DRAFT

June 17-19, 1998

National Science Foundation

4201 Wilson Boulevard.

Arlington, Virginia 22230

NSF convened a LExEn workshop in June of 1998 to develop recommendations for future directions of the LExEn Research Activity. The agenda for the workshop and the list of participants can be found in Appendices B and C, respectively. The underlying principle sustaining this Activity is that the achievements should be greater than what could be attained by the routine award of single, investigator-initiated research grants. The organizing strategy for this Workshop was to form three Primary Questions for the participants to address, and to look at these questions through a LExEn lens. The Steering Committee for the LExEn Workshop recommended a preliminary set of Primary Questions to start the discussions, and all Workshop participants engaged in developing and defining the final set of questions for working group action.

Primary Questions

1. What conditions determine the limits of life?
2. What is the functional diversity of life?
3. How does life evolve from pre-biotic conditions?

Working Group Charge

Working groups were organized to address each Primary Question and each group was given two broad charges.

• Develop an "Investigative Strategy" directed at answering the Primary Question.
• Develop ideas about how to build a LExEn community of scientists, i.e. how to nurture and develop the interdisciplinary research teams necessary to make progress with LExEn objectives.

The Investigative Strategy was defined as a hierarchy of questions beginning with a single Primary Question and ending with a multitude of specific questions of sufficiently limited scope that they might be undertaken within a single, conventional research proposal. Between these two extremes is some undefined number of levels of questions of ever-increasing specificity. The working groups were asked to think of and develop significant questions that may be answered by long-term (a human lifetime), mid-term (a scientific career), and near-term (1-3 regular funding cycles) investigative strategies. The workshop participants were asked to develop questions strictly based upon the science of LExEn without regard to considerations of resource requirements.

The following report consists of summaries of the three working groups, each assigned to one of the Primary Questions. In their summaries, they present Specific Research Topics that include a wide range of general as well as specific questions to be answered through basic research. They also present Methodologies, Technologies, and Infrastructures that cover instruments and methods necessary for research in extreme environments, as well as community networking to encourage interdisciplinary action.

Considerations and Expectations

Working groups were asked to define the significance of the Primary Question being considered and construct an overview of a hierarchical structure for the representative questions developed for each time scale. At each level of the hierarchy the discussions were to produce representative examples of the most significant questions to be answered in order to gain new scientific understanding and insight.

Several specific points of guidance for the working groups were agreed upon at the outset:

• In choosing the representative questions, consideration should be given to the maintenance of an appropriate balance between the study of life forms and the study of the environments within which they exist.
• Where possible, questions should be posed in such a way as to optimize the potential benefit of interactions between disciplines.
• The importance of exploration and discovery-oriented research approaches should be recognized as a fundamental emphasis in addition to the more traditionally favored emphasis upon hypothesis testing.
• As appropriate, brief descriptions should be provided of the infrastructure, capabilities and technology required to achieve the stated research objectives.

A set of specific LExEn questions compiled by Hugh Van Horn following the initial workshop presentation was also provided to the working groups, and is included as Appendix D.

The expected outcome of the 1998 LExEn Workshop is new insights into the science that is LExEn, through a set of recommendations to establish ambitious and achievable goals that will guide this area of research well into the 21^st century.

A large population of organic molecules has been discovered in the interstellar gas with radiotelescopes. Space craft have recovered evidence that water was once plentiful on the surface of Mars and that the Jovian satellite Europa may be covered with ice, and there is the possibility that a fairly deep ocean lies beneath. Finally, over the last two years Jovian scale planets have been detected around at least a dozen nearby stars, with many more such discoveries probably soon to come. The astronomical setting for the origin of life is a scientific discipline likely to develop explosively over the coming decade.

On planet Earth, there is a recognition that contemporary environmental conditions are not the norm, and that habitats more hostile to life as we know it probably existed in the past. The geologic and fossil record offers an opportunity to gain insights into the history of extreme environments on Earth and may offer a perspective on the potential for life on other planets.

Life has successfully radiated into many diverse environments on Earth, including those that might be termed "extreme." This designation is anthropocentric and many extremophiles (organisms inhabiting unusual environments) cannot compete successfully, or even survive outside of these extreme habitats. An understanding of life processes in extreme environments will allow constraints to be placed on the limits of life.

