MICROBIAL DIVERSITY

Paul V. Dunlap University of Maryland Biotechnology Institute

I. Introduction quires for growth, extremes of temperature, pressure, II. The Scope of Microbial Diversity salinity, or other environmental factors. III. The Biological Significance of Microbial halophile An organism requiring high levels of salts Diversity for growth. IV. A New Era in Biological Sciences heterotroph An organism that obtains its carbon from V. The ‘‘Delft School’’ of General Microbiology organic carbon compounds. VI. The ‘‘Woesean Reformation’’ of Microbiology microbe Single-celled organisms, such as bacteria, VII. Major Groups of Microbes archaea, protists, and unicellular fungi. VIII. Concluding Comments phototroph An organism utilizing the energy of light, as in sunlight, for growth. psychrophile An organism that grows better at low temperature or requires low temperature for growth. GLOSSARY aerobe An organism that utilizes or requires the pres- ence of oxygen for growth. MICROBIAL DIVERSITY can be defined as the range anaerobe An organism able to grow in the absence of different kinds of unicellular organisms, bacteria, of oxygen. archaea, protists, and fungi. Various different microbes autotroph An organism able to utilize carbon dioxide thrive throughout the biosphere, defining the limits of as its source of carbon. life and creating conditions conducive for the survival barotolerant and barophilic Able to tolerate high pres- and evolution of other living beings. The different kinds sures and growing better under high pressure. of microbes are distinguished by their differing charac- bioluminescence Light production by living or- teristics of cellular metabolism, physiology, and mor- ganisms. phology, by their various ecological distributions and chemotroph Organisms that utilize chemicals as activities, and by their distinct genomic structure, ex- sources of energy. pression, and evolution. The diversity of microbes pres- cryptoendolithic Living within the surface of rocks. ently living on earth is known to be high and is thought elective culture The provision of appropriate physical to be enormous, but the true extent of microbial diver- and chemical conditions that elicit the growth of sity is largely unknown. New molecular tools are now specific metabolic types of microbes. permitting the diversity of microbes to be explored extremophile An organism that grows better at, or re- rapidly and their evolutionary relationships and history

Encyclopedia of Biodiversity, Volume 4 Copyright  2001 by Academic Press. All rights of reproduction in any form reserved. 191 192 MICROBIAL DIVERSITY to be defined. The purpose of this article is to define the functional and evolutionary foundation of the bio- the scope of, and highlight major themes in, our current sphere. understanding of microbial life and to describe recent progress in expanding knowledge of the evolution and biological significance of these organisms. II. THE SCOPE OF ‘‘The key to taking the measure of biodiversity lies in a MICROBIAL DIVERSITY downward adjustment of scale. The smaller the organism, the broader the frontier and the deeper the unmapped We live on ‘‘a microbial planet’’ (Woese, 1999) in the terrain.’’ (Wilson, 1994) ‘‘Age of Bacteria’’ (Gould, 1996). Microorganisms, the first cellular life forms, were active on earth for more than 3.0 billion years before the development of multi- I. INTRODUCTION cellular, macroscopic life forms. During that time and continuing into the present, through the invention of Rapidly accumulating evidence indicates that microbes a spectacular array of different metabolic and physiolog- most likely account for the vast majority of kinds of ical capabilities, microbes evolved to exploit the multi- organisms on earth. Microbes carry out a stunningly tude of environments and microhabitats presented by diverse array of metabolic activities, several of which the abiotic world. They thereby obtained the cellular were instrumental in creating conditions for the evolu- building materials and energy necessary for growth and tion of other life forms. Through their colonization of reproduction. In so doing, however, they progressively diverse and extreme environments, their geochemical altered the geochemical conditions of the planet, lead- cycling of matter, and their biological interactions ing to a continual development of new conditions and among themselves and with all other organisms, mi- habitats, abiotic and biotic. Those new conditions and crobes define the limits of the biosphere and perform habitats presented both challenges to survival and op- functions essential for ecosystem development and portunities to exploit, leading to continuing evolution health. However, because microorganisms are predomi- of distinct microbial types able to endure or take advan- nantly unicellular life forms that generally are smaller tage of the biogeochemical changes taking place on than can be seen with the unaided eye, they historically earth. Once cellular life began, it is likely that no place have received disproportionately little scientific atten- on earth containing the molecules and energy condu- tion compared to that given to animals and plants. This cive to life remained abiotic for long. lack of attention has begun to shift recently as awareness The evolutionary trend toward greater complexity, of the diversity of microbes and their biological impor- seen in the relatively recent appearance of multicellular tance has grown. Of the three presently recognized do- life forms (e.g., plants and animals), however, did not mains of life, two, the Bacteria and the Archaea, are cause microbes to be displaced. The appearance of entirely microbial, and the third, the Eucarya, through plants and animals did not shunt the unicellular mi- its vast array of protists and fungi, is primarily micro- crobes to forgotten corners of the biosphere to hang on bial. The essence and full scope of the diversity of mi- and eke out a marginal existence. Instead, multicellular crobes is revealed in the dramatic differences among organisms, which themselves can be viewed as highly these microorganisms in their phenotypic characteris- evolved, complex assemblages of microorganisms, have tics of cellular metabolism, physiology, and morphol- provided unicellular microbes with a wide variety of ogy, in their ecological distributions and activities, and new habitats to colonize and exploit. Consider the vari- in their genomic structure, expression, and evolution. ous microbes whose growth is favored by the different Appreciation for the true extent of microbial diversity and changing habitats provided by the growth and se- is growing rapidly through the development and use of nescence of roots, stems, leaves, flowers, and fruits dur- molecular phylogenetic approaches, which are enabling ing the life of plants. Consider the multitude of physico- rigorous analysis of the origins and evolution of micro- chemically distinct habitats of the human skin, of our bial life. In combination with classical methods of elec- mucous membranes, and the changing environments tive culture, isolation, and phenotypic analysis, the ap- of our complex intestinal system. Along with these habi- proaches of molecular phylogeny are stimulating the tats, colonized often by assemblages of several different discovery of multitudes of new microorganisms, open- kinds of microbes, consider the species-specific devel- ing up their biology for study, and providing a clear opmental and metabolic symbioses certain bacteria have understanding of the importance of microorganisms as established with plants, such as nitrogen-fixing Rhizo- MICROBIAL DIVERSITY 193

