also begin immediately. Sincegenomic approaches generate vast amounts of data,improvements in information technology, databasearchitecture, and data management strategies will sig-nificantly accelerate scientific progress. Outstandingunanswered questions include the amount and natureof within-population genetic diversity, the dynamics ofgenome evolutionary processes, and the levels of genetransfer in microbial communities.

A number of well recognized educational needs existwith respect to microbial genome science and are particularly critical for community genomics. Theseinclude cross-disciplinary training at the graduate andpost-graduate levels, in fields that meld biology andcomputer science or mathematics.

Microbial genomics holds great promise for improvingour world. By enabling a predictive understanding of theeffects of perturbation on the microbial communitiesthat impact human health and the environment,genomics could hold the key to treating diseases andmanaging the precious natural resources and processesthat sustain life on this planet.

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Introduction

It is astonishing but true—microbes are responsible formaintaining life on Earth. By driving global cycles ofoxygen, carbon, and other essential elements, microbeshave created the atmosphere, soil, and sediment thatsupport the remarkable diversity of animals and plantsthat have existed for millions of years. Microbes haveplayed an essential role in the creation of the fertile landthat sustains crops and livestock and, therefore, sustains human populations. Even within our own bodies, bacteria are necessary for digestion and absorp-tion of nutrients and for educating our immunesystems. Life on earth has thrived because of the activi-ties of microorganisms, and in their absence that lifewould cease to exist.

Despite the pivotal roles they play in life on Earth,microbes are only beginning to be well understood.From the beginning, the study of microbes has beendependent on advances in technology, and discoveriesin microbiology come only at the pace allowed by themethods available. Microbiology was born less than 350years ago when Antonie van Leeuwenhoek groundglass into magnifying lenses and identified “animal-cules” thriving in almost every environment he couldimagine. Then, in the 1860s, Louis Pasteur showed thatthese organisms can cause disease and can be purgedfrom food and medical instruments using heat. Ferdi-nand Cohn discussed the role that microorganisms playin cycling natural elements in 1872. Robert Koch grewbacteria on solid media 10 years later, allowing bacteriaand fungi to be cultivated and studied more easily in thelab. In 1924, Albert Jan Kluyver wrote about the common metabolisms of many different microbes andwas the first to point out that life on earth would beimpossible without their actions.

In this past century, the development of molecular biology allowed great advances in understandingmicroorganisms. In 1944, microbiologists Oswald Avery,Colin MacLeod, and Maclyn McCarty showed thatdeoxyribonucleic acid, DNA, held the information thatdrives the activities of living things and is the basis ofheredity. The polymerase chain reaction (PCR), first con-ceived in 1983, enabled scientists to take a smallamount of genetic material from microbial cells andamplify it for faster, more accurate analysis. Since then,molecular biology has evolved rapidly and scientistshave developed a range of powerful techniques foruncovering the activities of microscopic organisms,allowing microbiologists to pinpoint the causes andidentify the cures of elusive diseases, to better exploitthe beneficial microbes used in food and industrialprocesses, and to characterize the myriad organismsthat cycle global nutrients.

In recent years, scientists have opened a new windowon the world of microbes: genomics. The genome of anorganism is all of the DNA or genetic material that theorganism holds. It is the set of instructions that directsthe growth, metabolism, and reproduction of every living creature. In short, the genome is a blueprint forlife. “Genomics” rose to prominence with the firstdescription of a complete genome sequence of an inde-pendent life-form in 1995. The term describes the studyof all or part of the genome to answer a question aboutan organism. Genomic techniques are powerful, andthus far they have enabled scientists to explore themetabolic capabilities and genetic identities of manymicrobial strains, leading to insights about the rolethose organisms play in disease and environmentalprocesses. In this report, the term “genomic tech-niques” includes approaches that entail genomesequencing and analysis as well as functionalapproaches, in which the production of gene productsin individuals or microbial communities is monitored.

Molecular methods for studying microbes, includinggenomic techniques, have allowed researchers toexamine many genes and functions, but our ability tounderstand the microbial world has been limited by thedifficulties inherent in studying microbes in their naturalsetting: microbial communities. Microbial communitiesare formed by organized groups of microbial specieseach having different, often complementary, functionsor activities. In aggregate, the microbial community hasemergent properties greater than the sum of its individ-ual members. Outside pure laboratory cultures andexceptional symbioses, virtually all microbes exist incommunities. Whether in soil, water, attached to envi-ronmental surfaces, or in the gut of a human being,microorganisms live in close proximity to othermicrobes, and the implications of this closeness areprofound. For example, microbes in communitiesexchange nutrients, taking the substances they requireand excreting those for which they have no use. Thesemetabolic activities alter the microenvironments inwhich microbes live, so microbial interactions candefine the impact of a community on its environment.The spatial arrangement of cells in a community canamplify microbial interactions and may be responsiblefor many of the important processes communities carryout. Community-level processes may also be influencedby cell-to-cell signaling or quorum sensing—enablingmany microbial communities to take a census of theirown numbers and growth state and to respond to theirenvironment in a coordinated fashion. In dense micro-bial communities, genetic material may be exchanged,allowing the various members to acquire and relinquishdifferent metabolic capabilities and growth characteris-

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tics. Microbial community members can be highlydiverse, including members from each of the threedomains of life and can display metabolic capabilities ofevery sort. Alternatively, they may consist of only a fewtypes of members living in close association and relyingon simple associations for their needs and sustenance.

The result of dynamic community interactions atgenomic, metabolic, and structural levels is an interact-ing network of microbial species whose collectivefunctions are often greater than the sum of its parts. Forexample, certain microbial communities are capable ofbreaking down hazardous chemicals that individualspecies cannot degrade. Likewise, consortia of bacteriaacting together are required to satisfy the nutritionalrequirements of many larger organisms. Microbial com-munities are potent, and they accomplish many of thefunctions that are necessary for higher organisms toexist and for sustaining our planet’s habitability.

Microbial communities are many and varied, and theirinteractions, diversity, and structure make these assem-blages complicated systems indeed. This complexitymakes it difficult to tease out the function of individualmicrobes in their natural settings when communitiesare examined as a whole. Given these difficulties, whyshould scientists study microbes in communities? Theanswer is obvious: microbes are inextricably linked to,and defined by, the communities in which they arefound. To extract individual members from a commu-nity, thrust them into controlled, aseptic laboratorymedium, and monitor their behavior in completely for-eign nutrients and physical circumstances is of dubiousutility to understanding microbes in the real world.Microbes do not exist alone; they exist in communities.Understanding the role that microbes play in the worldrequires that we understand them at the communitylevel. Hence, efforts to describe the activities ofmicroorganisms should focus on studying them withinthe context of communities wherever possible.

The pressures of community living, including populationdynamics, recombination, and lateral gene transfer, areemerging as critical drivers of ecosystem evolution. By using genomic techniques to approach these phenomena, microbiologists can now build on the vastdata resources from genome sequencing projects andcarry the science into a new era. In this new stage,genomics will be used to describe the evolutionaryforces that drive the microbial communities that impacthuman health and the environment. Thus, among thechallenges before us is the need to determine how thetechniques, tools, and perspectives of genomics can bemost effectively used to understand better wholemicrobial communities—as single units of study.

The Genomic Landscape on Earth Today:Its Evolution, Diversity, and Dynamics

It is instructive to view the biological world through thelens of the genome. Most enduring ecological changesare mediated at the level of genes or groups of genes,where the forces of natural selection are brought tobear. Hence, the biosphere can be thought of as agenomic landscape, where the interactions betweengenomes and the environment dictate the outcomes ofevolution and the formation and stability of the biosphere. This view of the world has been referred toas “gene ecology.”

