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Microbial Diversity and the Biosphere

Norman R. Pace
Departments of Plant and Microbial Biology and Molecular and Cell Biology, University of California, Berkeley, CA (Current address: Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309-0347)

Introduction

Microorganisms occupy a peculiar place in the human view of life. They receive little attention in our general texts of biology. They are largely ignored by most professional biologists and are virtually unknown to the public except in the contexts of disease and rot. Yet the workings of the biosphere depend absolutely on the activities of the microbial world (Madigan and others 1996). And a large bulk of global biomass is microbial (Whitman and others 1998). Our texts articulate biodiversity in terms of large organisms: insects usually top the count of species. Yet if we squeeze out any insect and examine its contents under the microscope, we find hundreds or thousands of distinct and unidentified microbial species. A handful of soil contains billions of microorganisms, of so many types that accurate numbers remain unknown. At most only a few of these microorganisms would be known to us; only about 5,000 noneukaryotic organisms have been formally described (Bull and others 1992) in contrast with the half-million described insect species. We know little about microbial biology, a part of biology that looms large in the sustenance of life on this planet.

The reason for our poor understanding of the microbial world lies in the fact that microorganisms are tiny, individually invisible to the eye. The mere existence of microbial life was recognized only relatively recently in history, about 300 years ago, with Leeuwenhoek's invention of the microscope. Even under the microscope, however, the simple structures of most microorganisms, usually nondescript rods and spheres, prevented their classification by morphology, through

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which large organisms had always been related to one another. It was not until the late 19th century and the development of pure-culture techniques that microorganisms could be studied as individual types and characterized to some extent, mainly by nutritional criteria. However, the pure-culture approach to the study of the microbial world seriously constrained the view of microbial diversity because most microorganisms defy cultivation by standard methods. Moreover, the morphological and nutritional criteria used to describe microorganisms failed to provide a natural taxonomy, ordered according to evolutionary relationships. Molecular tools and a perspective based on gene sequences are now alleviating these constraints to some extent. Even the early results are changing our perception of microbial diversity.

A Sequence-Based Map of Biodiversity

Before the development of sequence-based methods, it was impossible to know the evolutionary relationships connecting all of life and thereby to draw a universal evolutionary tree. Whittaker, in 1969, just as the molecular methods began to develop, summarized evolutionary thought in the context of the “five kingdoms” of life: animals, plants, fungi, protists (“protozoa”), and monera (bacteria) (Whittaker 1969). There also was thought to be a higher, seemingly more fundamental taxonomic distinction between eukaryotes, organisms that contain nuclear membranes, and prokaryotes, predecessors of eukaryotes that lack nuclear membranes (Chatton 1937). Those two categories were considered independent and coherent groups. The main evolutionary diversity of life on Earth, four of the five traditional taxonomic kingdoms, was believed to lie among the eukaryotes, particularly the multicellular forms. These still-pervasive notions had never been tested, however, and they proved to be incorrect.

The breakthrough that called previous beliefs into question and brought order to microbial, indeed biological, diversity emerged with the determination of molecular sequences and the concept that sequences could be used to relate organisms (Schwartz and Dayhoff 1978; Zuckerkandl and Pauling 1965). The incisive formulation was reached by Carl Woese, who, by comparing ribosomal RNA (rRNA) sequences, established a molecular sequence-based phylogenetic tree that could be used to relate all organisms and reconstruct the history of life (Woese 1987; Woese and Fox 1977). Woese articulated the now-recognized three primary lines of evolutionary descent, termed “urkingdoms” or “domains”: Eucarya (eukaryotes), Bacteria (initially called eubacteria), and Archaea (initially called archaebacteria) (Woese and others 1990).

Figure 1 is a current phylogenetic tree based on small-subunit (SSU) rRNA sequences of the organisms represented. The construction of such a tree is conceptually simple (Swofford and others 1996). Pairs of rRNA sequences from different organisms are aligned, and the differences are counted and considered to be some measure of “evolutionary distance” between the organisms. There is no consideration of the passage of time, only of change in nucleotide sequence. Pairwise differences between many organisms can be used to infer phylogenetic trees, maps that represent the evolutionary paths leading to the modern-day sequences.