FIGURE 1 Light-micrograph of a section of the light organ of the sepiolid squid Euprymna scolopes. The animal harbors a dense population of the luminous marine bacterium Vibrio fischeri extracellularly within a ventral tissue complex, the light organ, and uses the light produced by the bacteria in predator avoidance. Reprinted from Claes and Dunlap (1999). Copyright  1999 Wiley-Liss, Inc. bium with legumes, and with animals, such as biolumi- we do, most, however, grow best at lower oxygen levels, nescent Vibrio with marine cephalopods and fishes (Fig. and anaerobic microbes of many different types require 1). Instead of passing on the torch of preeminence in the strict absence of oxygen to survive, as found, for life to the developing multicellular organisms and then example, in sediment and gut tracts. Temperatures that politely withdrawing from the life’s center stage, mi- from a human perspective are extreme—from just be- crobes were full participants and driving forces in that low the freezing point of water in some oceanic waters development, they have continued to diversify, and they and sea ice, to several degrees above its boiling point in remain fully dominant. Plants and animals provide a waters near hydrothermal vents and in hot springs—are highly visible but thin multicellular skin over a biologi- not extreme at all to the bacteria that colonize these cally rich and complex microbial world. A sense of the habitats. Indeed, water temperatures considered cold biological dominance of microbes is given by estimates for humans (i.e., 15ЊC) can be lethally hot to true cold- of the total number of living bacteria, roughly 5 ϫ 1030 loving, psychrophilic bacteria. Barotolerant and baro- cells, with a collective biomass, despite their small size, philic bacteria, active at and requiring the extremely possibly equal to that of all other life forms (Whitman high pressures of the deep sea, exist at pressures that et al., 1998). We live in the once-and-always age of mi- would crush the human body, while other microbes, crobes. the halophiles, found in salt lakes and solar evaporation Microbes of one kind or another, especially bacteria, ponds, require salt concentrations that would quickly survive and grow almost everywhere on earth. Whether pickle our tissues. Add to this the cryptoendolithic mi- widely distributed, having been spread globally by crobes living just below the surfaces of sandstone in winds, water currents, and animals, or occurring only the Antarctic, the acid-tolerant and acid-requiring bac- in localized areas where they have adapted to grow teria of acid mine drainage and sulfur springs, and the under specific environmental conditions, microbes dra- alkalinity-requiring bacteria of desert soils and alkaline matically extend our perception of the limits of the lakes that live at pH levels that would be caustic to our biosphere. Previously, that perception was shaped to a skin. This array of microbial attributes gives a sense of large extent by our notion of the conditions under the physical and chemical extremes at which certain which plants and animals can live. As aerobic organ- bacteria survive and grow (Madigan et al., 1999). These isms, we require the high levels of oxygen present in organisms have no ‘‘protection’’ from the environment air for survival. While many bacteria utilize oxygen as other than their inherent metabolism and physiology, 194 MICROBIAL DIVERSITY which, instead of protecting them, exquisitely suit them III. THE BIOLOGICAL SIGNIFICANCE to living in these extremes, on which they are depen- dent. Major research initiatives around the world seek OF MICROBIAL DIVERSITY to expand knowledge of ‘‘extremophile’’ biology identi- fying these ‘‘exotic’’ microorganisms and bringing into Microorganisms are ‘‘the foundation of the biosphere’’ study the metabolic and physiological attributes that (Staley et al., 1997), providing its ‘‘essential, stable un- adapt them to life at the physical and chemical limits derpinnings’’ (Woese, 1999). Microorganisms have of the biosphere. played and continue to play fundamental roles in the What about microbial diversity in less extreme envi- evolution of higher life forms on earth. They have done ronments, away from the limits of the biosphere? Many so and continue to do so through the essential ecological environments, such as garden soil, coastal seawater, processes they carry out in obtaining the materials and and lake sediments, do not exhibit such dramatic ex- energy needed for growth and reproduction. A primary tremes of temperature, acidity, pressure, or other fac- example of the evolutionary role microbes have played tors. Microbial life in these environments is strikingly is oxygenic photosynthesis, invented by phototrophic diverse. While many different types of microbes can be bacteria more than 2 billion years ago and which re- isolated and grown in laboratory culture from these leases oxygen as a by-product of energy generation. environments, most so far cannot. A typical expectation Over time, that release of oxygen led to a gradual change is that much less than 1 percent to a few percent of the in the earth’s atmosphere from reducing to oxidizing. microbial types seen in an environmental sample will The oxidizing atmosphere, as it developed, allowed en- grow in culture (e.g., Amann et al., 1995). That means ergetically more efficient aerobic organisms to evolve the vast majority of microbes, even from commonly and provided a protective shield of ozone against ultra- studied environments such as coastal seawater, have violet radiation for terrestrial and aquatic organisms. not been brought into study or identified. Thus, discov- Equally striking examples are the bacterial endosymbio- eries of new types of microbes are waiting to be made, tic origins of chloroplasts, light-harvesting organelles and they are being made. Examples of the unexpected in plants, and of mitochondria, energy-generating or- include the discovery of magnetotactic bacteria, which ganelles, major events in the evolution of plant and synthesize intracellular chains of magnetic granules that animal lineages in the Eucarya. Furthermore, the fixa- orient the cells to magnetic fields (e.g., Amann et al., tion of atmospheric nitrogen, reducing nitrogen gas 1995) and the occurrence of new members of presum- to ammonium and converting it into organic nitrogen ably anaerobic Archaea in oxygenated seawater at shal- forms, is an entirely bacterial activity, carried out by low depth (e.g., DeLong, 1998). These reports demon- various symbiotic and free-living microbes (Madigan et strate that microbial life in more accessible and al., 1999). commonly encountered environments is still poorly un- The ecological processes carried out by microorgan- derstood and far more diverse than presently known isms are equally fundamental. For example, global bio- or expected. Therefore, most habitats that are known geochemical cycles of major elements, carbon, nitrogen, to be rich in microbial life and that have been sampled, sulfur, and iron, essential components of all living cells, such as seawater, soil, and animal gut tracts, have not operate through microbial activity. Specifically, degra- yet yielded for study anywhere near their full comple- dation of complex carbohydrates such as chitin, form- ment of microbes. And what about easily accessible ing the exoskeleton of arthropods, and cellulose, hemi- habitats likely to be rich in microbial life but that have cellulose, and