3

Detecting Extant Life

INTRODUCTION

After the Viking and Apollo missions of a quarter-century ago, biologists largely turned away from developing research programs designed specifically to look for life on another planetary body. Only in the last 6 years, with more insight from reconnaissance missions and the controversy over the martian meteorite ALH84001, has interest in extraterrestrial life detection—and sterilization of spacecraft—been renewed. This rising interest comes, in a sense, on the heels of a revolution in the field of microbiology, extraordinary progress in understanding the geobiological history of Earth, and detection of life in extreme environments. In the past decade alone, more than 1,500 new species of microorganisms have been discovered and genetically sequenced. In the next decade, it can be expected that phylogenetic trees will be redrawn and restructured and that genomics—the listing of an organism's entire genetic code—will become economical for all molecular microbial geologists to undertake. What remains to complete the picture regarding terrestrial life is to understand what microorganisms are doing, where and when they are doing it, and how these organisms and their metabolisms have evolved within Earth's environment over geologic time.

The detection of extremely low levels of microorganisms after spacecraft sterilization involves increasing refinement of laboratory techniques designed to detect known types of terrestrial organisms. It also requires research into the possibilities and consequences of failure to detect unknown or poorly known microorganisms that exist within Earth's environment. The detection of extant life on samples returned from another planet, or analyzed in situ, is a much less well-defined venture. It requires a set of assumptions about the fundamental nature of life that might exist on another planet. It also requires selection of techniques that will work well in difficult quarantine laboratory environments or in miniaturized and automated form on an extraterrestrial body.

This chapter summarizes the workshop session focused on in situ life detection. Whereas previous chapters and corresponding workshop sessions address more general considerations regarding what life might be like beyond Earth and what targets are most promising, this chapter and the associated workshop papers are technique based. The committee attempted, within the confines of a reasonable length to the workshop, to cover a broad spectrum of types of techniques and to characterize each technique in adequate depth to indicate its capabilities and limitations. For those reasons, the papers are not comprehensive in terms of addressing all possible techniques. The techniques selected include those that are very specific and powerful in their detection capability but are hard to generalize beyond terrestrial organisms because of their specificity (e.g., polymerase chain reaction



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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques 3 Detecting Extant Life INTRODUCTION After the Viking and Apollo missions of a quarter-century ago, biologists largely turned away from developing research programs designed specifically to look for life on another planetary body. Only in the last 6 years, with more insight from reconnaissance missions and the controversy over the martian meteorite ALH84001, has interest in extraterrestrial life detection—and sterilization of spacecraft—been renewed. This rising interest comes, in a sense, on the heels of a revolution in the field of microbiology, extraordinary progress in understanding the geobiological history of Earth, and detection of life in extreme environments. In the past decade alone, more than 1,500 new species of microorganisms have been discovered and genetically sequenced. In the next decade, it can be expected that phylogenetic trees will be redrawn and restructured and that genomics—the listing of an organism's entire genetic code—will become economical for all molecular microbial geologists to undertake. What remains to complete the picture regarding terrestrial life is to understand what microorganisms are doing, where and when they are doing it, and how these organisms and their metabolisms have evolved within Earth's environment over geologic time. The detection of extremely low levels of microorganisms after spacecraft sterilization involves increasing refinement of laboratory techniques designed to detect known types of terrestrial organisms. It also requires research into the possibilities and consequences of failure to detect unknown or poorly known microorganisms that exist within Earth's environment. The detection of extant life on samples returned from another planet, or analyzed in situ, is a much less well-defined venture. It requires a set of assumptions about the fundamental nature of life that might exist on another planet. It also requires selection of techniques that will work well in difficult quarantine laboratory environments or in miniaturized and automated form on an extraterrestrial body. This chapter summarizes the workshop session focused on in situ life detection. Whereas previous chapters and corresponding workshop sessions address more general considerations regarding what life might be like beyond Earth and what targets are most promising, this chapter and the associated workshop papers are technique based. The committee attempted, within the confines of a reasonable length to the workshop, to cover a broad spectrum of types of techniques and to characterize each technique in adequate depth to indicate its capabilities and limitations. For those reasons, the papers are not comprehensive in terms of addressing all possible techniques. The techniques selected include those that are very specific and powerful in their detection capability but are hard to generalize beyond terrestrial organisms because of their specificity (e.g., polymerase chain reaction