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Figure 1
Universal phylogenetic tree based on small-subunit ribosomal RNA sequences. Sixty-four
rRNA sequences representative of all known phylogenetic domains were aligned, and a tree
was produced with FASTDNAML (Barns and others 1996; Maidak and others 1997).
That tree was modified to the composite one shown by trimming lineages and adjusting
branchpoints to incorporate results of other analyses. The scale bar corresponds to 0.1 change
per nucleotide. (From Pace 1997, reprinted with permission.)

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The tree in figure 1 is largely congruent with trees made by using any molecule in the nucleic acid-based, information-processing system of cells. But phylogenetic trees based on metabolic genes, those involved in manipulation of small molecules and in interaction with the environment, commonly do not concur with the rRNA-based version; see Doolittle and Brown (1994), Palmer (1997), and Woese (1998) for reviews and discussions of phylogenetic results with different molecules. Incongruities in phylogenetic trees made with different molecules can reflect lateral transfers or even the intermixing of genomes in the course of evolution. Some metabolic archaeal genes, for instance, appear much more highly related to specific bacterial versions than to their eucaryal homologues; other archaeal genes seem decidedly eukaryotic; still others are unique. Nonetheless, recently determined sequences of archaeal genomes show clearly that the evolutionary lineage of Archaea is independent of both Eucarya and Bacteria (Bult and others 1996; Smith and others 1997).

Interpreting the Molecular Tree of Life.

“Evolutionary distance” in the type of phylogenetic tree shown in figure 1, the extent of sequence change, is read along line segments. The tree can be considered a rough map of the evolution of the genetic core of the cellular lineages that led to the modern organisms (sequences) included in the tree. The time of occurrence of evolutionary events cannot be extracted reliably from phylogenetic trees, despite common attempts to do so. Time cannot be accurately correlated with sequence change, because the evolutionary clock is not constant in different lineages (Woese 1987). This disparity is evidenced in figure 1 by the fact that lines leading to the different reference organisms are not all the same length; these different lineages have experienced different extents of sequence change. Nonetheless, the order of occurrence of branchings in the trees can be interpreted as a genealogy, and intriguing insights into the evolution of cells are emerging.

A sobering aspect of large-scale phylogenetic trees like that shown in figure 1 is the graphic recognition that most of our legacy in biological science, historically based on large organisms, has focused on a narrow slice of biological diversity. Thus, we see that animals (represented in figure 1 by Homo), plants (Zea), and fungi (Coprinus) constitute small and peripheral branches of even eukaryotic cellular diversity. If the animals, plants, and fungi are taken to make up taxonomic “kingdoms”, we must recognize as kingdoms at least a dozen other eukaryotic groups, all microbial, with at least as much independent evolutionary history as that which separates the three traditional eukaryotic kingdoms. The taxonomic termkingdom has no molecular definition. I use it to indicate main lines of radiation in the particular domain; 14 such “kingdom-level” lines are associated with the eucaryal line of descent in figure 1 (see also Sogin 1994).

The rRNA and other molecular data solidly confirm the notion stemming from the last century that the major organelles of eukaryotes—mitochondria and chloroplasts—are derived from bacterial symbionts that have undergone specialization through coevolution with the host cell. Sequence comparisons establish mitochondria as representatives of Proteobacteria (the group in figure 1 including Es-

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cherichia and Agrobacterium) and chloroplasts as derived from cyanobacteria (Synechococcus and Gloeobacter in figure 1) (Sapp 1994). Thus, all respiratory and photosynthetic capacity of eukaryotic cells was obtained from bacterial symbionts;the “endosymbiont hypothesis” for the origin of organelles is no longer hypothesis but well-grounded fact. The nuclear component of the modern eukaroytic cell did not derive from one of the other two lineages, however. The rRNA and other molecular trees show decisively that the eukaryotic nuclearline of descent extends as deeply into the history of life as do the bacterial and archaeal lineages. The mitochondrion and chloroplast came in relatively late. This late evolution is evidenced by the fact that mitochondria and chloroplasts diverged from free-living organisms that branched peripherally in molecular trees. Moreover, the most deeply divergent eukaryotes lack even mitochondria (Cavalier-Smith 1993). These latter organisms, little studied but sometimes troublesome creatures—such as Giardia, Trichomonas, and Vairimorpha—nonetheless contain at least a few bacterium-type genes (Bui and others 1996; Germot and others 1996; Roger and others 1996). That might be evidence of an earlier mitochondrial symbiosis with' Eucarya that was lost (Palmer 1997) or perhaps other symbiotic or gene-transfer events between the evolutionary domains.