lignin, structural polymers in plants, is not yet been examined at all or at best have been sam- essential. Without microbial conversion, these poly- pled only minimally? One type of habitat is the gut mers would accumulate, removing huge amounts of tracts of the several hundred thousand known insect carbon from the biosphere and blocking a multitude of species, only very few of which, such as the termite, biological processes that allow micro- and macroorgan- have been examined microbiologically. Yet the gut tract isms to live. In the absence of these microbial degrada- of each species—because of the different foods the ani- tive processes, life on earth would soon falter. Besides mal eats, the gut’s specific morphology and physiology the microbial degradation of complex organic com- for digesting that food, and the environmental condi- pounds, consider the range of metabolic diversity in tions under which the animal lives—is likely to host microbes, from oxygenic and anoxygenic photosynthe- its own very different kinds of microbes. Our ‘‘microbial sis, sulfate reduction, methanogenesis, denitrification, planet’’ is largely unexplored. iron oxidation, nitrite oxidation and nitrate reduction, MICROBIAL DIVERSITY 195 hydrogen and methane oxidation, and so on, all ways played by microbes, awareness of microorganisms re- by which microbes obtain the energy necessary for mains limited. The diversity and scientific importance growth and reproduction. These considerations form of microbes have been largely passed over in human in part the basis for a commonly held view that bacteria society, in science, in biology, and even in discussions and other microbes, in carrying out these processes, of biodiversity (Hawksworth, 1991), overshadowed by serve humans and other higher organisms as environ- attention to macroscopic forms. The prevailing and er- mental recyclers and bioremediators. That view, while roneous view for many biologists is that bacteria are essentially correct, overlooks an essential point—these ‘‘primitive, simple and relatively uniform’’ (Pace, 1996). activities and processes are the fundamental biology of This view developed naturally from early technical and this planet. Microbes are the biosphere; their activities scientific limitations for discovering and studying bacte- create and provide the foundation for all other life. ria and other microbes in the 18th and 19th centuries, To gain a perspective on the significance of microbial such as the need for high-resolution microscopy and diversity, imagine a biological survey crew tasked with the need to understand cellular structure, biochemistry, discovering and documenting life forms on a newly polypeptides, and nucleic acids. Such limitations did encountered planet. Consider that upon landing, the not hinder as starkly the beginnings of macrobiology. crew found, remarkably, no macroscopic life. However, Later, as limitations to the study of microbial life were suppose that an initial sampling of a cubic centimeter overcome in the first three-quarters of the 20th century, of the planet’s surface revealed the presence of millions the bias against microbes as scientifically important bio- of discrete microscopic cellular entities. Imagine that logical systems was nonetheless maintained and rein- with much additional analysis these entities were found forced through the lack of a comprehensive phylogeny by the crew to represent thousands of different kinds of microbes. This situation was especially true for bacte- of organisms, distinguishable by their morphology or ria, which generally lack distinctive morphological their dramatically different ways of obtaining the energy characters and for which sexual reproduction like that and nutrients necessary for metabolism and reproduc- of plants and animals is absent. That bias remains tion. Would the survey crew be surprised? What if upon largely extant today. For example, most college and analysis of the genetic material from the different types university departments of biology and biological sci- present, these microbial life forms were confirmed to be ences are staffed predominantly with animal and plant differentand werefound inmany casesto bedramatically biologists, with relatively few if any microbial biologists. more distinct from each other evolutionarily than are Yet ‘‘the incongruity [between the scientific perception seaweeds and humans, would the crew be impressed? of microbiology and the preeminence of microorgan- What if the crew continued sampling, spreading out and isms in the real world] is astounding; it is worrisome; choosing other locations of the planet’s surface, and it cannot be scientifically justified or tolerated’’ (Woese, found similar ‘‘species richness’’ wherever they looked, 1999). Fortunately, the bias and incongruity are begin- but often with little or no overlap in the types of entities ning to be eliminated. present from one environment to the next. Would that start the crew thinking about, to paraphrase E. O. Wilson (1994), ‘‘a strange and vastly complex living world virtu- ally without end’’? It would, of course. However, there is V. THE ‘‘DELFT SCHOOL’’ OF no need to invoke new planets. This imaginary scenario GENERAL MICROBIOLOGY describes the reality of microbial diversity on earth. The only difference is the microbially driven evolution of The confluence of two distinct but complementary ap- macroscopic life forms on earth, giving rise to the plants, proaches in biological science, one classical and one animals, and macroscopic fungi. more recent, is leading to a shift in awareness about microbial diversity and its scientific importance. The classical approach is that of elective culture, also re- ferred to as enrichment culture, by which new microbial IV. A NEW ERA IN types are brought into culture and isolated for pheno- BIOLOGICAL SCIENCES typic analysis. The elective culture approach, through the careful design of growth media and conditions, Despite the complexity and richness of microbial life seeks to provide an appropriate physical and chemical and the essential evolutionary and ecological roles environment that, when inoculated with an environ- 196 MICROBIAL DIVERSITY mental sample (mud, for example), will elicit the they have been thought to play key roles in the insect’s growth of specific metabolic types of bacteria postulated nutrition, which is based on microbial degradation of to be active or present in the sample. For success, the cellulose and conversion to acetate, a major carbon and approach requires a thorough knowledge of biochemis- energy source for the insect. However, no spirochetes try, good observational skills, and sensitivity to poten- from the termite gut previously had been isolated in tial novelty in microbial metabolism and physiology. pure culture. That inability limited knowledge of the A recent example of this approach, demonstrating its contribution these morphologically distinct and numer- central importance and value in microbial research, is ically significant bacteria make to host animal nutrition. the isolation of acetogenic sprirochaete bacteria from Leadbetter and coworkers, however, successfully estab- the hindguts of termites (Leadbetter et al., 1999). Spiro- lished culture conditions that favored the growth of chetes are major members of the diverse microbial con- spirochetes over other bacteria and that simultaneously sortium resident in the termite hindgut (Fig. 2), and encouraged the growth of bacteria able to form acetate,