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques [PCR]). They also include more general approaches that could be useful in characterizing a broad range of life forms very different from those of Earth, but for which the interpretation could be ambiguous (e.g., time-of-flight mass spectroscopy). In this sense some of the papers could arguably have been included in the session on extinct life (e.g., see the Session 3 paper by Kirschvink on magnetic biomineralization), and indeed a corresponding paper on iron biomarkers is given in Session 4 (see the paper by Anbar). These papers have been split between the two different sessions, along with the papers on microbe-mineral interactions, to emphasize the utility of such techniques in the search for either extant or extinct life. The distinction between extant and extinct life detection becomes arbitrary for the most general techniques that rely on mineral biomarkers or other morphological characterizations. Most of the participants who presented techniques suitable for assessing the degree of sterilization also emphasized their application to extraterrestrial life detection. In large measure, this reflected the emphasis of the workshop on the search for life elsewhere. To correct this imbalance the committee prepared Table 5.2 (see Chapter 5), which lists techniques discussed at the workshop and gives their sensitivity (typically, when applied in terrestrial laboratories rather than extraterrestrial environments), limitations, and some indication of development needed. Finally, the committee's narratives in this chapter and in Chapter 4 do not attempt a detailed discussion of each technique presented in the sessions. In particular, it was not the purview of the committee to rank in some way the applicability of the techniques. The workshop discussions and the session papers represent a snapshot of the kinds of techniques and their capabilities available at a time when interest in finding life beyond Earth has reached a new crescendo. BIOMOLECULES AND MOLECULAR TECHNIQUES New technologies originating in the biomedical community will be available for use in detecting extant life on other planets. Testing these methods on Earth, where many biological communities can be studied with sophisticated laboratory setups, is crucial. The ambiguities associated with metabolic tests for extant life, demonstrated in the prolonged (some would say continuing) controversy over the Viking lander results, demand a broader suite of sensitive detection approaches. The evolution of molecular techniques is toward much higher specificity, in the sense that one can look at all the major components of cellular structures. At issue, of course, is how to generalize from the characteristics of these structures to those that might differ from the terrestrial. Imaging techniques, although less reliant on specific biochemical assumptions, are more subject to misinterpretation. To apply molecular techniques to the search for life elsewhere requires deciding how geocentric one must be. Must one look for a different set of biochemical molecules, perhaps based on compounds that are structured differently from those of terrestrial life? The Session 3 papers by Stahl; Meller and Branton; and Ruvkun, Finney, Gilbert, and Church present approaches that will work for life forms that use the basic biopolymers found in all terrestrial life—specifically, RNA and DNA. Stahl focuses on the use of molecular methods coupled with microscopic techniques, which provides a powerful combination of sensitivity and generality. The paper by Meller and Branton and that by Ruvkun, Finney, Gilbert, and Church offer two techniques capable of single-molecule detection that are miniaturizable for in situ deployment: nanopore technology and robotic PCR detection. An alternative imaging approach, described in the Session 3 paper by Jacobsen, allows one to image hydrated organic samples in an organism, thus potentially obtaining the structure of the ribosomes in the organism, and simultaneous structural and compositional data. Highly sensitive techniques for measuring a variety of organic molecules, such as proteins, complex lipids, and carbohydrates, are active areas of investigation. Although there are simple monomeric organic molecules that occur both in terrestrial biology and in abiotic organic phases of meteorites, the macromolecular nature of biologically produced versus abiotically produced molecules should be diagnostic. Cotter describes (see Session 3) improvements in time-of-flight (TOF) mass spectrometry, for which significant technological advances have been made. TOF mass spectrometers can now be miniaturized to allow in situ analysis of biomolecules on an extra-terrestrial surface. The versatility and sensitivity of this technique are high. Protein sequencing has been demonstrated in terrestrial laboratories using this technique.

