Norman R. Pace is a Professor in the Departments of Plant and Microbial Biology and of Molecular and Cell Biology at the University of California Berkeley.

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Microbial Ecology and Diversity

Late in the 20th century, our appreciation of the complexity of microbial ecology began to change dramatically

Norman R. Pace

Advances throughout microbiology have brought microbial ecology to a remarkable threshold. It has long been recognized that microorganisms are the foundation of the biosphere. Only now, however, do we have the conceptual basis and the technical capacity to study the microbial world comprehensively, and to understand the makeup and interactions of the microbial communities that mold our environment. That is our task in the next century.

This centennial tribute offers selected "pivotal ideas" in microbial ecology over the past century. Because the notion of a microbial world is not much older than a century, these advances span the history of microbial ecology as a scientific discipline. These ideas have changed mightily the perspective on microbes with which we entered the century, and they identify agendas for the next. My list of pivotal ideas is not exhaustive, and the order of its items is roughly chronological.

The Microbial World as the Underpinning of the Biosphere

Leeuwenhoek invented the microscope and discovered the microbial world in the 17th century, but studies of specific microbes did not begin until the 19th century, mainly in a medical context. Although the early methods were crude and perspectives on microbes were limited, many early bacteriologists recognized the looming importance of that hidden world. The great microbiologist Ferdinand Cohn, for instance, emphasized in the 1870s the role of microbes in rejuvenating the environment through decay processes.

Since then the evidence bearing on the extent and essential nature of microbial contributions to the biosphere has grown substantially. It is, therefore, remarkable that our K-12 and even university textbooks of general biology provide such scant acknowledgement of how microbial organisms affect the environment.

A major problem is that microbes are too small for human sight. Consequently, even many biologists consider microorganisms to constitute only a minor component of global biomass. The reality is far different. For example, according to William Whitman and colleagues at the University of Virginia, Charlottesville, microbial communities may constitute more than half of the biomass on Earth, with plant materials accounting for most of the remainder (animals do not contribute significantly to global biomass). Although the uncertainties in such calculations are huge, even the rough estimates indicate that microbial life permeates not only our familiar world, but also the depths of the ocean and Earth's crust, wherever physical conditions permit adaptation and survival.

Autotrophy as a Microbial Way of Life

Autotrophy, the fixation of CO₂, is the essence of primary productivity. The early microbiologists, whose main focus was disease, evidently did not think much about primary productivity. Winogradsky, through his studies of sulfur-oxidizing organisms, and Beijerinck, with his nitrogen-fixers, identified the concept of chemo-autotrophy a century ago. That concept paved the way for understanding many mineral transformations and beginning to think about a role for microbes in primary productivity. Although we have learned much since then, our understanding of autotrophic metabolism remains limited; such organisms tend to be difficult or impossible to culture for study in the laboratory. Iron, hydrogen, and sulfur metabolisms, for instance, are not well studied, yet probably contribute significantly to primary productivity in the crust of this planet and perhaps elsewhere in our solar system. Autotrophy based on photosynthesis arose in bacteria. Since the plant chloroplast is fundamentally a bacterium-derived from a cyanobacterium-primary global productivity derives ultimately from microbial processes.

Enrichment Culture Technology

Enrichment culture techniques, pioneered by Winogradsky and Beijerinck in the early part of this century, provide a way to tease apart the complexity of the natural microbial world and ascribe the chemical basis of environmental transformations to the activities of specific organisms. Culture techniques have resulted in the formal description of the relatively small collection of only about 5,000 microbes that provides most of our knowledge about microbial diversity. Such studies also led to many insights into the biochemistry, physiology, molecular biology, and, more recently, genomic sequences of microbes.

The precision and controls possible with pure-culture studies are seductive to microbiologists. Hence, the main experimental investment has fallen on relatively few, easily grown, "model" organisms, such as Escherichia coli and Bacillus subtilis.

Compared to the rapid advances that come from laboratory research focused mainly on model organisms, progress on deciphering the roles of a far wider variety of microbes in the environment has come more slowly. The precision of laboratory experimentation does not extrapolate simply to the environment. Thomas Brock laments that "microbial ecology is microbial physiology under the worst possible conditions." Moreover, because most environmental organisms are not cultured by standard techniques, microbial ecology has been slow to develop.

Interest in microbial diversity was largely diverted and overshadowed in the mid-century explosion of information that came from a focus on only a few organisms. Although interest among some academic scientists in microbial diversity and ecology continued and to some extent expanded, funding to support those interests, compared to support for research in biomedical areas, was typically scarce.

In addition, articulating what "diversity" means proves conceptually challenging. Before investigators developed the metaphor of molecular evolutionary trees, there was no convenient way to describe diversity in qualitative terms and no quantitative means to gauge the extent of diversity among two or more organisms under comparison.