from H2 and CO2, breakdown products of cellulose deg- radation. In this way, acetogenic spirochetes from the termite hindgut were brought into pure culture for the

first time. H2/CO2 acetogenesis, a type of metabolism previously unknown among spirochetes, reveals an im- portant way, formation of acetate, in which these bacte- ria contribute to termite nutrition. The ability to design culture conditions that elicit the growth of specific bac- terial types known or suspected to be present in an environment is central to development of our under- standing of microbial diversity and microbial ecology. Once a novel microbial type has been brought into culture, that organism’s special or unique cellular me- tabolism, physiology, and genetics can then be studied in detail. This now classical elective culture approach devel- oped through the insights of Sergei Winogradsky, a Russian soil microbiologist, and Martinus Beijerinck and later Albert Kluyver, Beijerinck’s successor, in Delft, Netherlands. Their work was instrumental in forming the foundations of general microbiology and microbial ecology. When Cornelis van Niel, a student of Kluyver, moved in 1928 from Delft to the Hopkins Marine Laboratory in Pacific Grove in California, he brought with him the ‘‘Delft School’’ tradition in microbiology, which he continued and further devel- oped through his research and through a course he taught in general microbiology and comparative biochemistry (van Niel, 1949). Through his course van Niel trained a generation of microbiologists. Those individuals have gone on to use the Delft School, van Niel approach, in their research, and they in FIGURE 2 Phase contrast micrograph of the contents (diluted) of the hindgut of the termite Reticulitermes flavipes (Kollar). The microbial turn have taught other generations of microbiologists, assemblage of this common eastern subterranean termite includes further disseminating the Delft School tradition. Most several types of microbes, including a variety of different kinds of notably today, the elective culture and isolation ap- spirochetes, both free and attached to cellulolytic protists. H2 and proach to the study of metabolically and ecologically CO2 , formed during microbial cellulose digestion, is converted by diverse bacteria is fostered in young microbiologists certain of the spirochetes to acetate (Leadbetter et al., 1999), a major source of nutrition for the animal. The different spirochaetal cells and other scientists through the Microbial Diversity range in length from approximately 5 Ȑm to over 30 Ȑm. Courtesy summer course of the Marine Biological Laboratory of J. Breznak, Michigan State University. in Woods Hole. MICROBIAL DIVERSITY 197