A working definition of an extreme environment is one where physical and chemical conditions approach or exceed the tolerances for life. However, it should be emphasized that some organisms actually thrive even under selected extreme environmental conditions. Common examples include high temperature fumeroles and high salt content evaporite deposits. More subtle extreme environmental conditions may also include habitats that contain only dilute concentrations of life sustaining nutrients or those with unusually high concentrations of the same solutes. Many of these unusual environments are extreme by more than one criterion. For example, the deep-sea is a high pressure, low temperature habitat, and some hypersaline lakes in Antarctica have high salt concentrations and very low temperature. Many extreme habitats are inaccessible and have not been adequately characterized or even explored. A key requirement of any new program of life in extreme environments is the need to integrate life processes with a detailed study of the environmental conditions.

There are both intellectual and practical reasons for undertaking ecological studies of extreme environments. First and foremost relates to the origin of life itself. While even a comprehensive study of life in extreme environments will not necessarily provide the answer, scientific investigations in the full range of habitats in our universe will provide a better understanding of how our planet functions from geological through biological processes. These additional insights will provide much needed data on chemical fluxes and climate variability as well as the biological consequences of climate variability. There are also many potential practical applications of this information from biologically-mediated materials production, to natural resource preservation, to "biochip" manufacturing and other biotechnological applications.

Microorganisms possess qualitative and quantitative requirements for diverse resources that are essential to support life. Requirements vary according to species, metabolic state and micro-environmental conditions. Water, C, N and P are presumed to be basic to all life, although required levels vary. Other resources, such as some enzyme trace metal requirements, can be organism-specific and substitutions are possible. In considering resource requirements, it is critical to explicitly distinguish between resource classes and levels that are required for growth and those required for survival alone. Progress in research related to the "resource" questions will benefit greatly from environment-specific geochemical models. Examples of questions related to life’s resource requirements include:

The ability to monitor extreme environments and the response of these environments and of their inhabitants to natural perturbations would be a valuable outcome of the development of in situ measurement and sampling techniques. Sampling of material present in space and improved (higher spatial resolution) detection and characterization of planetary environments are also desired goals of the LExEn program that can be addressed in concert with other government sponsored programs.

Investigation of the factors that limit life will also require laboratory studies carried out in appropriate conditions. Instrumentation for thermodynamic and kinetic studies and for laboratory spectroscopic studies of reactive molecules will need to be upgraded. Technology required for reproduction of extreme environments in the laboratory, and for studies of extremophile physiology, will need to be developed. Modeling efforts also need to be enhanced.

Improvements and innovations are also needed in methods used for in situ sampling in extreme environments to allow characterization of habitats and inhabitants. This includes development of:

A major goal of LExEn will be sampling of the full range of extreme environments, requiring improvements in current methods of obtaining in situ samples. Examples include:

• Enhancement of Ocean Drilling Program (ODP) drilling technology to allow:

- prevention of contamination of drill-core

- retention of volatiles

- recovery of cores from extreme ocean depths

- recovery of material from directly beneath active vent sites

• Installation of a microbiological laboratory on board the ODP drilling vessel
• Development and enhancement of drilling and coring techniques for ice and terrestrial habitats
• Adaptation of existing CORK (Circulation Obviation Retrofit Kit) technology for subsurface microbiological studies
• Alternative methods of obtaining samples from extreme environments, such as use of smaller sea-floor drilling platforms
• Sampling of event plumes and of the range of hydrothermal effluents exiting the seafloor as a means of sampling sub-surface environments
• Development of platforms for aerobiology
• Planetary sample return and analysis

Many new insights into understanding life at its extremes are likely to come from sharing of knowledge amongst disciplines that have not traditionally interacted, e.g. microbiology, planetary science and astronomy, geology, geochemistry, paleontology, molecular biology and engineering. To ensure multidisciplinary potential is realized we recommend the following:

Community, organismal, and molecular processes in extreme environments will be examined in diverse ways including: bioenergetics (light, chemical energy), uptake of substrates (carbon compounds, other nutrients), physiology (metabolic processes), community interactions (syntrophy, symbiosis, competition, genetic exchange, controls on levels of activity), preservable indicators of biological processes (biomarkers), and evolutionary adaptation of metabolic processes to extreme environments.

The processes of life in extreme environments parallel those in ‘normal’ environments yet require specialized adaptations to achieve those ends. Extreme environments impose extraordinary constraints upon the organisms that live there. This section addresses specific mechanisms at each of the levels of functional diversity discussed above (molecular, biochemical, cellular, multicellular/consortial, ecological and evolutionary) that allow organisms to inhabit these environments. The reciprocal interaction between environments and living systems (at the entire range of scales) needs to be documented using interdisciplinary approaches. Laboratory and field investigations of these questions necessitate the development of new technical and analytical methodologies due to the extreme nature of these environments. Information gleaned from these studies will be useful for the recognition of life in other contexts (on Earth and other planets), the identification of commercially useful products, biotechnology and the utilization of basic technology and instrumentation developed in this area. We need to learn the general principles involved with life in extreme environments due to the extreme range of environments, species, and communities.