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques More general biomolecular approaches to the search for extant extraterrestrial life involve looking for biomolecules or biological structures that do not exist on Earth. In his Session 1 paper, Benner argues the case for looking for biological systems that do not rely on our dual-biopolymer world of DNA and RNA as information carriers and proteins as structural or catalytic molecules. However, it is unclear how amplification of non-DNA or RNA information-carrying molecules could be accomplished or, alternatively, whether single-molecule techniques (e.g., involving nanopores or other nanotechnologies) can be sensitive to and diagnostic of a broad range of extraterrestrial biomolecules. As discussed in Chapter 1, analysis of smaller organic molecules (e.g., amino acids) can be broader but also far more subject to ambiguity in the interpretation of a biological or abiotic origin. Time-of-flight mass spectroscopy will be useful in analyzing whatever molecules are encountered in the mass range of sensitivity, but again interpretation in terms of a biological origin remains problematic. OTHER TECHNIQUES DISCUSSED DURING THE WORKSHOP An even broader approach eschews looking for biomolecules and instead looks for mineralogical traces of organisms through geochemical structures that have been altered by biological processes. Electron microscopy at the organism-mineral interface would be pursued on returned samples or, after an aggressive technology development, on candidate rocks at the landing site. More terrestrial data are required to fully understand how living organisms affect the geochemistry and morphology of mineralogic substrates. As they explain in their Session 3 paper, Barker and Banfield have characterized the microbe-mineral environment, and potential mineralogical biosignatures, in lithobiontic microbial communities. From these observations, they developed a model intended to be predictive of mineralogical and textural properties that could be indicative of biological processes. This work not only provides an important context for examining signatures of organisms in extraterrestrial samples, but also leads to specific protocols for sample preparation that avoid altering or destroying the evidence. These considerations for morphological detection of extant organisms should also be compared with the approaches to morphological characterization of extinct organisms discussed in Session 4 (see the papers by Cady, Moldowan, and D. McKay). Some terrestrial organisms produce their own minerals with uniquely biological properties. For example, the crystal structure and other structural or chemical aspects of biologically produced magnetite crystals can be diagnostic, as described in the paper by Kirschvink. He argues that a detailed and systematic comparative analysis of biogenic magnetite from terrestrial bacteria leads to the conclusion that the magnetite crystals in the SNC meteorite ALH84001, generally accepted to have come from Mars, are the signatures of martian biology. The conclusion itself is clearly controversial and led to a very vigorous workshop discussion. Kirschvink's conclusions highlighted an important point coming out of the workshop debate—the need for multiple techniques and multiple groups to analyze precisely the same portions of and phases in retrieved samples. Stable isotopic measurements are yet another approach to discerning the presence of life (or previous life) in a sample. Stable isotopic measurements of organic and inorganic compounds have been widely used on Earth for selecting critical biological reactions in complex ecosystems or determining when in Earth's history important biological mechanisms evolved. Fogel argues (see her paper in Session 3) that protein-chip technology provides a new, potentially miniaturizable approach to the isotopic detection of candidate biomaterials. SUMMARY OF METHODS FOR DETECTING EXTANT LIFE Given the presentations at the workshop and the committee's subsequent deliberations, there appear to be a variety of techniques currently available for the detection of viable microorganisms and, if organisms are present, for determining their biomass, growth rates, and metabolic and enzymatic activities. Most of these methods have been applied to both liquid and solid samples and thus could be useful in detecting viable cells from spacecraft and martian samples. Some of the imaging systems, including flow cytometry, measure total numbers of cells in a liquid medium. While the molecular and some of the biochemical methods are designed for detection of terrestrial organisms, other methods, including microscopy coupled with the use of macromolecule-specific fluorescent dyes, uptake of radio-labeled organic and inorganic compounds, microcalorimetry, and stable isotope analyses,