VI. THE ‘‘WOESEAN REFORMATION’’ the principles and application of molecular phyloge- netic analysis in microbes and higher organisms. Nota- OF MICROBIOLOGY ble among them is the Marine Biological Laboratory’s course in Molecular Evolution, an intensive 3-week Despite the progress arising from the Delft School tradi- course dedicated to these topics. tion in exploring and defining the diversity of bacteria A second step in the Woesean reformation of micro- and their metabolic capabilities, a major problem lim- biology is the application of rRNA-based molecular phy- ited the development of general microbiology in the logeny to microbial ecology (e.g., Hugenholtz et al., first three-quarters of this century: the lack of a unifying 1998; Pace, 1996). Sequence analysis of 16S rRNAs, for phylogeny of microorganisms. Various efforts at classi- example, extracted from natural environments provides fying bacteria and systematizing relationships among direct access to the diversity of bacteria in that environ- them, based on phenotypic characters of morphology ment, bypassing the need for culturing microbes and and biochemical growth attributes, were attempted and giving a rapid and potentially comprehensive assess- abandoned. The frequent lack of characters, the insta- ment of microbial community composition. rRNA- bility or the shared nature of many characters, and based approaches have opened up for study many mi- the awareness that phenotypic characterization was not crobial activities and associations. The result is a rapidly grounded necessarily at the genetic level left these ef- expanding awareness of the diversity and ecology of forts with major flaws. The inability to place microbes, microbes, both culturable and not-yet-cultured (Amann especially bacteria, in an evolutionary context caused et al., 1995; Hugenholtz et al., 1998; Pace, 1996; general microbiology and microbial diversity largely to Pace, 1999). languish as mainstream sciences at a time when other The strengths of the elective culture and molecular areas of biology and microbiology (e.g., clinical micro- phylogeny approaches make them naturally comple- biology and biotechnology) were developing rapidly mentary. The cultivation of a new microbe leads to (Woese, 1999). What was needed was a unifying phylo- acquisition of the organism’s 16S or 16S-like rRNA se- genetic framework, founded at the genome level, which quence in the context of data on its metabolism, physi- would allow the true evolutionary relationships among ology and habitat. That sequence provides a defined microbes to be analyzed critically and defined. point of phylogenetic reference and a highly specific The use of informational macromolecules, begun tool with which to examine the organism’s distribution more than 30 years ago, is now fulfilling that need for in the environment. Equally exciting is the opportunity a unifying phylogeny of microbes (Woese, 1987; 1999) to examine and explore the environment for the full, and is reforming our view of the evolution and diversity natural microbial diversity present, without concerns of microbial life. Analysis primarily of ribosomal RNA about the bias inherent in and finesse required for cul- (rRNA) sequences, especially for the small subunit 16S turing. Those explorations identify and define more and 16S-like rRNAs, has created ‘‘the first valid micro- deeply within a unified phylogenetic framework the bial phylogenetic systematics’’ (Jannasch, 1997). The diversity of microbes present while also offering poten- functionally constant 16S and 16S-like molecules, com- tial insights into their metabolism and physiology. That mon to all organisms, contain evolutionarily highly con- information, in turn, can motivate more refined or novel served regions, suitable for comparing less closely re- attempts at cultivation of the sequence-identified mi- lated organisms, and more variable regions, suitable crobes. Each approach nurtures and magnifies the for assessing evolutionary relationships in more closely strengths of the other. For microbiology in the next related organisms. The universality of ribosomal RNA century truly to ‘‘emerge as the primary biological disci- extends the value of this sequence comparison approach pline’’ (Woese, 1999) the continuing confluence of to all life forms, but importantly for bacteria it has these approaches must be encouraged. established a unified phylogenetically based system with which to begin defining bacterial evolutionary rela- tionships, a ‘‘first step in microbiology’s reformation’’ VII. MAJOR GROUPS OF MICROBES (Woese, 1999). Along with 16S rRNA, other molecules, such as elongation factor Tu, 23S rRNA, and F1F0 AT- Through analysis primarily of 16S and 16S-like rRNA Pase, also provide substantial phylogenetic information genes, microorganisms can now be placed in a poten- and can serve as alternative markers for inferring rela- tially comprehensive phylogenetic framework, one that tionships (Ludwig and Schleifer, 1999). A variety of includes all living organisms and therefore is universal. opportunities exist at various institutions for learning Examination of the 16S and 16S-like rRNA-based uni- 198 MICROBIAL DIVERSITY