• What are the unique physiological features of individual extremophiles that yield an advantage for growth and survival in an extreme environment?
• What are the shared features within a group of extremophiles (i.e., hyperthermophiles) from one type of extreme environment?
• What are the shared features between groups of extremophiles (i.e., between psychrophiles and barophiles)?
• Do extremophiles evolve convergent mechanisms to solve a common environmental problem?
• How were these specialized physiological features acquired?
• What are the energetic costs and benefits of adaptation to extreme environments?

These studies require the development of methods to detect and analyze cellular components that are not routinely examined in biology.

• Are there unique examples of the following in extremophiles: cofactors, proteins, cytosolic solutes, membranes, electron carriers, energy currencies, metabolic pathways, nutrient acquisition mechanisms, transcriptional and translational mechanisms?
• Are special life history strategies necessary in extreme environments?

One of the challenges encountered by microorganisms in extreme environments is the physiological and structural adaptations required for basic physiological processes including growth, reproduction, and cell maintenance. These questions must be approached at several levels of cell metabolism.

• What is the full range of potential energy sources, including expanded ranges of known sources and new sources, and the bioenergetic mechanisms for harvesting these sources? Also, what is the metabolic basis for hierarchical, complimentary, or competitive energy utilization within and among microorganisms?

Microorganisms also have requirements for nutrients that serve as building blocks for the synthesis of cell monomers and polymer.

• Are there nutrients used by extremophiles that have not been previously identified as biological components and new biochemical paradigms for assimilating nutrients into cell components?

Cell structure may also have a critical role in the specific adaptation to extreme environments.

• Are there specific structural adaptations (i.e., intracellular macromolecules, cell envelop, etc.) that cells have developed to survive and proliferate in extremes and during fluctuations between optimal and non-optimal environmental conditions?
• Are there behavioral responses to extreme environments such as attachment mechanisms and motility (i.e., chemotaxis, thermotaxis, phototaxis)?

Research on these topics requires identification of metabolic pathways and mechanisms of control at both the biochemical and genetic levels. Specific physiological extremes include: high and low temperature, low water activity (desiccation, halophily), high and low pH, high ionizing radiation, high pressure, low nutrient concentrations, extremely low and high redox potentials, environmental fluctuations.

Identification and characterization of novel metabolism will require multidisciplinary approaches. Examples of possible collaborations for conducting research on microorganisms with not previously described metabolisms would include:

1. Group and species interactions

The following topics will be discussed: the transfer of specific nutrients among community members, the degree of specificity of interactions within a community, the extent to which species are trophic generalists or specialists, the presence or absence of ‘keystone’ species, endosymbiosis and the degree to which the community is necessary for the survival of individual species, and the implications of the presence or absence of specific physiologies upon community structure.

The degree to which species interactions and community function in extreme environments differs from that in more benign environments is not known. The energetic costs associated with adaptation to extreme environments may limit the number of trophic levels. Conversely, the exclusion of many larger organisms from extreme environments may release communities from ‘top down’ control (structure in the environment imposed by predation and grazing). Specific metabolites produced in extreme environments may be a currency of exchange among species or serve as alternative nutritional resources as fluctuations in the environment occur. Specific physiological adaptations may structure interactions among species in extreme environments. Communities may become more or less structured and functional relations among species more or less coupled as life approaches the limits. For example, approaches to the limits of life may require symbiotic interactions and consortia (sets of organisms achieving ends that are attained by single organisms in other environments) to carry out ecological functions. The degree to which ‘keystone’ species play an important role in extreme environments is unknown. It is not clear whether the boundaries to life in terms of physiology coincide with the ecological boundaries (can extremophiles in pure culture survive closer to the limits of life than can a consortia or in situ ecosystem or vice versa?) Little is known about community ecology in any extreme environment. Investigation of species interactions in extreme environments should elucidate general underlying principles of ecological organization and how they are constrained by the physico-chemical environment. Qualitative assessment of communities (who is there) needs to have an explicit link with rigorous quantitative analysis of abundance within and across ecosystems (at a single location, between locations and globally). The number of species, their identities, biomass and a frequency distribution need to be documented.