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques should be useful in detecting viable life from extraterrestrial environments that might have different biochemical characteristics. The most effective approach for detecting extant life is to use multiple techniques that can give some indication of growth and activity of specific taxonomic or physiological groups of microorganisms. Measuring Biomass The biomass, an index of the number of cells in a sample, is measured either directly by using imaging technologies or cultivation to quantitatively enumerate the cells in a sample or indirectly by using a biochemical proxy for the number of cells. The best biochemical methods can be used to approximate the number of cells in a sample and are very useful in detecting low numbers of “free-living” cells or cells attached to solid substrates. The current limitation for enumerating microorganisms in sediments by epifluorescence microscopy is approximately 100,000 cells per cubic centimeter.1 Lower numbers of cells can be detected using fatty acids as a proxy for biomass. In some cases it is advantageous to dislodge microbes from particles, sediment grains, and rocks in order to obtain quantitative biomass results. Microbes are dislodged from solid material by grinding the samples with a mortar and pestle and/or by using detergents and mild sonication.2,3 Other methods are available for isolating a single cell from a sample. These involve the use of micromanipulators and lasers to direct single viable cells into capillary tubes for subsequent culturing in defined media or for single-cell PCR analyses.4,5 So far, three methods have been applied to liquid samples, and there is no method reported for the removal of a single cell that may be attached to a solid substrate. There is a pressing need to develop methods for the detection in single cells of evidence of metabolic activity and of specific macromolecules, including an analysis of their chemical structure and isotopic signature. Direct Measurements of Biomass The following methods involve direct imaging of intact cells or their cultivation on specific media. The direct techniques include observations of general morphological characteristics of intact cells, identification and quantification of phylogenetic groups of microorganisms, and specific physiological biomarkers. Fluorescent dyes— specific for different macromolecules such as proteins, lipids, and nucleic acids —are routinely used to identify organisms in natural samples using epifluorescence microscopy. These techniques include the following: Light microscopy. This technique is generally limited to samples with more than than 105 organisms per milliliter or gram of sample. It is generally used in conjunction with various stains (Gram stain, lipid and protein stains, and so on) or to observe motility of viable cells.6 (Technique not discussed during the workshop.) Epifluorescence microscopy. Acridine orange, 4,6-diamidino-2-phenylindole (DAPI), and other dyes can be used for enumerating organisms with DNA, RNA, and protein. This technique can determine the presence of microorganisms on surfaces of solid substrates, in soil, on rocks, and in biofilms. It also allows for the concentration of low levels of microorganisms from liquid samples by filtration. It can be used to enumerate bacteriophages and other viruses (from 30- to more than 50-nm diameter).7−9 (Technique not discussed during the workshop.) Autofluorescence with epifluorescence microscopy. This technique can be used for enumerating organisms with autofluorescing compounds such as flavins (methanogens) and chlorophyll.10,11 (Technique not discussed during the workshop.) Image-analyzed fluorescence microscopy. This technique gives the numbers, size, and distribution of microorganisms. 12 (Technique not discussed during the workshop.) Electron microscopy. Scanning electron microscopy (SEM) with electron diffraction (EDX) can be used to identify microorganisms on surfaces, along with elemental analyses of organisms and the surrounding environment. Environmental SEM allows observation of samples without the use of fixatives. Transmission electron microscopy (TEM) is most important when using ultrathin sections of microorganisms because internal membranes, cell walls, and ribosomes can be readily observed. These techniques can be combined