FIGURE 3 The universal tree of life. The three domains of life, Bacteria, Archaea, and Eucarya, represented by small-subunit rRNA sequences of various organisms within each domain, are shown. The domain Bacteria (Eubacteria) and the domain Archaea (previously the archaebacteria) are entirely microbial, and the domain Eucarya is predominantly microbial. From Pace (1999), with per- mission.

versal phylogenetic tree (Fig. 3) shows the three cur- dominance of microbial life forms. The true diversity rently proposed domains of life, Bacteria, Archaea, and of life is microbial. Eucarya. Future analyses, using expanding sequence data sets and markers of phylogenetic relationships other than 16S and 16S-like rRNA (Ludwig and A. Domain Bacteria Schleifer, 1999) will serve to test and refine the validity The unit of biological diversity is the species. The classi- of this evolutionary grouping. Regardless, the current cal concept of a biological species, a reproductively universal tree reveals in a simple, compelling way the isolated interbreeding or potentially interbreeding pop- MICROBIAL DIVERSITY 199 ulation of individuals, however, does not work for bac- species (Staley et al., 1997). Presumably, the external teria and other microbes that do not interbreed, that surfaces of these organisms, and also the mouths, gut have undefined or cosmopolitan distributions, and that tracts, and internal tissues of the animals, provide myr- generally lack distinguishing morphological characters. iad types of microhabitats for bacterial colonization by So, in discussing bacteria, members of the domains assemblages of different microbes (Amann et al., 1995) Bacteria and Archaea, how is a species defined? The and in many cases by specific bacterial symbionts, as phylogenetic species concept, a group of individuals in the various nutritional endosymbioses of between for which phylogenetic analysis has demonstrated a bacteria and insects. Consequently, these estimates of shared genealogical relationship (Alexopoulos et al., bacterial species must be too low. When the approxi- 1996), seems more workable for bacteria and other mately 2 million species of fungi and protists, many of asexually reproducing microbes. The question then be- which undoubtedly also have largely different assem- comes the extent of genealogical relationship between blages of microbes as well as specific bacterial symbi- two individuals necessary to designate them as members onts, also are factored in, then the total number of of the same species. Operationally for bacteria, if the bacterial species easily could exceed tens of millions. genomic DNA of two strains is 70% or more similar, Future generations of microbiologists will likely find as determined by DNA-DNA hybridization analysis, even this estimate to be conservative. Regardless, how- they are considered the same species. With respect to ever, of what the actual total number of bacterial species rRNA, 16S rRNA sequence, similarity values below 97% turns out to be, bacteria are stunningly diverse. are a strong indication that the two bacteria are different Previously, all bacteria were grouped together taxo- species (Amann et al., 1995; Madigan et al., 1999). The nomically as prokaryotes. That grouping was based pri- combination of these two approaches, both based at the marily on the common lack of a membrane-bounded genomic level, is powerful. When further combined nucleus in bacteria. Prokaryotic organisms, however, with the identification of distinguishing phenotypic are now separated phylogenetically into two domains, characters, such as cell morphology, motility, and flag- the Bacteria (or Eubacteria) and the Archaea (formerly ellation, response to oxygen, requirement for organic the archaebacteria). The Archaea exhibit many similari- growth substrates, growth temperature range, special ties to the Eucarya, demonstrating that the prokaryotic metabolic attributes, characteristics of the habitat, and body plan is not a phylogenetically definitive character. so on, a character-rich, biologically meaningful descrip- The bacteria exhibit a wide variety of ways of obtaining tion of a bacterial species can be obtained. for growth the necessary carbon, as in the various kinds Conservative estimates place the total number of of heterotrophic and autotrophic microbes, and energy, species of bacteria at 50,000 to 3,000,000 (e.g., Staley as in phototrophic and chemotrophic microbes. et al., 1997). As of 1999, approximately 5000 bacterial The domain Bacteria presently contains well over species had been described (Pace, 1999), a very small two dozen divisions, or kingdoms, of organisms (Fig. portion of the estimated total number, though many of 4). Madigan et al. (1999) suggest, however, that the these descriptions do not yet include rRNA sequence true number of kingdoms in the Bacteria may be 50 or information. Most easily accessible environments, even more. As the number of these groups indicates, physio- those commonly sampled for microbial life, are likely logical diversity within the Bacteria is profound. The to contain a multitude of as-yet-uncultured bacteria. diversity is especially striking in two of the kingdoms, Another important consideration in estimates of the the Proteobacteria and the Gram-positive bacteria number of bacterial species is the extent to which micro- (Madigan et al., 1999), members of which exhibit a habitats, habitats relevant from the microbe’s viewpoint, wide range of different metabolisms, physiologies, mor- have been sampled. A reasonable assumption is that phologies, and habitats. Examples among the Proteo- most biotic and many abiotic surfaces are colonized by bacteria include the human enteric bacterium Esche- bacteria. Each surface presents a different microhabitat richia coli, pathogenic pseudomonads, light-producing and therefore is likely to be colonized by a different marine photobacteria, the fever-causing rickettsias, individual bacterial type or assemblages of types, with gliding bacteria and fruiting-body formers, stalked and the ‘‘many different microenvironments creating an al- budding bacteria, nitrogen-fixers, sulfate reducers, and most infinite variety of selective conditions’’ (Palleroni, so on. Within the Gram-positive kingdom are staphy- 1994), that is, conditions selecting for the specific meta- loccoccal parasites of humans, the milk-sugar fer- bolic and physiological types. An estimated total num- menting lactobacilli, and spore-forming soil bacteria, ber of plant and animal species is approximately 9 mil- among many others. This diversity, however, probably lion, with insects making up the majority of those reflects more the relative ease of cultivation and long- 200 MICROBIAL DIVERSITY