• Biofilm architecture – Role of cellular aggregates and exopolymers in stabilizing the community and facilitating the harvesting of energy and the flow of chemical species among community members. There is a need to understand the response of this architecture as a part of the adaptation to particular environments.
• Syntrophy, symbiosis, competition – Need for baseline studies of such interactions within extreme environments as a basis for understanding their role in the adaptation and survival in extreme environments.
• Genetic exchange – Need for baseline studies of transformation, etc. processes as factors in developing the genetic ensemble in extremophiles. For example, delineate the roles of gene transfers in the diversification and dispersal of molecular adaptations of extremophiles.
• Controls on levels of activity – Need to delineate "who is active and inactive" in these communities, as a basis for discriminating between modes of growth and survival in extreme environments.

Extreme environments are frequently patchy, ephemeral and unpredictable. This structure requires organisms to evolve novel mechanisms for persistence during times of environmental change or for the colonization of new areas. Many organisms from extreme environments have also been detected in a wide variety of environments outside of their ‘typical’ habitat. The ecological tolerance of these organisms may be extremely broad, or there may be special adaptations to allow persistence in a resting state.

Denizens of extreme environments appear to be highly adapted to specific sets of conditions, but these habitats tend to be patchy, or even widely separated, and sometimes ephemeral. Some adaptations seem to be at the ecosystem level.

• How then do these extremophiles manage to find and colonize appropriate sites?
• It would seem inefficient to blanket the earth with germ plasm at random, yet what mechanisms could guide individuals or community members to various extreme sites?
• How much genetic information can be dedicated to efficient dispersal mechanisms?
• It has been suggested, for example, that hydrothermal vents would act as infrared light beacons, and some community members appear to have infrared detectors. Does this mechanism guide dispersal?
• To what extent are stable survival forms of extremophiles different from those that have been studied?

Microorganisms interact with and also process substantial quantities of materials that, as a result, acquire characteristics that become signatures of the biological processes that altered these materials. For example, a sandy sediment surface that was stabilized by a biofilm might preserve the distinctive surface of that biofilm long after its demise. Organic molecules can be biosynthesized for light-harvesting, and their distinctive molecular structures can persist in the sedimentary rock record as legacies of specific photosynthetic bacteria. Thus careful assays of remnant biological indicators can not only reveal the former existence of an ecosystem, they can paint a rather detailed picture of its membership and principal biogeochemical processes.

The following categories of preservable process indicators collectively can offer this picture:

• Cellular morphologies - the traditional evidence of life, recognizable cells and fragments can offer definitive evidence of former life, yet are often nonspecific regarding cellular function. A need for a better understanding of morphological properties (e.g., pleiomorphism) in extreme environments.
• Microscale rock textures - the structure and behavior of cells and cell aggregates can belie their function (e.g., photosynthesis, taxis) and also serve as templates for mineralogical and sedimentary textures that survive into the sedimentary rock record. A more systematic understanding of microbial textures in extreme environments that are amenable to geologic preservation (e.g., hydrothermal systems).
• Community level structures - These represent an aggregation of microscale textures and their integration into larger-scale structures (e.g., stromatolites, reefs) that can become even more enduring geological legacies of community structure and function.
• Organic compounds - beyond merely indicating the former existence of life, structurally distinctive molecules can identify particular ecosystem processes (e.g., photosynthesis). There is a Need for further development of extremophile biomarker systematics.
• Stable isotopes - Stable isotopic patterns arise by isotopically-selective enzymes acting upon, for example, networks of carbon pathways in organisms and their ecosystems. Isotopes thus necessarily reflect the dominant processes and pathways in a microbial community. Furthermore, isotopic patterns of biological activity typically survive well in the geologic rock record. There Need for a broader inventory of measurements of maximum possible isotopic discrimination of carbon, nitrogen and sulfur by assimilatory and dissimilatory enzymes in extremophiles.
• Light from extrasolar systems - To the extent that a distant planet’s atmospheric composition has been altered by its biosphere, the light reflected from that planet can telegraph that biosphere’s existence to astronomers. To the extent that distant biospheres occupy "extreme environments," reflected starlight might someday reveal the abundance and diversity of extraterrestrial extremophiles. It might also enable production of better models of the range of atmospheric compositions in earth’s pre-oxygenated world.

Phylogenetic trees by themselves do not provide information on abundance activity or function. Efforts need to be directed, in the context of phylogenetic data towards quantitative assessments, spatial and temporal variability, and functional assessment. These include:

• What are the commonalities and differences within and between specific extreme habitats?
• What is the biological diversity (intraspecific, interspecific, functional) in particular extreme habitats past and present?
• What is the environmental diversity/variability in different extreme habitats?
• How does diversity change along environmental gradients that approach biological limits?
• What are indicators of biological activity in terrestrial extreme environments that could be diagnostic for extraterrestrial life?