Sex and Lateral Transfer in Ecology and Evolution

Genetic exchange in microorganisms was recognized about 50 years ago and quickly became a fundamental tool for conducting modern research in biology. DNA transfer in natural settings probably figures highly in community adaptation to environmental situations such as habitat change and must be a powerful driver of the evolutionary process. Early on, prominent microbiologists such as Joshua Lederberg acknowledged the potential importance of gene transfer in the environment; indeed, environmental transfers of traits such as drug resistance and metal metabolism are now well known.

Most microbiologists accept the emerging concept of physiological "islands," patches of DNA that migrate by transposition and contain multiple genes required for a particular complex trait, such as pathogenesis or symbiosis. However, not so long ago, most of them instead would have considered genomes of microorganisms to be fairly stable over very many generations. That belief has given way to the view that some, if not many, microbial genomes can be remarkably volatile.

For instance, sequence comparisons of genes isolated directly from microorganisms in their native habitats suggest that members of natural microbial taxa readily exchange DNA between nonidentical but close relatives. Microhetero- geneity in particular gene sequences indicates that environmental communities are collections not of singular species but of "clusters" of closely related organisms that possibly have similar physiologies and probably exchange genes frequently. Thus, our description of the microbial "species" needs to address not a singular type of organism, but a phylogenetic relatedness group, a "phylotype."

The Molecular Tree of Life

Until the late 1970s, thought on evolutionary diversity was framed in the context of an organism being either prokaryotic or eucaryotic. Then Carl Woese, through molecular phylogenetic studies of microorganisms, revolutionized our understanding of biological diversity and evolution.

When Woese began analyzing ribosomal RNA (rRNA) sequences, he expected those sequences from diverse organisms to fall into two, not three, fundamentally distinct groups. To account for this discrepancy, he reasoned there were three primary lines of evolutionary descent, or "domains," now termed Archaea (formerly archaebac- teria), (eu)Bacteria, and Eucarya (eukaryotes). His announcement of that fundamental organization of biology touched off a flurry of critical responses from other biologists who defended the prokaryote-eukaryote or the five-kingdoms notions to account for biological organization. However, these familiar notions had not been tested against such data. Sequence analyses with rRNA and other genes proved them basically incorrect.

Meanwhile, the shift in public and textbook treatments of this issue is far from complete. Microbiologists, however, long without a way to relate the evolutionary biology of microorganisms meaningfully, came to welcome this concept of the three evolutionary domains. In particular, it brings a broad sense of natural order to the classification and identification of microbes.

Figure 1

Woese writes elsewhere in this issue about the impact of this phylogenetic perspective on the conceptual foundations of microbial biology. On the practical side, the phylogenetic framework with its numerical sequence comparisons provides a conceptual approach to microbial identification and taxonomy. The sequence-based universal phylogenetic tree provides a metric for the otherwise nebulous concept of biodiversity (Fig. 1).

Moreover, this tree provides a rough map of the evolutionary course that led to the modern organisms being described. Thus, it goes well beyond the predecessor approach to describing relations among microorganisms that is based primarily on anecdotal information, such as physiological tests.

Molecular Ecology and Microbial Diversity

Because only a small proportion of microorganisms, usually <1%, proves culturable with standard techniques, microbiologists developed a severely limited view of the microbial world and the interactions that go on within it. Moreover, the behaviors of organisms that can be successfully cultivated may not provide a representative picture of all that occurs within natural communities in particular environments.

In fact, the microorganisms that tend to be abundant in natural settings typically have evaded efforts to culture them. The reasons for such failures are many, reflecting an underlying inability to recreate appropriate conditions. This routine failure at culturing microorganisms from diverse environments is further complicated by "syntrophy," the interdependence of two or more organisms. Marvin Bryant, Ralph Wolfe, and their colleagues recognized this phenomenon in the late 1960s when they discovered interspecies hydrogen transfer. Syntrophy is probably a common theme in microbial ecosystems.

Molecular biology is providing new ways for studying microorganisms in diverse environments without a need to cultivate them. For example, by analyzing relevant genes obtained directly from environmental samples, microorganisms from particular habitats may be not only identified but also counted and otherwise evaluated. Surveying microbial ecosystems in this way amounts to much more than a taxonomic exercise.

Thus, for instance, the microbial DNA sequences become resources for designing additional experimental tools, such as hybridization probes, that can be used to identify, monitor, and further analyze the inhabitants of particular ecosystems. Additionally, the sequences identify environmental targets for specific cultivation studies. These molecular methods are enabling microbiologists to realistically assess the natural makeup of microbial communities. Moreover, when coupled with other imaging and microchemical technologies, they offer accurate new ways to view microbial landscapes. Although gene-based microbial surveys have only begun, the results are revising our perception of the types and distribution of microbes in the biosphere. Sequences obtained from microorganisms in natural environments seldom correspond exactly to those of cultivars. Indeed, the dominant constituents of most environmental communities were not encountered in a century of culture studies.