The root of the universal tree in figure 1, the point of origin of the modern lineages, cannot be established by using sequences of only one type of molecule. However, recent phylogenetic studies of gene families that originated before the last common ancestor of the three domains have positioned the root of the universal tree deep on the bacterial line (Doolittle and Brown 1994). Therefore, Eucarya and Archaea had a common history that excluded the descendants of the bacterial line. The period of evolutionary history shared by Eucarya and Archaea was an important time in the evolution of cells during which the refinement of the primordial information-processing mechanisms occurred. Thus, modern representatives of Eucarya and Archaea share many properties that differ from bacterial cells in fundamental ways. One example of similarities and differences is in the nature of the transcription machinery. The RNA polymerases of Eucarya and Archaea resemble each other in subunit composition and sequence far more than either resembles the bacterial type of polymerase. Moreover, whereas all bacterial cells use sigma factors to regulate the initiation of transcription, eucaryal and archaeal cells use TATA-binding proteins (Marsh and others 1994; Rowlands and others 1994).

The Metabolic Diversity of Life

The molecular-phylogenetic perspective, as depicted in figure 1, is a reference framework within which to describe microbial diversity; the sequences of genes can be used to identify organisms. That is an important concept for microbial biology. It is not possible to describe microorganisms as is traditional with large organisms, through their morphological properties. To be sure, some microorganisms are intricate and beautiful under the microscope, but mainly they are relatively unfeatured at the resolution of routine microscopy. Therefore, to distinguish different types of microorganisms, early microbiologists turned to

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metabolic properties of the organisms, such as their sources of carbon, nitrogen, and energy. Microbial taxonomy accumulated as anecdotal descriptions of metabolically and morphologically distinct types of organisms that were essentially unrelatable. Molecular phylogeny now provides a framework within which we can relate organisms objectively and through which we can interpret the evolutionary flow of the metabolic machineries that constitute microbial diversity.

Laboratory studies of microbial metabolism have focused mainly on such organisms as Escherichia coli and Bacillus subtilis. In the broad sense, such organisms metabolize much as animals do; we are all “organotrophs,” using reduced organic compounds for energy and carbon. Organotrophy is not the prevalent form of metabolism in the environment, however. Autotrophic metabolism—fixation of CO2 to reduced organic compounds—must necessarily contribute to a greater biomass than the organotrophic metabolism that it supports (a principle long appreciated by ecologists). Energy for fixing CO2 is gathered in two ways: “phototrophy” (photosynthesis) and “lithotrophy” (coupling the oxidation of reduced inorganic compounds—such as H2, H2S, and ferrous iron—to the reduction of a chemical oxidant, a terminal electron acceptor, such as oxygen, nitrate, sulfate, sulfur, and CO2). Thus, metabolic diversity can be generalized in terms of organ otroph or autotroph, phototroph or lithotroph, and the nature of the electron donor and acceptor.

The phylogenetic patterns of types of carbon and energy metabolism among different organisms do not necessarily follow the evolutionary pattern of rRNA (figure 1). Presumably, that is because of past lateral transfers of metabolic genes and larger-scale symbiotic fusions. Nonetheless, domain-level tendencies might speak to the ancestral nature of the three domains of life (Kandler 1993). The perspective, here, is limited mainly to Archaea and Bacteria. Such broad generalities cannot yet be assessed for the Eucarya, because so little is known about the metabolic breadth of the domain and the properties of the most deeply divergent lineages. There is considerable information about one pole of eukaryotic diversity—that represented by animals, plants, and fungi. We know little about the other pole—the amitochondriate organisms that spun off the main eucaryal line early in evolution (Sogin 1994). The known instances of such lineages—represented by Trichomonas, Giardia, and Vairimorpha in figure 1—are primarily pathogens. Pathogenicity in humans is a rare trait among the rest of eukaryotes and bacteria, and no archaeal pathogen is known. That correlation might indicate that nonpathogenic, deeply divergent eukaryotes are abundant in the environment but not yet detected. They should be sought in anaerobic ecosystems, possibly coupled metabolically to other organisms. A driving theme of the eucaryal line seems to be the establishment of physical symbiosis with other organisms. Beyond that, the general metabolism of the rudimentary eukaryotic cell seems simple and based on fermentative organotrophy. By virtue of symbiotic partners, however, eukaryotes are able to take on phototrophic or lithotrophic lifestyles and to respire using the electron-acceptor oxygen (Smith and Douglas 1987).