7. Evolutionary adaptation to extreme environments

Adaptations that allow organisms to exist in the context of extreme environments are attained through the acquisition of novel genes. Evolution in extreme environments may utilize novel or typically rare evolutionary mechanisms. Genetic characterization of organisms from extreme environments, phylogenetic comparisons across the tree of life and the analysis of genetic variation through time and space will allow the elucidation of the mode of evolution in extreme environments. Since the phylogenetic patterns of single genes are commonly incongruent, genome level analyses are fundamental to these questions. A balance of coverage over the entire tree of life and the detailed analysis of several model systems are needed. Informational macromolecules of extremophiles are frequently difficult to extract and isolate from extreme environments so novel approaches will undoubtedly be required. Input from the engineering community will be especially valuable in all aspects of the isolation (without contamination) of organisms from extreme environments. In addition, the adaptations of organisms through time to extreme environments have not been static. The geological record needs to be used as a way of determining how, when, and why (the physical environmental context) these changes have occurred.

Microbes appear to be able to evolve very rapidly, since they have short generation times, can tolerate high mutation rates, and engage in promiscuous genetic exchange. Their ability to populate extreme environments affords opportunities to measure these rates. It raises the question of what maintains identifiable microbial species. One might expect microbes to show only very limited central tendencies, whereas the few studies on this topic that have been completed show strong clustering.

Experimental measurements of genomic changes during the relatively short term ‘evolution’ of hydrothermal vents, for example, might illuminate these questions. Such measurements would require either field or laboratory sampling systems that could be used over time in either closed or open systems. Complete population profiles of the communities would need to be developed and complete genomic sequencing information on selected members obtained over time. Detailed physiological characterization of the same targeted organisms should accompany the genomic analyses. The null hypothesis might be that no new traits would evolve, but rather new species would move into the niche.

• What is the genetic and molecular basis of adaptation to extreme environments?
• How do molecules/organisms/communities adapt functionally to the harsh (detrimental) aspects of their environment?
• How do molecules/organisms/communities take advantage of opportunities unique to or created by their environments? (e.g. infrared photosynthesis near smokers, unusual chemotrophies)
• What are the mechanisms that generate these adaptations? (vertical and horizontal transfer of information, symbioses/syntropy, etc) How do these mechanisms constrain the limits of life processes?
• What is the geologic record of evolution of life processes in extreme environments?
• What geologic traces remain of life processes in extreme environments? How can the details and generalities of life processes be inferred from the geologic record?
• What is the distribution of extreme environments over time? Has this driven evolutionary transitions between extremophily and mediocrephily? Has the definition of ‘extreme environments’ changed at the scale of geologic time, and if so how could this impact evolutionary processes?

The diversity and extent of life along a stable high temperature thermal gradient would be examined. The current limit of life at high T in a pure culture is 113C. However, little is known about the diversity of life at high T, and how it changes in along thermal gradients above 100C. This proposal seeks to address the question "How does diversity change along environmental gradients that approach biological limits?"

The following are physical and chemical measurements which will be used in this research. One of the requirements of our study is that the predominant gradient sampled is thermal, and not significantly different in terms of chemical composition or other physical and chemical parameters. It will be necessary to establish, in situ, a suite of instrumentation to measure and continuously monitor pH, temperature, inorganic ions, eH, etc. along the gradient. This will engineering and ground truthing chemical and physical probe development and data logging, and deployment strategies. The biological process measurement (this requires high temperature methodological development instrumentation) includes: CO2 fixation, heterotrophic potential, N2 fixation, and Ch4 production. Sampling will include harvest of in situ biomass from solid biofilm scrapings, as well as colonization slides. Requires instrumentation development and deployment strategies.

Diversity will be assessed from a variety different levels from intraspecific to functional levels. The first pass will include characterization of rRNA gene diversity at specific points along the gradient. Major rRNA groups will be quantified by quantitative pcr methodologies. Dominant "species clusters" will be identified, and the intraspecific diversity (microheterogeneity) will be assessed by comparison of rRNA spacer regions.

The research team requires a range of expertise including engineering, biogeochemistry, microbiology and molecular biology and population biology.