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques with methods that remove microbes from solid materials such as soils and rocks, where they can be concentrated, fixed, and prepared for thin sections.13 (Technique not discussed during the workshop.) Immunofluorescence (FA). This involves specific antibodies for specific physiological groups of bacteria such as nitrifiers, methanogens, and methylotrophs, or specific protein antigens such as RuBPCase and nitrate reductase. It can also be adapted for the general detection of proteins or cellular membranes from any organism.14−16 (See workshop presentation by Fogel in Session 3.) Coulter counter. Use of this device has limited application with natural populations of bacteria, although new laser models might be used to measure organisms that fluoresce or that can be stained to fluoresce. It is useful for determining growth rates of pure cultures of bacteria and phytoplankton and for measuring protozoan grazing rates of bacteria. It requires large volumes of water.17 (Technique not discussed during the workshop.) RNA or DNA probes. These are used to identify specific species of bacteria or genes using epifluorescence microscopy (fluorescent in situ hybridization; FISH), dot blots, and quantitative PCR. General 16S rRNA fluorescent probes of unique sequences can differentiate eubacteria, eukaryotes, and archaea. Additional probes are also available for detecting specific taxonomic groups of microbes. The FISH technique is dependent on cells having sufficient amounts of rRNA and hence a full complement of ribosomes.18 (See workshop presentation by Stahl in Session 3.) Quantitative PCR. This technique quantifies microbial communities at cluster to species levels; it could be developed into a useful procedure to enumerate specific groups of organisms that are not amenable to FISH because of low amounts of rRNA.19,20 (Technique not discussed during the workshop.) Analytical flow cytometry. This is a technique for rapidly characterizing, quantifying, and sorting particles based on simultaneous, multiple measurements of cellular light scatter and fluorescence. Recent experiments couple flow cytometry with fluorescence and fluorescent antibodies or fluorescently labeled DNA or RNA probes. Large volumes of water are required. 21−23 (Technique not discussed during the workshop.) Plate counts and “most probable number” (MPN). These methods are based on determining the number of viable bacteria capable of growing on specific media. They are useful for isolating specific physiological groups of bacteria or for estimating the numbers of coliforms and other bacteria important to public health. Most environmental microorganisms have not been cultured, so culturing methods have limited application in characterizing the diversity and biomass of microbial communities from most environments.24,25 (Technique not discussed during the workshop.) Laser-scanning confocal microscopy (LSCM). This approach enables two- and three-dimensional images of environmental samples to be obtained without fixation.26,27 (Technique not discussed during the workshop.) Indirect Measurements of Biomass All organisms have specific biochemical characteristics that serve as biosignatures. Some of these characteristics can also be used to estimate the number of cells present in a sample. Lipids are the most versatile of these biosignatures in that they can be used to estimate the number of cells in a sample, provide taxonomic information, and indicate the physiological condition of the microbial community (starved, stressed, or dormant). All of the indirect measurements can be used to detect very low levels of cells and have the added feature that these compounds can be extracted and concentrated from solid samples. These techniques include the following: Adenosine triphosphate (ATP). This is a useful indicator of biomass, although the ATP content of cells can vary depending on their metabolic state. The procedures are easy and do not require expensive equipment. Actively growing cells have a total carbon to carbon in ATP ratio of 1:273.28,29 (See the paper by Soffen in Session 1.) DNA and RNA. These have limited applications as biomass indicators and depend very much on the size and metabolic state of cells. Moreover, the RNA content of cells can vary greatly in different species. The genome size of different phylogenetic groups of microorganisms can vary by a factor of 3. Quantitative

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques procedures are tedious, particularly if the sample contains low levels of microorganisms.30,31 (Technique not discussed during the workshop.) Muramic acid. This specific biosignature for bacteria is best applied when measuring the biomass of attached bacteria or bacteria associated with animal guts and other environments having a complex multikingdom flora. Quantitative correlation between muramic acid and bacterial biomass depends on the absence of significant numbers of Gram-positive bacteria and a good determination of the average size (volume) of the bacteria in the population. The quantitative procedures for muramic acid assay are tedious.32,33 (Technique not discussed during the workshop.) Lipopolysaccharide. The Limulus assay is specific for Gram-negative bacteria and very sensitive (detects <10 cells per milliliter). The method has wide applications in medical microbiology, particularly for detecting bacteria in urinary tract infection. Too much contaminating lipopolysaccharide in aquatic environments prevents this method from being practical. 34 (Technique not discussed during the workshop.) Pigments. Chlorophyll, phycocyanin, and other pigments are generally measured using fluorescent imaging systems.35 (Technique not discussed during the workshop.) Lipids. Ester- and ether-linked fatty acids are used for differentiating eubacteria from archaebacteria. Also, unusual lipids, such as long-chain polyunsaturated fatty acids, esoteric branched chains, and so forth, can be diagnostic for specific groups of bacteria and their metabolic state. Some investigators have made the assumption that lipids represent some fixed percentage of cell carbon (usually 1 percent of total cell carbon) so as to use lipids as biomass indicators. A fatty acid database is available for many bacteria, and fatty acid profiles are used to identify specific groups of bacteria, particularly those having public health significance.36,37 (Technique not discussed during the workshop.) Other biochemical parameters. Esters, specific enzymes, and combinations of compounds can also be used as indirect indicators of biomass. (Technique not discussed during the workshop.) Determining Growth Rates The ultimate test of the viability of cells is their ability to grow and divide. All of the methods described below can be applied to natural populations of microorganisms. The direct methods rely on microscopic observations of dividing cells and measurements of increasing numbers of cells in growth chambers or on glass slides in situ. Radio-labeled substrates are used to make indirect measurements of growth rates based on the rate of DNA, RNA, and protein synthesis. These methods are very sensitive and measure growth rates in environmental samples containing low number of cells. Recently, the combination of molecular methods with microautoradiography has proven useful in estimating the growth rates of specific taxonomic groups of microorganisms. At present, there are no methods for measuring the growth rates of microbial communities or specific taxonomic groups of micro-organisms in situ. Direct Measurements of Growth Rates The direct methods for estimating microbial growth rates are not particularly quantitative but can provide some information about the viability of a community and its potential for growth. Laboratory culture methods rarely provide the conditions necessary for growth of all the different taxonomic groups of microorganisms within a community. These techniques include the following: Frequency of dividing cells. This involves correlation of the number of dividing cells in a natural population of bacteria with growth rate, diffusion plates with synchronous cultures, time experiments that prevent separation of dividing cells, and so forth. Evidence of cell division (chains of cells, fruiting bodies, budding cells) is a first-order observation for the presence of viable cells in a sample.38,39 (Technique not discussed during the workshop.) Microautoradiography. This technique is used mostly to differentiate active from nonactive cells but has