Genomes evolve over time, changing in subtle or radicalways, constantly adapting to the surrounding environ-ment. When two organisms with identical genomes areseparated and exposed to different selective pressuresover many generations, their genomes can accumulategenetic differences, causing the organisms to divergeand resulting in a branch-like pattern of evolution. Thedivergent evolution of previously identical genomes isone way in which diversity can be created. Alternatively,new genomic combinations can evolve through lateralgene transfer, by which a cell can acquire genes fromits neighbors or lose genes rapidly. Hence, genomescan evolve gradually through vertical transmission ofmutations, gene duplications, deletions, and rearrange-ments. Alternatively, they can evolve more suddenly andsporadically via horizontal transfer of genetic informa-tion between different microbial species.

Understanding the different mechanisms of genomeevolution offers interesting insights into the process.For example, it is possible to view evolution from theperspective of the organism that gains and loses genesand functions or from the perspective of the gene orgroup of genes, which can evolve gradually over time orincrease their influence by moving to new hosts.

The structure of the genome—meaning the organizationof genes with respect to one another—may also play animportant role in the evolution of microbial systems. Ithas been found that the genomes of closely relatedorganisms can actually exhibit a great deal of variabilityin their structure. That variability can increase over time.Genome heterogeneity has been documented in strainsof Thermotoga, for example, and in a number ofpathogens, such as Helicobacter pylori, but it isunknown what precise role this heterogeneity plays inthe evolution of communities of organisms.

Lateral gene transfer is the exchange of genes betweenmicrobial strains, and intragenic recombination refers tothe re-arranging of elements within a gene. Together,these processes, known as gene transfer, have been

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found to be significant driving forces in the evolution ofmicrobial systems. This contradicts prior beliefs, whichheld that these processes were important only in a lim-ited number of microbial habitats. Gene transfer allowsindividual organisms access to novel genetic material,and hence, to novel metabolic and structural capabili-ties that may offer an evolutionary advantage. Lifestyleand local habitat affect the rates and mechanisms ofgene transfer, and some bacterial species appear toabsorb genes from other organisms so readily that theyare likened to a “gene vacuum.” For example, Heli-cobacter pylori readily incorporates genes from theDNA pool to which it is exposed. It can take up genesfrom a plethora of different organisms, acquiring thecapability to metabolize previously excluded substratesor tolerate adverse conditions. It is worth noting, how-ever, that named prokaryotic species have been shownthrough population genetics analysis to include manydiscrete subpopulations, each of which might have theproperties of species. Until we better understand howgenomic variation is organized into populations, we can-not be certain that our impressions of genomic variationwithin a species are correct. In the case of related

groups of organisms that freely exchange genes, theconcept of a group-wide genome is useful. The pres-ence of agents that mediate gene exchange, namelyphages and plasmids, also has an influence on the ratesand mechanisms of gene exchange. Until now, the bar-riers to studying gene transfer and to quantifying theeffects of gene transfer on evolution and communitydynamics have been daunting. Genomics-based com-munity approaches are well suited to exploring thesegaps in the current understanding of the microbialworld, and the results of such investigations promise tobe fruitful.

Applying Genomics to MicrobialCommunities—The Most ImportantUnanswered Questions

Advantages of Taking a Community-Based ApproachCommunity-based genomics approaches offer a key tounderstanding the world of microbes. Previously, mean-ingful research in genomics often demanded thatindividual microbial community members be cultivatedin the laboratory, in isolation from the forces and rela-tionships of the communities that sustained them intheir natural habitats. The requirement of cultivation hasimposed a number of constraints on research, not theleast of which is the problem of culturing microbes thathave yet to be cultured. Currently, we are only able tostudy thoroughly at best one percent (1%) of knownbacteria and archaea, as the culture requirements areeither unknown or impractical to implement for the vastmajority of prokaryotic microbes. Other limitationsinclude the difficulty of extrapolating the results of culti-vation-based investigations to the wide world outside

the lab and differences between the pure-culture behav-ior of microbes and the behavior of microbes incommunities. Community-level genomics approacheshave enabled scientists to take a step back from theapproaches that involved dissecting communities intotheir individual member cells and enabled them to stepsimultaneously into new realms of research where theoverarching processes, interactions, and relationshipsat work in communities can be more directly observed.It is also important to note that the most significant out-standing questions in microbiology are related toecological and evolutionary processes, which can onlybe studied in the relevant context of the communitiesand environments in which they occur.

Genomics and other molecular methods are extremelypowerful, and they eliminate many of the problemsassociated with cultivation-dependent techniques. Animportant and critical place still exists, however, for thecontinued exploration of microbial cultivation, whichplays a role in understanding the metabolic and struc-tural feats of which microbes are capable. Genomicscould even help to guide these investigations by allow-ing researchers to identify the organisms appropriatefor cultivation and the likely combination of culture tech-niques that would enable their growth in the lab.

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Genomes evolve over time, changing in subtle or radical ways, constantly adapting to the surrounding environment.

Genomics approaches applied to communities offerunique promise for answering many outstanding ques-tions—from identifying the minimum required gene setin defined communities to fundamental questions aboutthe evolution of all ecological systems. General areas ofinquiry where genomics can be of particular value inleading to new discoveries include:

• Cataloguing diversity of microbial communities;• Understanding how genomic diversity relates to

species and/or functional diversity;• Deciphering how genome composition and dynamics

relate community stability;• Elucidation of the nature extent, and relative signifi-

cance of lateral gene transfer; and• Mapping the functions, dynamics, and interactions

of individuals and communities within specific envi-ronmental settings.

Outstanding Questions, Unique Opportunities

DiversityIt has long been acknowledged that the functional diver-sity of the microbial world is staggering. From thetermite gut to deep-sea hydrothermal vents to unseengroundwater plumes of noxious chemicals, microbescarry out forms of metabolism and respiration that areentirely beyond the capabilities of larger organisms.Within the last 20 years, scientists have found that thegenetic diversity of microbes is also vast. Microbesoccupy most of the branches of the tree of life, repre-senting the majority of the genetic potential in thebiosphere. However, this extensive functional andgenetic diversity, even within defined microbial commu-nities, has confounded both efforts to characterize theforces that drive diversity and to identify and interpretdiversity patterns. Scientists using genomic techniqueshave begun to tease apart the rules of microbial diver-sity, but many questions remain.

Molecular analyses of microbial communities based onconserved genes and associated phenotypic propertiessuggest that diversity is structured in discrete popula-tions that organize along environmental gradients. Theterm “ecotype” has been suggested in reference tothese ecologically specialized populations. For example,closely related “ecotypes” of the most abundant photo-synthetic organism on Earth, marine Prochlorococcus,are partitioned along light and nutrient depth-relatedgradients in the sea. The levels of molecular resolutionare currently too low for most microbial groups to inves-tigate whether other organisms (and their genomes) areorganized into species-like ecotype populations.Genomic approaches can address the issue of whetherindividual microorganisms form populations of organ-isms of like function or genetic relatedness. If so,efforts can also be made to go beyond statistical char-acterization of these groupings to determine theunderlying biological processes that might drive thephenomenon. Evolutionary theories, such as periodicselection theory, can be used to formulate testablehypotheses to begin to understand these processes.On the other hand, ecological theories developed forplants and animals might not apply to microbial popula-tions, especially if lateral gene flow is frequent, so thattheory-independent approaches to discovering theprocesses influencing how microbial genomes evolvemust also be considered.

Efforts can also be directed toward understanding thedegree of variability in microbial community diversityand the changes in the relative abundance of genes andgenomes that accompany community perturbation.

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Box 1: Community Genomics and the Metagenome

“Community genomics” refers to the application of genomics tools and techniques to the study ofentire microbial communities as a single unit. Thisapproach begins with pooled DNA or RNA repre-sentative of the whole community and involves thecreation of an inventory of genes of the communityin aggregate, using random shotgun sequencingmethods, or targeted sequencing of large genomefragments. It may involve the assembly of individualgenome sequences that correspond to singlestrains, species, or genomic variants. In a manneranalogous to efforts in single organism genomics,community genomics projects seek to define theexpression patterns and function of the gene poolunder a variety of conditions. Variation in content orexpression in relation to time, space, and perturbation help reveal the biology and evolutionof the community as a whole. In discussing community genomics, the term “metagenome” hasbeen coined to refer to the entire set (or inventory)of genes that belong to a given community.