· Microbial communities in permanently ice covered lakes in Antarctica

Several different analogues of extraterrestrial life exist on the continent of Antarctica. One type of environment, lakes, that are permanently isolated under the Antarctic ice sheets for millions of years, may be an analogue for potential subsurface aqueous reservoirs similar to those thought to exist on Europa. Besides first order information gained on diversity of life on Earth at low temperatures, this project will involve the development of general methods, procedures, and instrumentation for sampling deep aqueous reservoirs buried under thick layers of ice -- technology that may be useful in guiding potential future explorations of Europa. We propose to develop and apply methods to aseptically recover microbiological samples in waters deep beneath the Antarctic ice sheets.

Microbial cells have been reported at densities of 1e4/gram of sediment 500 m below the seabed in a variety of geographic locales. However, most data are either isolates recovered from these sediments or total cell numbers. A major question is "are these microorganisms alive and active, or simply preserved after burial?" We propose here to assess the activity of microbial cells, in situ, 1000m below the sea surface. Radioisotopic tracer methods, in tandem with an in situ drilling/sampling/injection system, will be developed and applied to assess the activity of microbial cells in situ.

• In situ monitoring (cellular/ecological): Methods for observing microbial communities on the scale of microns; determining the composition of organisms in order to interpret and integrate biogeochemical measurements; quantitative methods for assessing microbial species variability and activities; non-destructive physicochemical techniques for assessing the energetics of cells and discrete biochemical processes; metabolic pathway monitoring in genetically engineered extremophiles; molecular-level probes that indicate biological and chemical processes during real time measurement

• Isolation and cultivation (sustained growth): Generic techniques for the isolation of microbes from extreme conditions and difficult-to-access environments; robotic systems; generic techniques for cultivation of key microbes; sustained growth bioreactors

• Biomarker analysis and systematics: an identification/development of suitable biomarkers indicative of life; an evaluation of these biomarkers for extraterrestrial utility; and remote-sensing biomarker technology

• Modeling: modeling of molecular, biochemical, cellular, multi-cellular and ecosystem processes

• Informatics: Information resources (databases) of relevance to major issues and research thrusts such as extremophile genomes of interest, cellular processes, extremophile inventory, and metabolic pathways; phylogenetic surveys of natural microbial diversity having significant numbers of samples (a useful resource and guide for further studies).

A first order question is simply, what organisms exist (qualitatively and quantitatively) in any specific habitat. In many habitats only a very small percentage of microorganisms can be cultivated by traditional means. A recent solution has been to retrieve and identify phylogenetically informative gene sequences (e.g., rRNA) from total population DNA, to assess microbial diversity. This is an important approach to characterizing microbial communities, and necessary in many instances to gain insight into the composition of naturally occurring microbial communities. The phylogenetic diversity of a community based on a single gene sequence, however, is of limited use unless viewed in the context of other studies. Single pass phylogenetic surveys will not shed light on the nature of extreme habitats or organisms as a stand-alone approach. However, phylogenetic surveys are a cornerstone for further quantitative, functional, and temporal assessments of microbial communities associated with extreme habitats.

Because of the inherently interdisciplinary nature of LExEn studies, and especially investigations of the functional diversity of life and extreme environments, it is particularly important to bring scientists from the associated fields together for cross-fertilization (and simply to teach each other the basics of the other fields and their languages), particularly people in the early stages of their careers.

- Life involves metabolism, reproduction, and biological evolution.

- We are dealing with life that is carbon and water based.

· Did origin of life take place in systems analogous to modern extreme environments?

· What was the early environment (and range of environments) on early earth?

· What role did impacts play in the origin of life?

• What are the physical and chemical prerequisites for the origin of life in extreme environments and how do these contribute to the formation and persistence of chemical precursors of life in these environments?

• What is the relationship between interstellar molecules (and cometary molecules) and the origin of life?

• Can an origin of life occur and result in sustained existence in modern extreme environmental conditions?

· What was the actual distribution of such environments on the Earth?

· What is the distribution of planets around stars?

4. What were the conditions under which life evolved?

• Are there extreme environments in existence today that emulate these conditions?

• Is short-term environmental change necessary to the origin or evolution of life?

• What are the feedback mechanisms between evolution of life and environment?

On the level of populations, we may find that genetic exchange among fairly distantly related organisms is more (or less, we don't know) common than for microbial populations in more benign environments. If genetic variation is produced exclusively by mutation, one would see closely related "clusters" of clones. The genome of extremophiles may also have characteristic features such as G+C content, etc.

• How does life evolve into or out of extremophily?

• What features are attributable to common ancestry vs. unique adaptation?