Symbiotic microorganisms commonly confer the lithotrophic way of life even on animals, although this was only recently recognized. The 2-m-long submarine vent tubeworm Riftia pachyptila, for instance, lives in the vicinity of sea-floor hy-

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drothermal vents and metabolizes H2S and CO2 by means of sulfide-oxidizing, CO2-fixing, bacterial symbionts (Tunnicliffe 1992). This invertebrate and metabolically similar ones might contribute substantially to primary productivity in the ocean (Kates and others 1993; Lutz and others 1994). It is not necessary to go to (from our perspective) unusual places, such as ocean-floor vents, to encounter equally fascinating H2S-dependent eukaryotes (Fenchel and Finlay 1995). Under foot at the ocean beach, for example, microbial respiration of seawater sulfate creates an H2S-rich ecosystem populated by little-known creatures, such as Kentrophoros, a flat, gulletless ciliate that under the microscope appears fuzzy because it cultivates a crop of sulfide-oxidizing bacteria on its outer surface (Fenchel and Finlay 1989); the bacteria are ingested by endocytosis and thereby provide nutrition for Kentrophoros. In other anaerobic environments, methanogens, members of Archaea, live intracellularly with eukaryotes and serve as metabolic hydrogen sinks (Embley and Finlay 1994). Still other symbioses based on inorganic energy sources are all around us and are little explored for their diversity of microbial life (Fenchel and Finlay 1995).

Many lithotrophic but comparatively few organotrophic representatives of Archaea have been obtained in pure culture (Kates and others 1993). There are primarily two metabolic themes, both relying on the use of hydrogen as a main energy source. Among the known members of Euryarchaeota, one of the two archaeal kingdoms known through cultivated organisms, the main electron acceptors are CO2 and the product CH4, “natural gas.” Most of the CH4 encountered in the outer few kilometers of Earth's crust or on the surface is determined by isotopic analysis to be the product of methanogenic archaea communities, past and present. Such organisms probably constitute a huge component of global biomass. They certainly offer an inexhaustible source of renewable energy to humankind.

The general metabolic theme of the other established kingdom of Archaea, Crenarchaeota, also is the oxidation of H2, but with a sulfur compound as the terminal electron acceptor. All the cultivated representatives of Crenarchaeota also are thermophiles. Consequently, such organisms have been referred to as thermoacidophilic or hyperthermophilic archaeons; some grow at the highest known temperatures for life, up to 113°C in the case of Pyrolobus fumaris (Stetter 1995). These crenarchaeotes might seem bizarre to us, capable as they are of thriving at temperatures sometimes above the usual boiling point of water on a diet of H2, CO2, and S and exhaling H2S. Yet, in terms of the molecular structures of the basic cellular machineries, these creatures resemble eukaryotes far more closely than either resembles our gut bacterium E. coli (Marsh and others 1994).

The metabolic diversity of microorganisms is usually couched in terms of the use of complex organic compounds. From that standpoint and on the basis of cultivated organisms, metabolic diversity seems to have flowered mainly among the Bacteria. Even here, however, reliance on organic nutrients probably was not ancestral. The most deeply branching of the cultured bacterial lineages, represented by Aquifex and Thermotoga in figure 1, are basically lithotrophs that use H2 as an energy source and such electron acceptors as sulfur compounds (Ther-

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motoga) or low concentrations of O2 (Aquifex) (Pitulle and others 1994). Cultivated organisms from these deeply branching bacterial lineages also are all thermophilic and thus share two important physiological attributes with the deeply branching and slowly evolving Archaea; a H2-based energy source and growth at high temperatures. That coincidence suggests that the last common ancestor of all life also metabolized H2 for energy at high temperatures; this inference is consistent with current notions regarding the origin of life—that it came to be in the geothermal setting at high temperature (Pace 1991).