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques been used to estimate the rate of division of bacteria in situ, and the activity of specific physiological groups.40,41 (Technique not discussed during the workshop.) Laboratory culture methods. These include chemostats, growth chambers, growth on filters and glass slides, and the formation of microcolonies on solid substrates. These methods have been useful in obtaining growth rates for microbes and microbial communities from oligotrophic environments. Other culture methods, such as the ability to grow in liquid nutrient and solid nutrient media, tissue culture, or animal hosts, are best suited for microorganisms that have complex nutrient requirements or are animal pathogens. Culturing methods including inoculation of samples into suitable hosts should be applied to martian samples for detection of viable microorganisms including animal pathogens.42−44 (See the paper by Soffen in Session 1.) Indirect Measurements of Growth Rates Most of these methods are widely used for measuring growth rates of heterotrophic and chemoautotrophic organisms, and organisms in aquatic environments. Some of these methods have been adapted for estimating microbial growth rates in sediments, biofilms, and detrital particles. The use of tritiated organic substrates allows for the measurement of uptake rates by low numbers of microorganisms (< 104 cells per milliliter) or with microbial communities having slow growth rates (less than one doubling per day). These techniques include the following: [3H]-Thymidine and/or[3H]-adenine uptake. This method is used to determine the rates of DNA and RNA synthesis and to correlate them with growth rates. It is used primarily for measuring growth rates of heterotrophic bacteria and is limited to microorganisms that are capable of assimilating thymidine or adenine. Thymidine uptake is very specific for heterotrophic bacteria, and most of the thymidine is incorporated into DNA. Adenine is assimilated by some phototrophic bacteria and eukaryotes; adenine is incorporated into both DNA and RNA. RNA content has been shown to vary considerably in bacteria from aquatic environments and thus is not useful for growth rate determinations. These methods can measure growth rates in samples with relatively low numbers of microorganisms (103 to 104 per milliliter).45−48 (Technique not discussed during the workshop.) [14C]- and [3H]-amino acid and [14C]-CO2 uptake. This approach measures the rate of incorporation into macromolecules and calculates a micro rate of increase in proteins, nucleic acids, lipids, and so on. The rate of [3H]-leucine incorporation into proteins as a proxy for growth rates has been shown to correlate well with growth rates determined by uptake of [3H]-thymidine into DNA.49,50 (Technique not discussed during the workshop.) Radio-phosphorus uptake. This approach measures [32P]-phosphate incorporation into phospholipids and nucleic acids.51 (Technique not discussed during the workshop.) DNA measurements. These involve quantitative extraction of DNA followed by measurement of DNA stained with DAPI (or related dyes) using a fluorometer. They are used in light and dark bottle in situ experiments to measure the growth rate of photosynthetic and chemoautotrophic organisms. 52 (Technique not discussed during the workshop.) Measuring Metabolic and Enzymatic Activity Many methods have been developed that estimate the rates of specific metabolic reactions in microbial communities from environmental samples or identify the metabolic potential of these communities. Since most of these methods require manipulation of the environmental sample, such as the addition of radio-labeled carbon or energy sources or substrates for specific enzymes, it is very important to re-create as many of the in situ conditions as possible during the incubation period. Other methods, including the use of microelectrodes and micro-calorimetry, can be performed in situ. Some of the molecular methods currently available or in the developmental stages allow for determination of specific metabolic activities associated with specific taxonomic groups of