One hypothesis resulting from studies of the evolutionary relatedness of microorganisms is that early life was thermophilic. Models of a high temperature Earth, at times when fossilized microbial life is first detected, are consistent with the proposal that early life evolved in extreme environments. In addition, interplanetary transport of life would also demand resilience to extreme conditions. It is conceivable then, that modern day extreme environments harbor forms of life that are similar to ancestral life. The demands on life in extreme environments might also spawn novel metabolisms that shed light on the pathways down which evolution has proceeded. The following questions represent the sort of guiding questions under which specific research projects might be developed.

• What do gene phylogenies tell us about the early history of life?
• Are extreme environments reservoirs of ancestral-like life forms?

· What role have extreme environments played in the origin of new metabolic pathways?

• Remote sensing

• In-situ sensing

• Laboratory reactors that simulate extreme environments

• Establish necessary infrastructure for continuous monitoring and sampling at observatories and for communication of results from these sites
• Support interdisciplinary proposals and workshops
• Support graduate students, postdoctoral fellows and sabbatical leaves for cross-disciplinary research
• Identify a few sites for intensive investigation (Extreme Environment Observatories)

• methods to isolate and culture microbes found in extreme environments;
• methods to study these microbes in their natural habitats and to describe their adaptive strategies from the molecular to the ecological level;
• technologies for non-contaminating sample recovery;
• sensors and sensing techniques to probe extreme environments on Earth or other planets;
• methods to study ancient microbial life and paleo-environmental conditions on Earth; and
• methods to investigate the potential for habitable environments on other planets (including theory and modeling).

9:00 a.m. Steering Committee Meets, Room 730

1:00 p.m. Workshop Opens- Chaired by Mike Purdy

3:30 p.m. Break

5:15 p.m. Discussion lead by John Delaney

6:30 p.m. Reception

7:00 a.m. Steering Committee Breakfast

8:00 a.m. Coffee and Doughnuts

8:30 a.m. Establish Working Groups and define charges

9:30 p.m. Break

10:00 a.m. Working Groups meet

12:30 p.m. Lunch (Steering Committee meets)