Often taken for granted, the correlation betweengenomic diversity and functional diversity within com-munities has not been thoroughly explored. With thepowerful genomic tools currently available, the time isripe to substantiate such intuitive connections withexperimental results, and model and predict genomeevolutionary dynamics with robust theory.

The boundaries of microbial diversity are also largelyunknown and could be effectively resolved usinggenomic techniques. For example, the minimum num-ber of genes necessary for survival as a self-sustainingorganism, outside of a symbiotic relationship, isunknown and could be investigated using these tools.Moreover, it is unknown whether the minimum gene setdiffers based on the metabolic lifestyle of a givenmicrobe. For example, do organisms that utilize organiccarbon have a smaller or larger minimum gene set thanorganisms that fix inorganic carbon? On a communitylevel, it is unknown whether communities operate closeto the minimum gene set or whether they retain manymore genes than the minimum, and what the biologicalmechanisms for retaining those extraneous genesmight be. Neither is it known whether a community’sminimum gene set varies across time and space.

Many ecologists agree that species diversity is corre-lated with ecosystem stability, but a consensus hascertainly not been reached. By using genomic tech-niques to examine the effects of system perturbationson genome content in microbial communities, microbi-ology can inform these and other debates relevant toclassical ecology.

There are multiple plausible theories about the nature ofmicrobial diversity. Under the ecotype concept of diver-sity, variation within each ecologically distinctpopulation (an ecotype) is purged recurrently by naturalselection. Such ecotypes would have the quintessentialproperties of species, in that each is genetically cohe-sive and each is irreversibly separate from all others.These ecotypes would be expected to coexist over evo-lutionary time, such that each would eventually bediscernible as a separate sequence cluster. Alternatively,in a species-less concept of diversity, what limits thevariation within an ecologically distinct population is notrecurrent selection but the population’s longevity. Underthe species-less concept, new ecologically distinct pop-ulations would arise at a high rate, owing to horizontaltransfer, but populations would also become extinct at ahigh rate. These two models make very different predic-tions for discovering ecological diversity throughmolecular methods. Under the ecotype concept, thereis a one-to-one correspondence between ecologicallydistinct populations and sequence clusters; under thespecies-less concept, the correspondence is many-to-

one. Genomic approaches can help to determine whichof these models most appropriately describes diversitywithin microbial communities.

Stability and Adaptation to PerturbationCommunity stability is another topic where genomicstools can be put to use. In combination with phyloge-netic and genomic approaches, researchers can beginto dissect the mechanisms of community adaptation toperturbation by separating the effects of (1) shifts inpopulation structure, (2) changes in gene expressionpatterns, and (3) gene transfer. Since genomic tech-niques can examine individual microbial groups at worktogether, the effects of perturbation and stress on com-munities can be gauged and extrapolated to thecommunities that impact the biosphere. This will allowresearchers to understand whether different stressorsinduce dormancy, community composition shifts, ecotype extinctions, stepwise or continuous changes,or other phenomena.

The question of whether similar niches support similarmicrobial occupants could be addressed usinggenomics as well. Of particular interest is the level ofresolution at which these occupants share genetic orfunctional similarities. For example, in a given type ofniche that is found in many different locations, theoccupants may belong to the same “species,” genus, orhigher phylogenetic cluster.

Genomics can help to bring microbiology into a new,predictive stage. Genomic tools could potentially beemployed to construct a framework by which certainfeatures of an organism’s genome might be predictedfrom the sequence of its small subunit rRNA gene (16Sor 18S) and the biochemical and physical characteristicsof the environment from which it was drawn. Alterna-tively, predictions can be made about the effects ofperturbation on microbial communities based on thefindings of genomic surveys. This is an especially criti-cal capability in light of its potential impact on managingcertain human diseases, which can be seen as distur-bances of the delicate balance that humans usuallymaintain with their microbiota or the microbiota in theirenvironment. (Examples of diseases that may be pro-voked or propagated by microbial communitydisturbances include inflammatory bowel disease, peri-odontal disease, “bacterial vaginosis,” and theovergrowth syndromes associated with antimicrobialuse.) Perturbations of microbial communities that comeinto contact with the human body can lead to disease,and if predictions could be made about the effects ofthese perturbations on human tissues, then therapiescould be more effectively targeted to counteract or pre-vent the deleterious effects.

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Lateral Gene TransferLateral gene transfer has recently been found to bemore prevalent in microbial communities than previ-ously thought. Hence, it is reasoned that gene transfermust play some significant role in community dynamicsand evolution, but it has been difficult to explore thisrole with the techniques available to date. Genomicsallows experimental approaches and hypothesis testingthat was impossible with older methods, and concernsrelevant to lateral gene transfer are particularlyamenable to testing by these methods.

Although it is widely acknowledged that lateral genetransfer is a large determinant of microbial communityfunction and evolution, the agents responsible for genetransfer (plasmids, phages, transposons, integrons, andother elements) have received relatively little attention.Not only are there numerous gaps in our knowledgeabout the agents of gene transfer, but the factors thatgovern this phenomenon are also little understood. Therules and trends underlying gene transfer are ripe forexploration using genomic techniques. With the intro-duction of genomic techniques, systematiccommunity-based surveys of mobile and transmissiblegenetic material are possible. As exchangeable packetsof information, these elements can be seen as “sub-genomic organisms” or “subcellular genetic entities.”Simple physical separation techniques can isolatephages, plasmids, and other genetic fragments andsequencing these elements would be very revealingand of only moderate cost.

The range of rates of gene transfer that can beobserved in different types of organisms or in differentenvironments is unknown, as is the relative importanceof the various mechanisms responsible for gene trans-fer. Limits on mechanisms of gene transfer are poorlyunderstood. For example, it is not known whetherexchange is limited more by phylogeny or proximity.Other unknowns include the average quantity ofgenetic material that is exchanged by these transfermechanisms, and whether there are “species cores” of genes that are excluded from lateral exchange and recombination.

Gene transfer is not only a way of acquiring capabilities,but it may also be a way of “cleaning house,” riddingcells of unnecessary genetic burdens. Whether or notthere are common strategies in balancing the ebb andflow of lateral gene transfer to optimize genome struc-ture is unknown. Similarly, the ability or inability to giveand receive genetic material in this way may be a deter-minant of evolutionary change, either enabling cells totake advantage of changing environmental conditions ordriving the extinction of cells that cannot adapt.

Each of these lines of inquiry can be effectively investi-gated using modern genomic techniques. Genomicresearch investigating lateral gene transfer will requirescientists to generate new theories about life in micro-bial communities.

Questions about gene expression can be addressedusing genomic techniques, as well. Although geneexpression has been explored in a myriad of cultivation-dependent studies, genomic investigations into thenature of gene expression in a microbe’s “natural” con-text, in the presence of its fellow community-members,promises to deliver a new understanding of how andwhen microbes put their inherited genetic codes intoaction. In sequencing the genomes of isolatedmicrobes, it is not uncommon to find that a third of thegenes have no known function. Through genomicsinvestigations that examine the expression of genes in acommunity environment, including coordinate geneexpression, great progress can be made in identifyingthe roles of these previously unknown, unidentifiedgenes. Other outstanding questions that are ripe forexploration using genomic techniques include thepower of environmental feedback on microbial commu-nity composition, function, and the tempo and mode ofgenome evolution.

Questions Relevant To All BiologyThe implications of applying genomic techniques tomicrobial communities are not limited to the realm ofmicrobiology; rather, they resonate in every aspect ofbiological science. Genomics can be applied to micro-bial ecosystems to test hypotheses of populationgenetics for larger organisms, for example, and couldhave implications for understanding the dynamics ofgenes in all ecosystems. Outstanding questions withrespect to evolution, including whether diversity isorganized by periodic selection, can also be approachedby use of these techniques and can inform discussionsof evolution with respect to larger organisms. Finally,the results of genomic studies that examine the influ-ences of microbial community members on othercommunity members via cell-to-cell interactions can beextrapolated to understand better the interactionsbetween the cells of multicellular organisms.