Chlorophyll-based photosynthesis was a bacterial invention. It seems to have appeared well after the establishment of the bacterial line of descent at or before the divergence of the line in figure 1 leading to Chloroflexus, a photosynthetic genus (Pierson 1993), and after the deeper divergences, such as those leading to Aquifex, and Thermotoga, which are not known to have photosynthetic representatives. Most bacterial photosynthesis is anaerobic, however. Oxygenic photosynthesis, the water-based photosynthetic mechanism that produces the powerful electron acceptor O2, arose only in the kingdom-level lineage of cyanobacteria. This invention changed the surface of Earth profoundly and is conventionally thought to be the basis, directly or indirectly, of most present-day biomass.

Anaerobic photosynthesis is widely distributed in the late-branching bacterial kingdoms. The more ancient theme of lithotrophy, metabolism of inorganic compounds, is also widely distributed phylogenetically, intermixed with organotrophic organisms. The pattern suggests that organotrophy arose many times from otherwise photosynthetic or lithotrophic organisms. Indeed, many instances of bacteria can switch between these modes of nutrition, carrying out photosynthesis in the light and lithotrophy or organotrophy in the dark. Particularly among bacteria, the type of energy metabolism seems highly volatile in evolution; bacteria that are closely related by molecular criteria can display strikingly different phenotypes when assessed in the laboratory through the nature of their carbon and energy metabolism. In the relatively closely related “gamma subgroup” of the kingdom of Proteobacteria (delineated by the genus Escherichia in figure 1), for instance, we find the phenotypically disparate organisms E. coli (organotroph), Chromatium vinosum (H2S-based phototroph), and the symbiont of the tubeworm R. pachyptila (H2S-based symbiont). The superficial metabolic diversity of these types of bacteria belies their underlying close evolutionary relatedness, giving no hint of the close similarities of their basic machineries. The versatility of Bacteria makes the metabolic machineries of Archaea and Eucarya seem comparatively monotonous. As the sequences of diverse genomes are compared, it will be possible to map the flow of metabolic genes onto the rRNA-based tree and see how metabolic diversity has been molded through evolution.

The molecular perspective gives us more than just a glimpse of the evolutionary past; it also brings a new future to the discipline of microbial biology. Because the molecular-phylogenetic identifications are based on sequence, not metabolic properties, microorganisms can be identified without the requirement for cultivation. Consequently, all the sequence-based techniques of molecular biology can be applied to the study of natural microbial ecosystems, heretofore little known with regard to organismal makeup.

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A Sequence-Based Glimpse of Biodiversity in the Environment

Knowledge of microorganisms in the environment has depended mainly on studies of pure cultures in the laboratory. Rarely are microorganisms so captured, however. Studies of several types of environments estimate that more than 99% of organisms seen microscopically are not cultivated with routine techniques (Amann and others 1995). With the sequence-based taxonomic framework of molecular trees, only a gene sequence, not a functioning cell, is required to identify an organism in terms of its phylogenetic type. The occurrence of phylogenetic types of organisms, “phylotypes,” and their distributions in natural communities can be surveyed by sequencing rRNA genes obtained from DNA isolated directly from the environment. A molecular-phylogenetic assessment of an uncultivated organism can provide insight into many of its properties through comparison with its relatives. Analysis of microbial ecosystems in this way is more than a taxonomic exercise in that the sequences provide experimental tools, such as molecular hybridization probes, that can be used to identify, monitor, and study the microbial inhabitants of natural ecosystems (Amann and others 1995; Hugenholtz and Pace 1996; Pace and others 1985).

Every nucleic acid-based study of natural microbial ecosystems so far performed has uncovered novel types of rRNA sequences, often representing major new lineages only distantly related to known ones. The discovery of rRNA sequences in the environment that diverge more deeply in phylogenetic trees than those of cultivated organisms is particularly noteworthy. It means that the divergent organisms recognized by rRNA sequence are potentially more different from known organisms in the lineage than the known organisms are from one another. The deepest divergences in both Bacteria and Archaea were first discovered in rRNAbased surveys of communities associated with hot springs in Yellowstone National Park (See Hugenholtz and others 1998, for review).