1:30 p.m. Working Groups meet

6:00 p.m. Adjourn

8:00 a.m. Coffee and Doughnuts

8:30 a.m. Writing and Working Group Meetings as required

11:00 a.m. Plenary Session

Jean Brenchley, Pennsylvania State University, jeb7@psu.edu

Jim Brown, North Carolina State University, jwbrown@mbio.ncsu.edu

Doug Clark, University of California, Berkeley, clark@cchem.berkeley.edu

Emily CoBabe, University of Massachusetts, ecobabe@geo.umass.edu

Jim Cowen, University of Hawaii, jcowen@soest.hawaii.edu

John Delaney, University of Washington, jdelaney@u.washington.edu

Ed Delong, Monterey Bay Aquarium Research Institute, delong@mbari.org

Dave DesMarais, NASA, ddesmarais@mail.arc.nasa.gov

Kathleen Duncan, University of Tulsa, BIOL_KED@centum.utulsa.edu

Jim Holden, University of Georgia, jfholden@arches.uga.edu

John Holloway, Arizona State University, john.holloway@asu.edu

Julius Jackson, Michigan State University, jhjacksn@pilot.msu.edu

Bruce Jakosky, University of Colorado, jakosky@orion.colorado.edu

Dave Karl, University of Hawaii, dkarl@soest.hawaii.edu

Marv Lilley, University of Washington, lilley@ocean.washington.edu

Douglas N.C. Lin, University of California, Santa Cruz, lin@ucolick.org

Barry Marrs, Fairville Products, Inc., barrym4212@aol.com

Betsy McLaughlin-West, Rutgers University New Brunswick, betsy@koros.rutgers.edu

Robert McMahon, University of Wisconsin, mcmahon@chem.wisc.edu

Anna-Louise Reysenbach, Rutgers University, alr@imcs.rutgers.edu

Thomas Schmidt, Michigan State University, tschmidt@pilot.msu.edu

Jeff Seewald, Woods Hole Oceanographic Institution, jseewald@whoi.edu

Bill Seyfried, University of Minnesota, wes@maroon.tc.umn.edu

Everett Shock, Washington University, shock@zonvark.wustl.edu

Loren Smith, University of Southern California, lhsmith@mizar.usc.edu

Lew Snyder, University of Illinois, snyder@astro.uiuc.edu

Kevin Sowers, University of Maryland, sowers@umbi.umd.edu

Diane Stoecker, University of Maryland, stoecker@hpel.umd.edu

Ron Swanson, Diversa Corporation, rswanson@diversa.com

Patrick Thaddeus, Harvard University, pthaddeus@cfa.harvard.edu

James Tiedje, Michigan State University, tiedjej@pilot.msu.edu

Meg Tivey, Woods Hole Oceanographic Institution, mktivey@whoi.edu

Jack Welch, University of California, Berkeley, wwelch@astro.berkeley.edu

Nick Woolf, University of Arizona, nwoolf@as.arizona.edu

Art Yayanos, University of California, San Diego, ayayanos@ucsd.edu

Oskar Zaborsky, Hawaii Natural Energy Institute, ozabo@hawaii.edu

Can life originate in more than one way?

What is the range of diversity of living organisms?

What is the range of carbon and energy pathways in living organisms?

How can we reconstruct metabolism from DNA sequences?

How large a piece of DNA is necessary to identify clustering?

Can a single gene have more than one function in different organisms?

How can gene fragments be assigned to specific organisms?

How do time changes in physical/chemical environments affect the evolution of organisms?

How will high-bandwidth data links affect scientific research, especially in remote/hostile/extreme environments?

Why might Mars or Europa have life or interesting prebiotic chemistry?

What locations might have extant or extinct life?

What would be a good indicator for the existence of life?

What metabolites might provide evidence for the (past) existence of life?

What's out there on Earth?

What is necessary to cultivate "uncultivatable" organisms in the lab?

How does population density affect organisms?

What are the environmental limits to life?

To what extent do extreme terrestrial environments represent possible extraterrestrial environments?

To what extent can extreme environments be engineered in the lab?

What microorganisms can survive cyclic exposure to extreme environments?

How does the fossil record reveal the history of evolution?

What are physical/chemical tracers of past life?

How interconnected are terrestrial environments?

How can evolutionary forces be understood within their ecosystems?

What role does gene transfer play in the evolution of organisms?

What are the "primary producers" in different ecosystems?

How can organisms be studied in their natural environments?

Can microorganisms be usefully modified by microprobes?

How does an organism become an extremophile? (Or vice versa?)

How do extremoophile populations adapt?

How are extremophile communities organized?

Is the rate of evolution faster in extremophiles?

How quickly can microbes adjust to rapidly changing environments?

What is the significance of subseafloor hyperthermophiles?

What are the intracellular energetics of hyperthermophiles?

Are there unique chemical signatures of living organisms?

How are different microbial organisms interdependent?

How does environment affect the organism, and how do organisms affect the environment?

Can we produce extreme environments in the lab?

Is there liquid water in planets and satellites?

Can dynamical evolution of planetary/satellite systems produce environments capable of supporting life?

Why is cryptobiotic soil clumpy?

How dry can you get and still have life?

What are the important energy sources for life in extreme environments?

What energy sources do we expect to be important on other planets?

"Who's there, and what are they doing?"

Is there a Europan ocean; if so, what is it like?

Are there biologically relevant molecules in interstellar clouds? Comets? Meteorites? Planetary atmospheres?

What chemical processes produce biologically relevant species?

What were the early atmospheric conditions for life?

Are there isotopic mechanisms capable of distinguishing the metabolic processes of fossil organisms?

How can we understand the patterns of microbial diversity in extreme environments?

How did life originate?

What processes of abiotic synthesis produced the chemical precursors of life?

When did "novel" metabolic processes arise?

How might astronomical events (e.g., comet/meteorite impacts) have affected the evolution of life?

What is the relationship between interstellar organic molecules and the origin of life?

What are the abundances of biologically relevant molecules in interstellar space?

What is the origin of complex interstellar molecules?

What strategies can enable us to discover and characterize novel microbes?

How do organisms sustain metabolic activity in extreme environments?

How can "consortia" best be studied in the laboratory?

How important is endosymbiosis?

What is the relation between the genomic "tree of life" and evolution?

What organisms live at very cold temperatures?

Are they active in situ at, e.g., -12C?

What adaptive strategies do they have?

How long can viable organisms live at very low temperatures?

How can we sample material from beneath deep ocean vents?

What are the spectroscopic signatures of life in planetary atmospheres?

What are the limits of habitable environments, e.g., in P, T?

Is liquid water necessary for life?

What is the spectrum of environments in which life can originate?

How did protein families and subunits of cells originate?

What prebiotic processes are relevant to the origin of life?

What determines the limits to life?

How does the fossil record shed light on the limits to life?

What processes allow life in extreme environments?

What prebiotic conditions exist in extreme environments?