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Issues and Challenges in ApplyingGenomics to Communities

Applying Ecological Principles to Microbial CommunitiesIn the visible world, living things are found in arrange-ments called ecosystems, in which the variousmembers establish a niche of specialty, interact withother members of the ecosystem, and arrange them-selves in patterns in which the various species utilizethe resources necessary for survival. Scientists havestudied ecosystems for many years, uncovering therules that govern them and the emergent propertiesthat come from combining many diverse organisms intoa single, functioning whole. Microbial communities helpstructure ecosystems on both small and large scales.Hence, experimentation in microbial community ecol-ogy may be used to inform the realm of classicalecology, and genomic techniques may be a particularlyimportant set of tools in making these links. For exam-ple, the phenomenon of invasive species in largeecosystems has a parallel in microbial communities: the

introduction of a new gene into the community. Untilnow, the available techniques in microbiology haveallowed researchers to track only the invasion of a givenmicrobe into a community, not the intrusion of theunique genetic material that that organism introduces tothe system. Genomic techniques now allow for trackingof genes, and the results of investigations into themovement of an invasive gene through a species, popu-lation, or community can contribute to our knowledgeabout the movement patterns of invasive plants,insects, or animals through the environment.

In other areas, the parallels between classical ecologicaland microbial community principles break down, andrules that apply to one apparently do not apply to theother. For example, the concept of biogeography, thegovernance of ecosystems according to climactic orgeographical environs, may or may not have a parallel inthe realm of microbial communities. Microbial distribu-tions may instead be ruled predominantly bybiogeochemistry, and the availability of niches and nutri-ents appropriate for community survival. In the areaswhere microbial ecology and classical ecology do notmeet, genomic information may not be useful in inform-ing the principles of ecology. However, a continued

dialog between classical ecology and microbial ecologywill often lead to a better understanding of our world,and attempts should be made to translate the results inone field in order to illuminate the other. Interactionslike these could lead to new insights into the dynamicsof both the biological systems that we can see andthose that work on the microscopic level.

Technical Issues, ChallengesGenomics as applied to microbial communities is a rela-tively new field. A number of challenges need to befaced and resolved in order to advance most effectivelyknowledge in the arena of microbial genome science.

COMPLEX POPULATIONS

Although genomics has introduced new ways toexplore the complexity of microbial communities, thatcomplexity may yet confound some lines of inquiry.Microbial communities exhibit a wide spectrum of intri-cacy. Some communities, intracellular parasites orsingle-species symbiont communities, for example,present relatively simple systems for study, and therange of the interactions at work is comparatively small.

However, other microbial communities are comprised ofwebs of inextricable interactions, in which nutrients,chemical signals, genes, and behavioral interactions areexchanged between different populations in an almosthopelessly complex network. Genomic work on thesecomplex communities can be very difficult indeed,since complexity confounds reproducibility. Genomicsurveys should be designed to allow for a range ofexpected complexities in the community being studied,without biasing the experimentation based on the levelof diversity anticipated by the researcher. The bestresearch will operate at several levels simultaneously,including the genomic level (in which surveys and somecomplete sequencing will be carried out), the geneexpression level, the metabolic level, and the physical-chemical level, and the population level.

LEVEL OF RESOLUTION

The interpretation of genomic data, and specifically thequestion of the level of detection achievable with thesemethods, remains a challenge in the field. Microbialcommunities may be comprised of thousands of distinctmicrobial taxonomic groups roughly equivalent to“species,” with most of these contributing to the overallfunction and character of the community. However, the

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Scientists have studied ecosystems for many years, uncovering therules that govern them and the emergent properties that come fromcombining many diverse organisms into a single, functioning whole.

vast majority of these groups are also be present at avery low frequency, contributing small signals to thegenomic data. In other experiments where genomics isused to compare communities, before and after treat-ment, for example, or from different areas of anexperimental site, small community differences may becritical to the results of the experiment. Without carefulplanning and experimental design, small, critical groupsand small, critical differences between communitiesmay go undetected. This could hinder the interpretationof experimental results, leading to false conclusions andimproperly directing future research efforts. Some priorknowledge of the diversity of the system at hand isrequired to tailor the questions that drive such genomicsexperiments, allowing proper experimental design toidentify minor populations and subtle differences.

CHROMOSOMAL COMPLEXITY

In most of the bacteria currently in culture, the majorityof the genome is kept in the form of a single, usually cir-cular chromosome that bears the genes necessary forsurvival. It is well known that the information found inextrachromosomal elements (such as plasmids), i.e.,the genetic information that is not included in this chro-mosome, also contributes to the biology manymicrobes. It is becoming increasingly clear, however,that many bacteria, such as Vibrio cholerae, bear morethan one chromosome. Moreover, eukaryotic microbes,such as fungi and protozoa, like their larger cousins theplants and animals, bear many chromosomes, each ofwhich carries a distinct set of genes, and mitochondrialDNA, which carries yet more genetic information. Thefact that the genome of a given microbe cannot alwaysbe found on a single, intact circle of DNA can be asource of difficulty in deconvoluting genomic data fromcomplex communities. In the future, researchers mustdesign experiments appropriately to overcome theproblems associated with accessing information onmultiple chromosomes.

GENOME ASSEMBLY IN COMPLEX COMMUNITIES

The amount of sequence data that is accumulated fromeven a modest genomics investigation can be enor-mous, and the computational handling requirementsrelated to analyzing microbial community data can bedifficult to overcome. When reconstructing thegenomes of organisms from community DNA samples,confounding factors such as insertions, deletions, poly-morphisms, and replicons that do not contain aphylogenetic marker like small subunit rRNA genes, cancause inappropriate alignments, creating genomicsequences that are actually agglomerations, orchimeras, of genome fragments from very differentorganisms. Simulation studies subjecting known wholegenome sequences to shotgun cloning and reconstruc-tion indicate that most mixtures of genomes fromdifferent microbial species can be assembled en

masse. However, the difficulties related to within-popu-lation insertions, deletions, polymorphisms, arepotentially substantial. The levels and types of geneticheterogeneity, and relative richness and evenness of dif-ferent genotypes, will have major impact on attempts atwhole community genome assembly. More data needto be collected on microbial genomic diversity withinand between populations, to understand, model andsolve these problems.

Normalizing genomic data to remove the dominantgenomes in the sample may be a key to accurategenome assembly. Normalization may cause the investi-gator to lose the very rare organisms in the sample(those present at a frequency of approximately 1 in10,000 or less), but this approach can allow the assem-bly of the average, or mean, community. Certainly, theinability to detect minor community members is not lim-ited to genomics techniques; it is a problem in manyapplications in microbial community analysis. It shouldbe remembered that genomics does not overcome allof the limitations of earlier methods and is not the defin-itive set of tools in understanding community function.

LATERAL GENE TRANSFER

Questions remain about the rate and extent of lateralgene transfer in microbial communities. As a result, itis unknown how stable genomes really are. The com-position of genomes may respond dynamically toevolutionary pressures over relatively short time peri-ods, complicating the interpretation of completegenome sequences. A genome sequence may be a“snapshot” of the genes that a given organism carriesand it may be subject to relatively rapid change due tolateral gene transfer in response to environmental con-ditions and other selective pressures. For example, itwas recently found that, although the genomes of twostrains of Escherichia coli, K12 and the deadlyO157:H7, share a similar structure and are of similarsize, a total of 1,915 genes are carried by one strainand not the other. Hence, as much as a third of thegenome of E. coli apparently is exchangeable anddynamic. Further work to delineate the significance oflateral gene transfer to microbial genomes should helpto illuminate this issue, guiding researchers in ways toconduct meaningful genomic surveys that take theinfluence of gene transfer into account.