FIGURE 4 Diversity and phylogenetic relationships among members of the domain Bacteria. The division level grouping are of two types: currently recognized divisions, represented by cultivated bacteria (black), and provisional divisions, represented to date by environmental sequences (outline). The scale bar represents 0.1 changes per nucleotide. From Pace (1999), with permission.

term scientific attention members of these two king- B. Domain Archaea doms have received than the true biological richness of the groups. One can anticipate that much more diver- The domain Archaea includes the majority of presently sity in other kingdoms of the Bacteria will be revealed known ‘‘extremophiles,’’ organisms that live at physical with time as bacteria are studied more intensively. Fur- or chemical extremes. Archaea increasingly are being thermore, as the fusion between elective culture and discovered, however, in less extreme types of environ- molecular phylogeny develops, many other types of ments, including the marine plankton, lakes, and sedi- bacteria, some entirely unexpected, will be discovered ments (e.g., DeLong, 1998; Vetriani et al., 1999). Diver- and will be found to warrant kingdom status. A recent sity within the Archaea is presently less well understood text (Madigan et al., 1999) highlights the different than in the Bacteria and Eucarya because the Archaea groups of bacteria, providing information on individual often require particular care to culture. Knowledge of types within a phylogenetic context. how to culture the Archaea has expanded in recent MICROBIAL DIVERSITY 201

FIGURE 5 Diversity and phylogenetic relationships among members of the domain Archaea. This maximum likelihood tree, based on small-subunit rRNA, shows representatives of the Crenar- chaeota (some extreme thermophiles and some marine environmental phylotypes) and of the Euryarchaeota (some extreme thermophiles, methanogens, halophiles, and marine environmental phylotypes). The environmental phylotypes are indicated with acronyms and numbers, with their environmental origins designated. T. maritima and A. pyrophilus form the bacterial out-group. The scale represents the expected number of changes per sequence position. See Vetriani et al., 1999 for details and references. Prepared and provided by C. Vetriani, Rutgers University.