Figure 2

However, since 1987, with these new approaches under way, investigators have tripled the phylogenetic divisions within the bacterial domain (Fig. 2) . More than one-third of these main bacterial divisions ("phyla," "kingdoms") contain no representative that has yet been cultured. Archaea and microbial eukaryotes are even more poorly represented in the culture collections. Even as this knowledge has rapidly expanded, our map of biological diversity, as represented by the universal phylogenetic tree, is far from complete.

Ecological Findings Identify Targets for Genomics

At the beginning of the 20th century, there was no clear concept of a genome. Just a decade ago, the possibility of determining a genomic sequence seemed a remote dream. Now, the expanding availability of genomic sequence information is profoundly influencing the discipline of microbiology. Our changing picture of microorganisms in the environment is helping us to identify important directions in which to pursue genome analysis. In turn, the information we can anticipate from those efforts will fuel studies of those organisms.

Figure 2 indicates the bacterial divisions with representatives that have been or are planned to be examined for genome sequence, and the number of sequences.

It is clear that the investment so far in genome analysis is directed toward a limited span of microbial diversity; only 12 of the approximately 36 bacterial phylogenetic divisions are represented in genomic sequencing activities. Some bacterial divisions with no representative genome sequence merit inclusion because of their prevalence in the environment. The planctomycetes, for instance, although observed in abundance in nature, are little-studied and consequently are little-known because of culture difficulties. Members of the Acidobacterium or the Verrucomicrobia divisions, for example, are as diverse phylogenetically as the well-studied proteobacteria (28 genome sequences), but are environmentally far more conspicuous, accounting for more than half of environmental rRNA genes in some studies. Other bacterial divisions, OP11 for example, have no cultivated representatives yet are highly abundant in nature.

For all the sequences being analyzed, however, we will still be far from an understanding of the cell and its place in the environment. The sequence of the genome will tell us the molecules, but not the interactions of those molecules that result in physiology, the ultimate basis of the biosphere.

Microbial Ecology Is the Future of Microbiology

Our concept of the microbial world is far better grounded and far more expansive than that of our predecessors from a century ago. In terms of our understanding of the makeup of microbial communities and their interactions, however, I am not so sure that we are much ahead of our scientific forebears. From laboratory studies we have learned a lot about a few organisms. However, we still know little or nothing about the specific nature and distribution of most of the organisms that dominate the environment, many revealed only in molecular studies over the past few years.

Moreover, we know little about the ways in which environmental organisms interact with one another, synergistically and in combat. Additionally, we know little about the diurnal, the annual, or the geological turnings of the microbial world. Questions related to these affairs of microbial communities, and the understanding and resources that the organisms offer, will provide agendas for studying microbial ecology over the next century. Technology has drawn us well ahead of our forebears, however. Our legacy of technology, only touched on here, opens opportunities to study the natural microbial world in situ, essentially for the first time.

Furtherance of our understanding of microbial ecology offers rewards beyond the academic. Some of the most vexing problems of society-environmental remediation, waste processing, public health, and control of epidemics, to name a few-involve fundamental problems in microbial ecology. If we understand the interacting organisms that make up the communities responsible for those processes, we can hope to channel some of their activities.

The natural microbial world and the organisms that comprise it promise endless resources. Humankind, of course, has a long history of reliance on the activities of microbial communities for making products such as bread, sausage, cheese, and wine. Only in this century, however, have humans pointedly gone to the microbial world for products such as antibiotics or other metabolites. Indeed, development of products has been the context for much of the development of microbiology as a discipline.

Traditionally, that focus on microbially based technology development has tended to be on single organisms, rather than the activities of communities of such organisms. The technology and principles that are applicable to microbial ecology also are relevant to the monitoring and manipulation of microbial consortia, coaxing from them useful products. The combinatorial nature of the activities of microbial communities holds promise for a wealth in future resources. Keys to those resources will emerge with deeper understanding of the natural microbial world.

ACKNOWLEDGMENTS

My research activities are supported by grants from NIH and NSF.

SUGGESTED READING

Amann, R. I., W. Ludwig, and K. H. Schleifer. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59:143-169.

Brock, T. D. 1987. The study of microorganisms in situ: progress and problems, p. 1-17. In M. Fletcher, T. R. G. Gray, and J. G. Jones (ed.), Symp. Soc. Gen. Microbiol., vol. 41. Cambridge University Press.

Hugenholtz, P., B. M. Goebel, and N. R. Pace. 1998. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. 180:4765-4775.

Pace, N. R. 1997. A molecular view of microbial diversity and the biosphere. Science 276:734-740.

Whitman, W. B., D. C. Coleman, and W. J. Wiebe. 1998. Prokaryotes: the unseen majority. Proc. Natl. Acad. Sci. USA 95:6578-6583.

Woese, C. R. 1987. Bacterial evolution. Microbiol. Rev. 51:221-271.

Woese, C. R. 1998. The universal ancestor. Proc. Natl. Acad. Sci. USA 95:6854-6859.

Woese, C. R., and G. E. Fox. 1977. Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proc. Natl. Acad. Sci. USA 74:5088-5090.