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COMPLEX ENVIRONMENTS

Many environments where microbial communities arefound are not amenable to experimentation with directgenomic techniques in which DNA from the entiremicrobial community is extracted and used in subse-quent analyses. Complex environments that containsoluble organic materials and inhibitory compounds areparticularly difficult. Soil, for example, is a notoriouslytroublesome environment in which to apply genomicsor other molecular techniques. Composed of a hetero-geneous mixture of organic and mineral, soluble andinsoluble materials, soil contains a number of sub-stances that may interfere with the extraction process.Furthermore, soluble materials may co-elute with theextracted DNA, contaminating the sample and inhibitingPCR, cloning reactions like ligation, or other processesin the analysis. Although advances in extraction andpurification techniques are moving the field forwardevery year, difficulties with environmental matrices per-sist and must be managed in many genomicsinvestigations of complex environments.

GENOMES IN CONTEXT

Microbial community genome dynamics can only beunderstood in the natural biotic and environmental set-ting in which it evolves. Genome diversity and structure,in part, captures natural historical events. Approachesthat integrate genomic techniques with other levels ofinformation include systems modeling, which couldfacilitate a predictive understanding of communities andthe functions of individuals. Mass spectrometry andnuclear magnetic resonance imaging could also proveuseful, as could geochemical techniques that can quan-tify physical and chemical parameters and track themover time. Ecological and environmental data and mod-eling are also natural complements to microbialcommunity genomic data.

Challenges in Community Genomics

Although genomic techniques can be a very powerfulset of tools in investigating the function, dynamics, andstructure of microbial communities, there are tradeoffsin using any given analytical approach. The drawbacksto using genomics, including problems of reproducibil-ity and expense, should be carefully considered prior toselecting the methods to be used in addressing a par-ticular question.

Ensuring reproducibility remains a challenge in applyinggenomic techniques to microbial communities, asmicrobial communities are extremely complex, and ithas long been acknowledged that complexity con-founds reproducibility. It may be unknown, for example,how representative a sample from a given communityis. In the case of an unrepresentative sample, one thatbears little resemblance to the larger community, subse-quent analyses can produce results that have littlerelevance to the community as a whole. Issues of scaleare also of major concern when sampling communitiesthat interact across distances of only a few microme-ters or less. Genomic surveys of microbial communitiesshould include sufficient independent replicate samplesand analyses to ensure that the results of the experi-ment can be reproduced faithfully. The questionremains, however, how much replication is sufficient?This can only be answered on a case-by-case basis, andresearchers need to closely scrutinize replication in theirexperimental design.

Another significant problem with current genomic tech-niques is their relative insensitivity in detecting minorcommunity members. This can pose a serious con-straint on experimental results, and should beconsidered carefully. Since minority community mem-bers can sometimes have disproportionately largefunctional impact, this is an important area to address.

Educational Requirements and Information DisseminationGenomic techniques have enabled great advances overthe past ten years, granting us insights into the func-tions carried out by microbial communities and howthese communities impact our world. In the future,progress will continue and new discoveries will revealhidden truths about microbial communities, allowingmedicine to derive novel health strategies, enablingecologists to predict the effects of perturbation on theprocesses that control greenhouse gases, and permit-ting engineers to optimize the degradation processesthat remove toxins from contaminated water and soil. Inorder to move the field forward quickly, however, anumber of needs must be fulfilled. Scientific needs

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Mobile Elements in Ocean MicrobesResearch has shown that as much as 10% of theDNA in the world’s oceans is found in phages, indi-cating that the potential exists for extensive lateral gene transfer among planktonic microbes. By quantifying, sequencing, or otherwise character-izing phage genes in seawater filtrate or any otherhabitat, researchers could take a “snapshot” of thegenes that are on the move at a given point in time.These results could then be compared to the genesthat are moving at different times or in differentlocations. This kind of investigation is already beingdirected at the phage community within human thehuman gut.

exist, including better tools for data management andmethods for analyzing rare members. In addition, edu-cation and training efforts need to be directed towardpreparing scientists for optimal use of genomic tech-niques.

Development and Characterization of Model Communities and EcosystemsModel microbial communities and ecosystems can helpfocus the efforts of separate research groups and facili-tate a more thorough understanding of complexassemblages. By providing fine-scale details about rep-resentative ecosystems, models allow researchers toconstruct informed, testable hypotheses about theirfavorite non-model communities, thus guiding researchinto productive areas. Moreover, model systems allowresearchers to focus resources and expertise on a fewuseful systems rather than on many different communi-ties that may or may not inform an understanding aboutother systems.

It is widely agreed that model microbial communities innatural contexts should be studied in order to move ourunderstanding of microbiology forward. Microbiologistsneed to form a consensus, not only on the value of par-ticular model systems, but also on the criteria forselection of more complex communities. Applyinggenomics techniques to suitable model ecosystemscould prove highly profitable and provide, for example,detailed information on the diversity, stability, and occur-rence of lateral gene transfer in a few systems that canthen inform an understanding of other microbial sys-tems.

Criteria for Model Selection A primary criterion for a model community or ecosys-tem is that it be of fundamental scientific value,theoretical interest, or practical importance. These mod-els can span the spectrum from the simple to thecomplex. Some simple systems may be amenable tolaboratory study and shared between different laborato-ries. Other model ecosystems might, instead, gatherthe focused interest of different investigators around aspecific geographic site or habitat. Models for use inunderstanding microbial communities should not onlyprove instructive in the realm of microbiology, but theyshould have relevance to other scientific or socialissues. In other words, a model should have “real-world” relevance. The more groups, or stakeholders,that have an interest in gaining knowledge about thesystem in question, the more suitable it is as a model.

Importantly, a model microbial community should betractable to continued study. The complexity or numberof different kinds of organisms should not be so greatthat genomics tools, in their current configurations, can-not be used to evaluate the dynamics of the system.

Models should also be robust, allowing researchers totest a wide range of hypotheses.

An ideal model microbial community would be derivedor would exist in an environment that is amenable toexperimentation. Environments can hinder research byvirtue of their complex matrix (i.e., soils), lack of readyaccess (i.e., remote deep sea environments), extremeconditions (i.e., volcanic sulfur springs), or any combina-tion of these challenges. Efforts should be made toensure that the environment from which a model sys-tem is derived does not prevent thoroughexperimentation or long-term study.

The “physiology” of a model system should be relativelysimple and/or informative, in order to facilitate modelingefforts and systems analysis approaches. By studyingcommunities that carry out fundamental physiologicalprocesses, research can be focused on those phenom-ena that are most pervasive in microbial communitiesand have the biggest impact on human health and theenvironment.

Finally, a model community or ecosystem should be sta-ble in terms of structure and function, in order to ensurethat the results of research are repeatable over time andin different laboratories.

Scale of Model SystemsA balance must be struck between the complementaryadvantages of using simple communities with relativelyfew members and using large, complicated ones withmany members. Although a model microbial commu-nity should be tractable and have a small enoughnumber of members so that the system can beapproached using genomic techniques and other meth-ods, this requirement does not mean that modelcommunities should be simple in all cases. The ques-tion of the appropriate scale of complexity for a modelcommunity can be answered by determining whathypotheses are being tested and/or what critical prob-lem needs to be solved. These are the ultimate driversthat will determine what value a particular model has foradvancing knowledge and human welfare. A range of

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Criteria for Model Selection• Fundamental Value or Practical importance• Tractable• Robust• Amenable environment• Simple physiology• Stable

complexities should be studied that span a spectrumfrom simple two member consortia to complex multi-species communities. Only by approaching a variety oflevels of complexity can the field be advanced rapidlyand limitations tested and overcome.

Simple communities, those with few members, offersome advantages to the researcher in these earlystages of development. The tractability and repro-ducibility of these systems may provide earlyunderstanding the dynamics of microbial communities.A community of limited size may also simplify genomicanalyses, enabling researchers to reconstruct thegenomes of the community with greater ease.

More complex communities, on the other hand, areprobably more representative of the way life is for mostmicrobes on this planet. They are also more likely to beself-organizing and self-sustaining, qualities that makethem a good model of ecosystems and of the earth’sbiosphere. For this reason, developing approaches fordeciphering the dynamics of complex communities rep-resents a high priority endeavor. It should be theultimate goal, and an immediately proximal activity, inmicrobial community genome analysis.