years, and additional habitats supporting their growth this group as substrates for methanogenesis, often with are being actively studied, so the disparity in the num- H2 as an electron donor. Methane, primarily from meth- bers of different kinds of Archaea compared Bacteria anogenic bacteria, is an important greenhouse gas, ac- should progressively diminish. The Archaea consists at counting for possibly as much as a few percent of total this time of three kingdoms, the Euryarcheaota, the primary production. Some methanogens, such as Meth- Crenarcheaota, and a provisional kingdom, the Korar- anococcus jannaschii, are hyperthermophiles. The ex- chaeaota (Fig. 5). treme halophiles, represented by Halobacterium sali- Two major types of bacteria, the methanogens and narum, are bacteria that require for survival and growth the extreme halophiles, are included within the Euryar- the exceptionally high salt concentrations found in salt chaeota. Methanogens are bacteria that produce meth- lakes and solar evaporation ponds. Other groups of ane as an end-product of energy conversion reactions; Euryarchaeota include a lineage of extremely acido- they occur in a variety of strictly anaerobic habitats, philic bacteria containing Thermoplasma and Picrophi- such as sediments, sewage sludge digestors, the rumen lus, and two lineages of hyperthermophiles, represented of cattle, and the termite hindgut. CO2 and similar com- by Pyrococcus and Archaeoglobus (Fig. 4). pounds, methanol and other methyl-containing com- The Crenarchaeota contains a large and physiologi- pounds, and acetate are used by different members of cally diverse group of hyperthermophilic, sulfur-metab- 202 MICROBIAL DIVERSITY olizing bacteria from terrestrial and marine hot springs database of 16S-like rRNA sequence information, is that and hydrothermal vents. Recently, several Crenar- the Eucarya consists predominantly of unicellular mi- chaeota (and Euryarchaeota) have been identified at croorganisms and that many phylogenetically deep, the 16S rRNA level as members of the plankton in kingdom-level divisions exist within the protists (Sogin cold oceanic surface and deep waters, coastal sediments, et al., 1996). Diversity within the protists dominates lakes, and in association with animals, indicating that that of the other eukaryotic lineages (Fig. 6). the Archaea are more cosmopolitan in their distribution than believed eariler (e.g., DeLong, 1998; Vetriani et 1. Protista al., 1999). The Protista is a large complex grouping of mostly A third kingdom of Archaea, the Korarchaeota, was unicellular eukaryotic organisms. They are morphologi- established provisionally based on rRNA sequences ob- cally diverse and can be found in most terrestrial, tained from samples of the Obsidian Pool hot spring aquatic, and marine habitats as free-living forms and in Yellowstone National Park and distinct from those as parasites of other protists, of fungi, and of plants of other Archaea (Pace, 1996). Attempts to bring these and animals. With their nutritional modes restricted hyperthermophiles into pure culture are underway. The primarily to osmo- and phago-heterotrophy and photo- discovery of this third Archaean kingdom and the recent trophy, protists are metabolically much less diverse than discoveries of Archaea in cold, oxygenated habitats Bacteria and Archaea. Along with various independent clearly indicate that the true diversity of Archaea is amoeboid groups, major groupings include the Alveo- likely to far exceed that based on presently identified lates, composed of ciliates (e.g., Paramecium), dino- species and sequences obtained from environmental flagellates (e.g., Alexandrium), and apicomplexans (e.g., samples. Implicit in this newly evolving view of Ar- Plasmodium), and the Stramenopiles, composed of the chaean phylogenetic diversity are substantially broader brown and golden-brown algae, diatoms, chrysophytes, metabolic capabilities and wider ecological roles for this oomycetes, and distinct groups of slime molds, among group of bacteria (DeLong, 1998; Vetriani et al., 1999). other groups. Cryptophytes, Rhodophytes, and Hapto- phytes are other major groupings of protists. Along with these groups are the diplomonads, trichomonads, C. Domain Eucarya microsporidia, amoeba-flagelates, and euglenoids Microbial groups in the Eucarya are the Protista and (Fig. 6). the Fungi, organisms that, in contrast to the Bacteria and the Archaea, have a membrane-bounded nucleus. 2. Fungi Endosymbiotic events are likely to have been major The fungi, sensu strictu, are commonly filamentous, driving forces in the evolution of eukaryotes. Bacteria multicellular heterotrophic organisms. Though pre- are thought to have diverged early from a universal viously thought to be similar to plants but lacking chlo- prokaryotic ancestor, followed by the Archaea, both of rophyll, fungi phylogenetically are not closely related which retained the prokaryotic body plan. Fusion of to plants. Instead, fungi are seen now to have diverged an archaean and a bacterium may have led to the nuclear from the animal lineage (Alexopoulos et al., 1996). line, which through symbiotic acquisition of phototro- Many fungi are saprobic, feeding osmotrophically on phic and nonphototrophic bacteria resulted in chloro- dead organic matter, and many are also parasites or plast- and mitochondria-bearing eukaryotic cells. Loss symbionts of animals. As such, they share the limited of one or both of these organelles, or failure to acquire range of metabolic capability of animals. Approximately them initially, along with secondary symbiotic events, 69,000 species of fungi have been identified, and more can be seen to account for much of the diversity of than 1,500,000 species are estimated to exist (Hawks- modern eukaryotes (Madigan et al., 1999). worth, 1991). Previous groupings placed eukaryotes into four king- Four major groups (phyla) of true fungi have been doms: animals, plants, fungi, and protists, with a fifth defined (Alexopoulos et al., 1996) (Fig. 7). The Chytrid- kingdom, Monera, to contain all the bacteria. Current iomycota contains a single class, Chytridiomycetes, understanding of phylogeny indicates that this five- which uniquely among fungi produces motile cells dur- kingdom system greatly underemphasized the diversity ing its life cycle. The motility organelle, a typical eukar- of bacteria while overemphasizing animals and plants, yotic flagellum, probably was retained from ancestral as described earlier. The five-kingdom system also un- protists (Berbee and Taylor, 1999). Chytrids play im- derrepresented the diversity within the protists. The portant ecological roles in decomposing organic materi- current, developing view, based on a rapidly increasing als. The Zygomycota contains two classes, the Zygomy- MICROBIAL DIVERSITY 203

FIGURE 6 Diversity phylogenetic relationships among members of the domain Eucarya. This evolutionary tree reveals the dominance of the protists over the fungi, animals, and plants. From Sogin et al. (1996), with permission.

cetes, which form thick-walled resting zygospores, and fungi. Plant pathogens in this phylum include the rust the Trichomycetes, obligate symbionts of arthropods. and smut fungi. Other fungi-like microbes commonly The Ascomycota, which form ascospore-carrying asci, studied by mycologists now are grouped with the pro- contains most of the lichen-forming fungi. The Basidio- tists. These include the Stramenopile groups of oomy- mycota, which produces sexual basidiospores on spe- cetes, hyphochytrids, and labryrinthulids, the plasmod- cialized basidia, contains many of the commonly recog- iophorids, and the dictyostelid, plasmodial, and acrasid nized fungi, such as mushrooms, puffballs, and bracket slime molds. The phylogenetic diversity and evolution FIGURE 7 Diversity of fungi and the timing of their divergences. See Berbee and Taylor (1999) for details. Species names are abbreviated with the first five letters of the genus followed by the first three letters of the specific epithet for these fungi (e.g., Dermocystidium salmonis ϭ Dermosal). From Berbee and Taylor (1999), with permission. MICROBIAL DIVERSITY 205 of the fungi is an area of active research (e.g., Berbee and Taylor, 1999). Bibliography

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