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques microorganisms along with identification of the specific genes being transcribed in situ by microbial communities. The combination of molecular methods and stable isotope analyses of membrane lipids has proven to be extremely useful for identifying the taxonomic groups of microorganisms involved in specific metabolic activities such as anaerobic methane oxidation in marine sediments.53 Metabolic Activity The identification of specific metabolic activity in environmental samples is particularly useful in sediments and biofilms that have steep spatial gradients of specific metabolic groups of microorganisms or in environments that are dominated by chemoautotrophic microorganisms. These activity measurements can be divided into those that measure rates of activity and those that identify metabolic groups involved in tranformation of various inorganic nutrients (e.g., those involved in nitrogen, sulfur, and metal cycles). Microelectrodes and other sensors are used in situ to measure various chemical species, temperature, pH, and Eh in submicron gradients of sediments and biofilms and may have some utility in searching for evidence of active life on Mars. Relevant techniques include the following: Heterotrophic potential. 14C- and 3H-labeled organic compounds are used in different concentrations along with application of Michaelis-Menten kinetic analyses. Uptake by natural populations is assumed to follow first-order kinetics. This technique is particularly useful with sediment or soil samples in which the rate of [14C]-CO2 evolution can indicate respiration rates of microbial communities. 54−56 (Technique not discussed during the workshop.) Radio-carbon uptake. This technique measures [14C]-CO2 uptake by photosynthetic and chemoautotrophic bacteria.57 (See the paper by Soffen in Session 1.) Isotopes for measuring specific metabolic activities. The uptake, oxidation, and reduction of a variety of 14C- and 3H-labeled carbon and hydrogen sources, isotopes of metals (54Mn), phosphorus (32P), and sulfur (35S) are measured. These methods are particularly useful for measuring rates of methane production and consumption, CO production and consumption, hydrogen oxidation, and incorporation of phosphorus into nucleic acids and sulfur into S-amino acids.58−62 (See the paper by Soffen in Session 1.) 35S-labeled energy sources. Uptake is used to measure the activity of sulfur-oxidizing and sulfur-reducing bacteria. This technique is very useful in submarine hydrothermal vent and salt marsh environments.63,64 (Technique not discussed during the workshop.) Respiratory activity. The respiratory-activity method is based on the reduction of dyes, including cyanoditolyl tetrazolium chloride (CTC).65−67 (Technique not discussed during the workshop.) O2, N2, and sulfur uptake and consumption. This involves in situ measurements with microelectrode probes and respirometer experiments. Microelectrodes can be used to detect micromolar levels of O2, nitrogen, and sulfur compounds (NO2−, N2O, H2S) through micrometer Eh and pH gradients in sediments, biofilms, and rocks.68 (Technique not discussed during the workshop.) Microcalorimetry. Since all biological processes are accompanied by heat production, it follows that all heat evolved during metabolism and growth is equal to the change in enthalpy. Enthalpy changes can be measured by microcalorimetry. This method has been used to detect evidence of active life in sediments and soils.69,70 (Technique not discussed during the workshop.) Electron transport system. This approach measures the reduction of dyes by natural populations of bacteria.71 (Technique not discussed during the workshop.) Microbial fractionation of stable isotopes of nitrogen, sulfur, and carbon. These analyses are particularly useful for nitrogen- and sulfur-cycle reactions and the production and consumption of methane. They can be used to follow carbon, nitrogen, and sulfur through food chains. It is assumed that extraterrestrial life would also fractionate these elements.72−74 (Technique not discussed during the workshop.)