Examples of Model CommunitiesModel systems are needed in order to make fasterprogress in understanding disease, the environment,and our global ecosystem. As a starting point for dis-cussions about which models should be recommendedto the microbiology community, a few examples of suit-able models were discussed.

MICROBIAL MATS

Thick layers of microbes, called microbial mats, oftengrow where geochemical conditions are appropriate,usually in thermal, hypersaline, or brackish marineenvironments. These communities have been shownto be stable, allowing repeatable experimentation.The chemical environments within microbial mats areamenable to study by microsensors, presentingdefinable, predictable gradients, and the number oforganisms usually found in these communities is wellwithin the range that is reasonable for carefulgenomic analyses.

SYMBIOTIC ASSOCIATIONS

Symbiotic systems, in which organisms apparentlycooperate to the benefit of all members, are also suit-able as model systems. Symbioses are frequentlyvery simple communities of as few as one or twomicrobial groups, and the physical boundaries ofthese communities are usually well defined. How-ever, the range of interactions in these communitiescan be limited, having little bearing on larger, morecomplex systems. For example, symbiotic microbial

communities may not be subject to invasion by non-native microbes, which limits the lessons thesesystems can offer for communities that are vulnera-ble to invasion.

OPEN OCEAN GYRES

Open ocean gyres, vast expanses of the oceans thatrepresent one of the biggest ecosytems on theplanet, may present a suitable model system foraddressing several outstanding questions aboutmicrobial community structure and function. Domi-nated by small planktonic bacteria and archaea,microbial communities in open ocean gyres havebeen found to have levels of microbial diversity thatmake them eminently tractable systems. Since thesecommunities largely control carbon and energycycling in the sea, they are of tremendous practicaland ecological significance as well.

HUMAN GASTROINTESTINAL TRACT AND ORAL CAVITY

At birth, the human gastrointestinal tract and oral cav-ity are sterile environments. Upon exposure to theworld, these environments are quickly colonized bymicroorganisms in what may be predictable, sequen-tial succession. As such, the gastrointestinal tract andoral cavity could serve as ideal model systems forstudying a number of phenomena, including commu-nity establishment and succession. An understandingof perturbation and its effects on community structurein these systems might lead to novel strategies forhealth maintenance and disease prevention.

MICROBIAL COMMUNITIES RESPONSIBLE

FOR CORAL DESTRUCTION

The microbial communities associated with destruc-tion of the world’s corals clearly engage a number ofconcerned stakeholders interested in knowing moreabout the mode of action, spread, and methods forcontrolling these destructive assemblages. Undoubt-edly, a systematic study of one or more of thesecommunities at many levels of resolution would be agood first step in making these advancements.

Managing DataBoth the power and the puzzle of genomic techniqueslie in the vast datasets generated by using these meth-ods. Large amounts of data can be derived even fromsimple genomic experiments, allowing researchers toreach conclusive, detailed conclusions about their sys-tem. Managing, analyzing, and integrating these data,however, remain a challenge for the field. Applyinggenomics to understanding microbial communitieswhose structure remains incompletely explored con-tributes even more complexity to the problem of datamanagement. For example, we do not yet have aproven concept of microbial species, and we do noteven know whether microbes fall into groups with

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species characteristics. However, if the ecotype con-cept of microbial diversity proves true, this constructcould prove useful for organizing a community ’sgenomic and metabolic diversity. Organisms from differ-ent ecotypes would be discernible by the sequenceclusters in which they fall, and then differences ingenomic content among organisms could be more eas-ily interpreted. For example, genes not shared amongdifferent ecotypes would be candidates for being thebasis of adaptive differentiation among ecotypes;genes not shared among members of the same eco-type would be ecologically meaningless, caused byrandom horizontal transfer events. On the other hand, ifthe species-less concept turns out to be correct, itwould be more difficult to determine which gene differ-ences are responsible for ecological diversity within thecommunity. Determining how microbial diversity isorganized will facilitate interpretation of genomic data.

A great need exists for better algorithms and com-puter software to handle the vast amounts of genomic data generated by community genomics applications.Mathematical and informatic formulations for handlingsequence and expression data, for example, arescarce to unavailable. Moreover, the expertise for cre-ating these tools is not abundant (See Needs andEducation Requirements).

Genomics databases are also lacking in number andquality. Historically, biologists have been ineffective inthe “care and feeding” of databases, an unfortunatephenomenon that now extends to poor maintenanceand tracking of genomics databases as well. By andlarge, diminished responsibility for biological databases,including genomics databases, is correlated with a lackof enthusiasm for funding these efforts. The reluctanceto support the necessary maintenance of genomic data-bases must be overcome if progress in genomics is tocontinue unabated.

Better integration between databases is a worthy goalfor informatics work in the future, as new data are gen-erated almost daily. It is particularly important to includerelevant physical, chemical, and biological data whenconstructing these databases. Often, these data areexcluded in compiling information about microbial com-munities, limiting the value of the resulting database forcomparing the results of different investigations.

As databases begin to proliferate, and the amount ofdata collected in investigations of microbial communi-ties continues to grow, making genomics informationaccessible to the scientific community has becomemore and more difficult. Efforts need to be focused oninforming scientists of the resources available to themand simplifying access to those resources, particularly

online resources, so that information can be effectivelydisseminated and utilized.

Microbiology has become an interdisciplinary science,and the application of genomics technologies to study-ing microbes is no exception. Unlike many prior areas ofresearch, however, genomics involves a strong infor-matics component, requiring microbiologists to takecomputing and mathematical lessons to heart. Collabo-rations between biologists, engineers, mathematicians,and professionals in other aligned fields are likely to bethe most productive efforts in investigating microbialcommunities.

Gene TransferThe fact that so many outstanding questions remainabout lateral gene transfer, a phenomenon that could bea significant determinant of community dynamics andevolution, stands in the way of making advancements inunderstanding life in microbial communities. Clearly, aneed exists to answer these fundamental questions. Byfocusing on questions about the rates, limits, agents,and preferred modes of lateral gene transfer,researchers can build a base of knowledge that canserve as a springboard for new theories and hypothesesabout population dynamics, evolution, and adaptation.

Analyses of IndividualsAlthough the focus of applying genomics in microbiol-ogy should be the characterization of microbialcommunities, a need still exists for identifying and ana-lyzing individual cells. Advancing our ability toaccomplish this should not be neglected. A great dealof our current knowledge about the interactions, func-tional capabilities, and limits of microbial communitieshas come from studies of individual isolates, studieswhich have offered views on the finer details of micro-bial life. Efforts to analyze single cells and microbialcommunity members should move forward in thefuture, allowing researchers to sequence the genomesof individuals and to observe the activities of thosecommunity members in situ. The technology to culti-vate previously uncultured microbes should also beencouraged, as microbes in cultivation can give con-crete evidence of the diversity of metabolisms andfunctions that microbes are capable of carrying out intheir natural environments.

Other NeedsIn order to move the field into a predictive stage,wherein the effects of microbial community perturba-tion on human health and the environment can beanticipated, efforts are needed to conduct coordinatedsurveillance of the impacts of disturbance on communi-ties at very basic levels.

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There is also a need for centralized repositories for archiving raw and processed materials from commu-nity analyses, which will allow easier verification andreproducibility of the results from these investigations. A federal facility would seem the best option for archive sponsorship.

The labs involved in microbial genomics are currentlyoperating with almost complete independence, oftenwith little knowledge of the efforts of other researchgroups—that is, until research results are published inthe peer-reviewed literature. In order to maximize therate of progress in the field, research projects should beconducted in a coordinated fashion, rather than com-petitively. Microbial genomics laboratories shouldassemble and participate in a research coordination net-work, in which the work of individual groups can becross-referenced with the work of other labs, maximiz-ing the effectiveness of their cumulative endeavors.