The gene-based studies of organisms in the environment have substantially expanded our view of the extent of microbial diversity, reflected in new branches in phylogenetic trees. Figure 2 shows a diagrammatic tree of known bacterial diversity. When Woese first summarized the phylogeny of the phylogenetic domain Bacteria, he could articulate about 12 main phylogenetic groups. These groups have been called “phyla,” “kingdoms,” or “phylogenetic divisions”; I use the latter term. The number of recognized bacterial phylogenetic divisions has expanded now to about 36 (figure 2). About one-third of these divisions, indicated by the outlined wedges in figure 2, have no known cultivated representative and were detected only by rRNA gene-based studies of environmental organisms. Some of the most abundant organisms in the biosphere fall into these divisions with no cultured examples. Their abundance identifies such organisms as worthy of future study (Hugenholtz and others 1998). Environmental surveys of rRNA genes also have expanded the known diversity of Archaea and revealed that such organisms, previously thought restricted to “extreme” environments (from the human standpoint), in fact are ubiquitous. Crenarchaeota, for instance, all of whose cultured representatives are thermophiles, is revealed by the

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Figure 2
Evolutionary-distance tree of the bacterial domain showing currently recognized divisions
and putative (candidate) divisions. The tree was constructed with the ARB software package
(with the Lane mask and Olsen rate-corrected neighbor-joining options) and a sequence
database modified from the March 1997 ARB database release (Strunk and others 1998).
Division-level groupings of two or more sequences are depicted as wedges. The depth of
a wedge reflects the branching depth of the representatives selected for a particular
division. Divisions that have cultivated representatives are shown in black; divisions
represented only by environmental sequences are shown in outline. The scale bar indicates 0.1
change per nucleotide. The aligned, unmasked datasets used for this figure are available
from  http://crab2.berkeley.edu/~pacelab/176.htm.(From Hugenholts and others 1998, reprinted with permission.)

molecular studies to be abundant in the marine environment and in soils (see Pace 1997, for review).

Microbial Diversity and the Limits of the Biosphere

Textbooks generally portray only a part of the global distribution of life—the part that is immediately dependent on either the harvesting of sunlight or the

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metabolism of the decay products of photosynthesis. The molecular phylogenetic record shows, however, that lithotrophic metabolism preceded and is more widespread —phylogenetically and geographically than either phototrophy or organotrophy. The lithotrophic biosphere potentially extends kilometers into the Earth's crust, an essentially unknown realm (Ghiorse 1997). These considerations suggest that lithotrophy contributes far more to the biomass of Earth than currently thought. If so, where is it?

Part of the lithotrophic biomass is in microhabitats all around us, usually away from light and O2. It is not necessary to look far to find such environments: the rumens of cattle and the guts of termites and humans, for example, are important sources of CH4, a signature of hydrogen metabolism. Most life that depends on inorganic energy metabolism, however, probably is in little-known environments, according to poorly understood geochemistry. The oceans, for instance, cover 70% of Earth's surface to an average depth of 4 km. Most life in the ocean is microbial, and the metabolic patterns of such organisms are not understood. Does the occurrence of a large standing crop of low-temperature crenarchaeotes, potentially H2 oxidizers, indicate an unsuspected, lithotrophy-based food chain in the oceans? Another little-studied environment with global importance is the deep subsurface (Fredrickson and Onstott 1996; Gold 1992; Lovely 1995). There is increasing evidence that the Earth's crust is shot through with biomass wherever the physical conditions permit. Metabolism of H2 is a dominant theme among organisms isolated from geothermal settings or deep aquifers (Pedersen 1993; Stevens 1997). H2 is generated readily by abiotic mechanisms, such as interaction of water with iron-bearing basalt, the main stuff of Earth's crust. Consequently, a food source is unlikely to be limiting in most subterranean environments; it is likely to be the oxidant, the terminal electron acceptor, that limits growth. Nonetheless, it seems possible that much, perhaps most, of the biomass on Earth is subterranean, a biological world based on lithotrophy. Although the metabolic rate of this subterranean biosphere is likely to be far lower than in the more dynamic, photic environment, life is likely to be as pervasive in occurrence, and perhaps in cellular diversity, as we experience on the surface.

The opportunities for discovery of new organisms and development of resources based on microbial diversity are greater than ever before. Molecular sequences have finally given microbial biologists a way to define their subjects—through molecular phytogeny. The sequences also are the basis of the tools that will allow microbial biologists to explore the distribution and roles of the organisms in the environment. Microbial biology can now be a whole science and can study the organism in the ecosystem.

Acknowledgments

I thank Sydney Kustu, Gary Olsen, and Carl Woese for helpful comments on the manuscript and Sue Barns and Phil Hugenholtz for assistance with figures. Research in my laboratory is supported by grants from the National Science Foundation and the National Institutes of Health. This article is based on an earlier one (Pace 1997).

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