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques Enzymatic Activity The activity of most enzymes can be measured in environmental samples if the sample contains sufficient levels of active enzymes. The methods that have been developed in microbial ecology focus on enzymes that indicate specific metabolic activity such as nitrogen cycle reactions and the rate of degradation of macromolecular organic compounds (e.g., proteins and carbohydrates) requiring enzymatic hydrolysis into soluble compounds that can be transported into cells. The new methods that utilize soluble fluorogenic compounds as a proxy for macromolecules are very sensitive and can detect low levels of extracellular hydrolases in environmental samples. These techniques include the following: Nitrogen-cycle reactions. These can be used in a number of different ways, including (1) acetylene reduction as a measure of nitrogen fixation, (2) acetylene block method for measuring denitrification, and (3) 15N uptake as a measure of both dissimilatory and assimilatory reactions. 75,76 (Technique not discussed during the workshop.) Calvin-Bensen cycle enzymes. These indicate phototrophy and/or chemolithotrophy and can be coupled with fluorescent antibody methods.77 (Technique not discussed during the workshop.) Exoenzyme activity. This is used frequently in sediments and biofilm samples as an indication of potential microbial heterotrophic activity. A number of new enzyme assays appear to be very specific and are currently being widely applied to studies of marine sediments. Probably the most important breakthrough is in the use of soluble fluorescent artificial substrates that allow measurement of rates of macromolecule degradation in situ (cellulose, lignin, chitin, protein, lipids, and organic phosphorus and sulfur compounds).78−81 (Technique not discussed during the workshop.) Molecular Methods At the present time, molecular methods are being developed to determine some specific genes that are being transcribed in situ, and new methods are being developed for determining the genes being transcribed by microbial communities in situ. These techniques include the following: mRNA analyses. Specific mRNA synthesized is indicative of specific activity expressed (e.g., mRNA for DNA polymerase indicative of cell replication, nif-mRNA indicative of nitrogen fixation). (See the paper by Stahl in Session 3.) Downstream sequencing. Sequencing downstream from 16S rRNA genes is used to help infer some physiological and metabolic potential and to clone and express unknown genes in Escherichia coli or other hosts. (Technique not discussed during the workshop.) Gene sequencing. This technique is used to amplify specific functional genes from natural populations, sequence, and construct trees. It works well if there is a large enough sequence database to infer specific phylogenetic groups of organisms.82 (Technique not discussed during the workshop.) Environmental genomics. This technique is under development and will elucidate the metabolic potential of organisms within an ecosystem and the genes being transcribed in situ. (See the paper by Stahl in Session 3.) Metabolic State The metabolic state can indicate whether in situ microbial communities or pure cultures of bacteria are actively growing, slowly growing, or not growing. Methods for assessing metabolic state can also indicate whether organisms are showing evidence of stress and other physiological states that are under genetic control and may or may not involve a dense population of cells (quorum sensing). These can be estimated by a variety of methods, including the following:

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Signs of Life: A Report Based on the April 2000 Workshop on Life Detection Techniques Energy charge. The ratio of ATP to adenosine diphosphate (ADP) and adenosine monophosphate (AMP) can be used to determine whether natural populations of microbes are active or dormant.83,84 (Technique not discussed during the workshop.) Ability of bacteria to divide. This approach exploits the use of antibiotics such as nalidixic acid, which allows bacteria to continue to grow but not to divide. 85,86 (Technique not discussed during the workshop.) Cyclic AMP and lactones. These serve as an index of primary or secondary metabolism or an indication of quorum sensing.87,88 (Technique not discussed during the workshop.) Bacterial size. Does small size indicate starvation or the presence of active oligotrophic microbial communities? The lower size limit for oligotrophic aquatic microorganisms is approximately 200 to 400 nm in diameter).89 (Technique not discussed during the workshop.) SUMMARY CONSIDERATIONS A combination of sensitive techniques is required to provide sample selection, compositional and structural determination at the molecular level, and identification of biogenic structures on the microscopic (but supra-molecular) scale. Mass spectroscopy is among the most robust and sensitive of molecular techniques and is likely to be on any life detection package. It has to be coupled to other molecular techniques to ensure that identification of specific compounds is possible from the sample mix. However, mass spectroscopy by itself is powerful for sample selection, in no small measure because it can be readily miniaturized. Imaging techniques, although potentially more powerful because of their generality, yield results that may not be definitive and could lead to prolonged debate regarding the biogenicity of particular structures. 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