Education RequirementsModels, data management, characterization of geneflow, and other issues are pressing topics in optimizing

the power of genomics, but the education and trainingof the researchers who will apply genomic methods isparamount. Without sufficient genomics training, effec-tive research would never come about. For example, ithas been noted that computing power is probablygoing to be less of a restriction in dealing with vastamounts of genomics data than the availability of ade-quately trained scientists. Efforts need to be made byuniversities, funding agencies, and individual scientiststo encourage development of new, interdisciplinaryfields, train scientists across traditional departmentalboundaries, attract professionals with cross-discipli-nary interests, and develop curricula that reflectadvances in genomics.

The application of genomics to microbial systems hasbrought to light novel, cross-disciplinary fields of spe-cialty, including computational biology. Infrastructure,financial support, and educational support are neededto ensure that these areas remain viable disciplines.Moreover, it is important to create the intellectual divi-sion between the relatively new fields that haverelevance to genomics and their parent disciplines. Forexample, computational biology is seldom seen as anindependent field of research, but is viewed as a subdi-vision of computer science or biology. Computationalbiology should be considered a necessary field of

research in its own right, with its own set of principles,goals, and methodologies.

Cross-disciplinary training is another area where the cur-rent educational system may limit the forwardmomentum of genomics. It is critical to train graduatestudents and postdoctoral associates in the disciplinesthat bridge traditional departmental boundaries.

One particularly important need is to train microbiolo-gists, molecular biologists, and biochemists inprinciples of evolution and ecology. These foundationsubjects in general biology are often omitted in thesemore modern disciplines. A poor understanding ofsuch basic biological laws may seriously reduce ourability to formulate sensible biological questions inmicrobial ecology and evolution. ASM should ensurethat students of microbiology base their views on natu-ral biological laws. Other examples of cross trainingbetween disciplines include training in both biologyand mathematics, or in molecular biology and com-puter science. Programs like these, in which studentsreceive in-depth training in aligned fields that have rele-

vance in the application of genomic techniques, shouldbe developed and supported at the university, state,and federal levels.

While the educational system acclimates to the currentneed for scientists trained both in biology and com-puter science or mathematics, research organizations,including universities, private companies, and otherentities, must compete to attract those few individualswho have an interest or are skilled in bioinformatics. Adialog must be established between biologyresearchers and informaticists to motivate the latter tobecome involved in genomics research. The mecha-nisms that encourage these interactions, namelycollaboration requirements by funding agencies, arefew and far between, and more routes for interactionsare needed. Moreover, career promotion mechanismsare needed as incentive for informaticists to stray fromthe realm of computer science and into the realm ofbiology. Biology is notorious for failing to adequatelyrecognize the contributions of computer scientists toresearch programs. This failure needs to be addressedif competent, motivated professionals are to beattracted to the field of genomics.

It is too often the case that undergraduate curriculareflect an out-of-date understanding of biology, and fail

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Cross-disciplinary training is another area where the current educational system may limit the forward momentum of genomics.

to represent recent advancements in science to stu-dents who are interested in the future of research. Therole of genomics in the future of science, and of micro-biological science in particular, needs to beemphasized, as advancements in our knowledge byusing these tools are likely to guide the cutting-edge ofresearch for years to come. For example, biology pro-grams should address the ways in which ourunderstanding and our modes of experimentation inevolution are likely to be affected by genomics. Ongo-ing partnerships between teachers and laboratories orinstitutions should be encouraged and redoubled sothat the powerful role of genomics in advancing scienceand broadening our understanding of life can be effec-tively communicated to students and the public.

Genomics in the 21st Century – What We Can Accomplish

Genomics has revolutionized the way biological scienceis executed. In microbiology, the tools and techniquesof genomics have an unprecedented power to eventu-ally enable the development of a new, predictivescience. In the future, advances in genomics will allowscientists to extrapolate microbial community functionand the impacts of disturbance on human health andthe environment from the results of straightforwardanalyses. Although a great deal of work stands betweenus and these future capabilities, the contributions thatmicrobial genomics is poised to provide for medicineand the environment are extraordinary.

Currently, our ability to predict the responses of a singlemicroorganism from the sequence of its genome canbest be described as feeble, and our ability to make pre-dictions for an assemblage of multiple organisms iseven weaker. However, we can make certain predictionsabout the inability of communities to carry out certainfunctions. For example, if genomic methods reveal thatan individual or a community lacks nif genes (the genesthat confer the ability to fix nitrogen), it can safely bepredicted that the individual or community is incapableof fixing nitrogen.

In the future, more powerful predictions will be basedon the assumption that genomically similar communi-ties have similar dynamics and carry out similarfunctions. This ecosystem predictive capability willcome from detailed work that links an understanding ofthe genome to an understanding of gene expression,protein function, and complex metabolic networks. Inthis way, by describing the parts of microbial communi-ties and then extrapolating from the model, scientistscan create predictive tools that will allow an under-

standing of ecosystem function. Ecosystem function,which is at the root of human disease as well as envi-ronmental stability, has fundamental biological, medical,and environmental relevance.

Predictive tools can enable novel health strategies inmedicine. The ability to predict disturbances in the com-munities present in the human body and in oursurrounding environments will allow doctors to avoid orcorrect the community imbalances that result in diseasein a predictable, repeatable fashion that bypasses cur-rent trial-and-error approaches. Antimicrobial therapymay be employed in a more discrete, less disruptive,and more informed fashion. In the future, “communitydiagnostics” will enable better prognostics and thera-peutics, all of which will be based upon microbialgenomics.

In the environment, advances in genomics will enablescientists to predict the impacts of community distur-bances. Of particular interest is the ability to predict theextinction of genes, organisms, and functions that arerelevant to environmental health, agricultural produc-tion, and global cycles of the elements. Microbialcommunities could be used as a source of diagnosticsignatures for environmental health, enabling bettermanagement of our limited natural resources. For exam-ple, the microbial communities of rice paddies have anunquestionable impact on world food supplies, andconcerns exist about the impacts of community pertur-bations on the global production of rice. Similarly, themicrobial communities of the oceans control globalcycles of the elements and strongly influence climate.Complex microbial communities in the environment ofthe human body are major factors in both health anddisease. In the future, using genomic information andanalyses, these impacts and influences will be tracked,predicted, and potentially manipulated. A deep genomicunderstanding of these integrated microbial processeswill provide a better understanding of our planet, ourinteraction with it, and our ability to predict and influ-ence its future behavior.

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Recommendations

Enhance research community access to productionDNA sequencing facilities

The scientific community needs more open access toproduction genome sequencing facilities and associ-ated bioinformatics tools. To date, there has beenlimited access to high throughput sequencing for thegeneral community. Research dollars for specific sci-ence projects are often disproportionately redirectedto maintain production laboratory infrastructure andoverhead. Better mechanisms need to be developedto create more opportunities and less “closed shop”scenarios in the production sequencing laboratories.More open mechanisms for providing productionDNA sequencing and open source bioinformaticsanalyses to the general scientific community need tobe available. Models for community sequencingaccess are already developing, such as the UnitedStates Department of Energy’s (DOE) Joint GenomeInstitute Community Sequencing Program. Even moreopportunities like this need to become available inthe future, as well as programs that supply the neces-sary follow-up funding for post-sequence analysis.

Create centers that facilitate research communityaccess to postgenomic analytical capabilities

Centralized infrastructures that can collaborate withthe community, and supply large scale “DNA chip” ormicroarray facilities, proteomic instrumentation andanalyses, imaging facilities, advanced fluorescenceactivated cell sorting facilities, and similar technologi-cal platforms are needed. National Laboratories,Centers of Excellence, and National Science Founda-tion (NSF) Science and Technology Centers representsome of the current mechanisms to create andenhance such structures. Community solutions thatfacilitate access to genome-based technologies willallow the research community to focus better onmerging the necessary disciplines and on the scien-tific questions.

Identify and nourish strategic collaborations across disciplinary boundaries

The trajectory of the scientific questions posed in thegenomic analysis of microbial communities necessi-tates transcending disciplinary boundaries.Computational biologists and bioinformaticians needto work side by side with environmental scientists.Microbial ecologists need to interface with theoreticalevolutionists and population geneticists. Medicalmicrobiologists would benefit from more ecologicalthinking. The blurring of disciplinary boundaries placeswell-known stressors on funding, peer review, aca-demic departmental, and tenure grantingmechanisms. These challenges need to be faced

openly by academic departments, university adminis-trations, academic journals, and funding agencies. Thechallenge is to foster integration, while maintainingstrong disciplinary foundations and high academicand disciplinary standards and quality. Areas thatwould stand to particularly enhance the analyses ofgenomic data in microbial community contextsinclude, but are not limited to, microbial ecology andpopulation genetics, bioinformatics and environmentalscience, microbial ecology and medical microbiology,and systems biology and environmental microbiology.

Develop community databases that integratemicrobial community genomics with relevant environmental, clinical, geographical, and geochemical data

We lack “biological information systems” that linkbiological datasets with associated physical, chemi-cal, and other environmental data. Such databasesand database standards should be developed. Devel-opment of such databases will need to be a largeinterdisciplinary community effort, requiring the inputof computational biologists, population geneticists,environmental scientists, ecologists, and microbiolo-gists. A plan will need to be developed to maintainsuch inter-operable databases, and this will requiretime and a broad community input.

Develop computational tools to analyze the com-plexity at all levels of biological information withinand between complex microbial communities

New sorts of computational tools and algorithms willbe required to inter-relate the large and complexdatasets that will arise from microbial communitygenome sequence analyses. One particular chal-lenge of significant magnitude will be the assemblyof whole genomes from sequences obtained fromcomplex mixtures of highly related organisms. The current DNA assembly strategies and algorithmswill need to be modified significantly to meet thesenew challenges. In addition, new ways to inter-relategenomic, environmental, clinical, biogeochemical,and biogeographical datasets will need to be developed and applied.

Encourage large efforts that focus on specific, naturally occurring model microbial communitiesand ecosystems

Establishing focused groups of researchers that teamto study particular microbial communities or ecosys-tems has potential to accelerate the development oftools and techniques for genome-enabled microbialcommunity analyses. Specific attributes of modelmicrobial communities might include widespread dis-tribution, geochemical importance, biomedicalimportance, tractability, or industrial importance.Those microbial communities that have already been

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extensively well studied using traditional methodshave a built-in infrastructure and historical datasetsthat would enhance the value of genome enabledapproaches. Model communities may come from anenvironment that is amenable to genomic experimen-tation and has a relatively simple physiology.

Establish research programs that investigate theprocess and dynamics of microbial communitygenome evolution in natural systems

Efforts to establish the relationships among genomicdiversity, ecological diversity, and ecosystem dynam-ics should be encouraged and accelerated. Inparticular, the processes and products of microbialspeciation, diversification and lateral gene transferevents need to be better documented and under-stood. The rate and extent of interspecies gene flowin microbial communities is largely unknown, andshould be investigated in order to understand betterthe stability of the microbial genome. The relativeinfluence of lateral gene transfer and other modes ofgenome evolution and diversification need to bemuch better quantified, modeled, and placed inappropriate ecological contexts.

Develop enabling technologies to dissect moreaccurately genomic content and dynamics in complex microbial communities.

New and better technologies to dissect the individualand collective genomes found in naturally occurringmicrobial populations need to be developed. Frontend purification techniques including novel cultivationstrategies and cell purification strategies such as flowactivated cell sorting, coupled with newly developedsingle genome amplification strategies, could greatlyaccelerate progress. Downstream analytical strategiesfor sequencing the collective genomes of whole com-munities also need development and improvement. Inparticular, algorithms for de-convoluting intraspecificand within-population genomic polymorphisms, needextensive development and improvement.

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Suggested reading

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Boucher, Y., C. J. Douady, R. T. Papke, D. A. Walsh, M. E. Boudreau, C. L. Nesbo, R. J. Case, and W. F. Doolittle. 2003. Lat-eral gene transfer and the origins of prokaryotic groups. Annual Review of Genetics 37:283-328.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14616063&dopt=Abstract.

Breitbart, M., I. Hewson, B. Felts, J. M. Mahaffy, J. Nulton, P. Salamon, and F. Rohwer. 2003. Metagenomic analyses of anuncultured viral community from human feces. Journal of Bacteriology 185:6220-6223.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=14526037&dopt=Abstract.

Cohan, F. M. 2002. What are bacterial species? Annual Review of Microbiology 56:457-487.http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12142474&dopt=Abstract.

Daubin, V., N. A. Moran, and H. Ochman. 2003. Phylogenetics and the cohesion of bacterial genomes. Science 301:829-832.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12907801&dopt=Abstract.

Delong, E. F. 2002. Microbial population genomics and ecology. Current Opinions in Microbiology 5:520-524.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12354561&dopt=Abstract.

DeLong, E. F. 2002. Towards microbial systems science: integrating microbial perspectives, from genomes to biomes.Journal of Environmental Microbiology 4:9-10. [No abstract available.]

Feil, E. J., and B. G. Spratt. 2001. Recombination and the population structures of bacterial pathogens. Annual Review ofMicrobiology 55:561-590.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11544367&dopt=Abstract.

Lerat, E., V. Daubin, and N. A. Moran. 2003. From gene trees to organismal phylogeny in prokaryotes: the case of the ?-Proteobacteria. PLoS Biology 1:101-109.http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12975657&dopt=Abstract. Full text at http://biology.plosjournals.org/plosonline/?request=get-document&doi=10.1371/journal.pbio.0000019.

Mann, N. H., A. Cook, A. Millard, S. Bailey, and M. Clokie. 2003. Marine ecosystems: bacterial photosynthesis genes in avirus. Nature 424:741. http://www.nature.com/cgi-taf/DynaPage.taf?file=/nature/journal/v424/n6950/abs/424741a_fs.html&dynoptions=doi1074264256.

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Rocap, G., F. W. Larimer, J. Lamerdin, S. Malfatti, P. Chain, N. A. Ahlgren, A. Arellano, M. Coleman, L. Hauser, W. R. Hess,Z. I. Johnson, M. Land, D. Lindell, A. F. Post, W. Regala, M. Shah, S. L. Shaw, C. Steglich, M. B. Sullivan, C. S. Ting,A. Tolonen, E. A. Webb, E. R. Zinser, and S. W. Chisholm. 2003. Genome divergence in two Prochlorococcusecotypes reflects oceanic niche differentiation. . 424:1042-1047.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12917642&dopt=Abstract.

Rodriguez-Valera, F. 2002. Approaches to prokaryotic biodiversity: a population genetics perspective. EnvironmentalMicrobiology 4:628-633.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12460270&dopt=Abstract.

Rondon, M. R., P. R. August, A. D. Bettermann, S. F. Brady, T. H. Grossman, M. R. Liles, K. A. Loiacono, B. A. Lynch, I. A.MacNeil, C. Minor, C. L. Tiong, M. Gilman, M. S. Osburne, J. Clardy, J. Handelsman, and R. M. Goodman. 2000.Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microor-ganisms. Applied and Environmental Microbiology 66:2541-2547.http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10831436&dopt=Abstract. Full text at http://aem.asm.org/cgi/content/full/66/6/2541?view=full&pmid=10831436.

Sullivan, M. B., J. B. Waterbury, and S. W. Chisholm. 2003. Cyanophages infecting the oceanic cyanobacterium Prochloro-coccus. Nature 424:1047-1051.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12944965&dopt=Abstract.

Tamas, I., L. Klasson, B. Canback, A. K. Naslund, A. S. Eriksson, J. J. Wernegreen, J. P. Sandstrom, N. A. Moran, and S. G.Andersson. 2002. 50 million years of genomic stasis in endosymbiotic bacteria. Science 296:2376-2379.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12089438&dopt=Abstract.

Ward, D. M. 1998. A natural species concept for prokaryotes. Current Opinion in Microbiology 1:271-277.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10066488&dopt=Abstract.

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