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EXTRAORDINARY GENETIC DIVERSITY AND VARIABILITY

Microorganisms differ from macroorganisms in a few fundamental ways and some of these differences have profound effects on the mechanisms and tempo of microbial evolution. Much of the early study of evolution depended on comparing physical characteristics (or phenotypes) — beak shape, the shape and relationship of bones, seed and leaf shapes and sizes, etc. Studying the evolution of microorganisms, however, was comparatively difficult. There were a limited number of phenotypes – like shape and nutrient requirements – that could be easily studied. With the discovery of the structure of DNA, it became clear that an organism's characteristics were related to underlying gene sequences (the organism's genotype). This launched the challenge of unraveling the genotype-phenotype connection, with implications not only for the study of evolution, but also development, physiology, disease risk and biodiversity. Advances in molecular biology opened the door to studying evolution in microbes in new and powerful ways. Microorganisms employ an impressive array of mechanisms to generate genetic variation and this makes the study of their evolution complex and its implications profound — both for understanding microorganisms themselves and evolution in general.

Microorganisms employ an impressive array of mechanisms to generate genetic variation and this makes the study of their evolution complex and its implications profound.

VAST TIME SCALE

Some of the apparent discrepancies between the evolution of microscopic and macroscopic life is due to differences in the temporal scales over which we compare evolutionary changes. In terms of the history of life on Earth, the adaptations discussed by Darwin arose in animals relatively recently. The various beak morphologies of Galapagos finches, for instance, evolved in response to the available food sources on the islands over the course of mere thousands of years.

In contrast, microorganisms have been evolving for billions of years, and many of the traits microbiologists study have evolved over vast time scales. Much of what we know about the earliest microbial evolution is based on a single gene – the SSU (short sub-unit) rRNA gene. Initially chosen by Carl Woese in the 1970s for his pioneering work using phylogenetics to understand the relatedness of bacterial species, the SSU rRNA gene has a number of characteristics that make it ideal for examining deep evolutionary history: it is found in every organism, it is highly conserved (the sequence is 50% shared between organisms as different as bacteria and mammals), and it does not undergo lateral transfer (so its history is reflective of the organism's history, a statement that would not be true for a gene that had moved between species), Woese's phylogenetic trees of cloned rRNA genes from microbes then available in laboratory culture revealed, among other things, a previously unknown domain of life—the archaea—whose rRNA sequences were as different from bacterial rRNA as they were from eukaryotic rRNA. SSU RNA has been an invaluable tool in building a universal tree of life, a tree that extends back to the earliest life forms, reflecting the evolutionary connections among all life forms. However SSU rRNA can only reveal so much. Indeed, when one is using individual genes to infer the earliest events in evolution, one must accept that some information is irrevocably lost; some of the bases in the gene will have mutated more than once, or mutated back to their original sequence, changes that are phylogenetically invisible. Therefore it would be highly useful to construct similar trees using other highly conserved genes, but at present few genes have been sequenced in a wide enough variety of organisms to allow in-depth analysis. Phylogenetics has enormous potential as a tool for unraveling the ancient relatedness of life if such data sets could be assembled.

MULTIPLE LEVELS OF SELECTION?

Darwin recognized that there is some relation between “all organic beings”, an idea that allows for the traits of one species to impact the success of another, Symbiosis, which involves direct genome-genome interactions, is clearly both an important driver of the evolution of countless microorganisms and a potential constraint. More recently, the “super organism concept”—the recognition that all organisms are actually a composite of their own cells and their co-evolved microbiota, and that evolution and selection may act on this composite rather than on the individual — has raised interesting conceptual questions. Studying microbial evolution at levels ranging from “selfish genes” to complex, interconnected species and niches will initially drive the development of models that describe evolution in the most basic ecosystems, but eventually what is learned about the drivers of evolution in simple microbial communities will have implications for evolutionary theory more broadly.

Darwin recognized that there is some relation between “all organic beings”, an idea that allows for the traits of one species to impact the success of another, Symbiosis, which involves direct genome-genome interactions, is clearly both an important driver of the evolution of countless microorganisms and a potential constraint.

ADAPTATION VS EVOLUTION

A complex issue in the study of microbial evolution is unraveling the process of evolution from that of adaptation. In many cases, microbes have the capacity to adapt to various environmental changes by changing gene expression or community composition as opposed to having to evolve entirely new capabilities

From experience with certain types of environmental changes, we have gained a general idea of how certain microbes will respond. In the case of oil spills, for example, we know to expect a reduction in diversity and a local increase in the abundance of organisms that mediate hydrocarbon metabolism. Over longer terms horizontal gene transfer can result in the distribution of genes that facilitate hydro-carbon breakdown and the creation and assembly of pathways that allow microbes to utilize hydrocarbons.

In the oceans, research has revealed clear evidence of physiological adaptation at the microbial community level, but we have yet to find evidence of innovation in an existing population as a result of changing conditions. Apparently, changing conditions generally enrich for new populations that are suited to the new niche, rather than for improvements or innovation in the extant populations. Research has revealed evidence of gene flow, but there is less evidence of evolutionary adaptation in response to specific pressures. In complex environmental communities – marine or terrestrial – searching for evidence of evolutionary change has rarely been attempted and is currently intractable. Communities of deeply buried, isolated sediments and other extreme environments may be more manageable for examining ecology and evolution in concert.

There are many examples in which microbes are undeniably evolving in the face of challenges created by humans. Xenobiotics, for example, are substances that come from biological sources, but make their way into environments where they are not normally present. For example, high concentrations of antibiotics are not normally found in humans; this is a recent, pharmacological innovation and has had unforeseen consequences for human health and the evolution of infectious agents. Human hormones and many pharmaceutical compounds that enter the water supply are also xenobiotics. Microbes have already evolved new catabolic pathways for the degradation of xenobiotics and synthesized compounds like herbicides or industrial pollutants. These pathways seem to be assembled by horizontal gene transfer of different genes from different organisms onto mobile elements such as plasmids, which are then shared and distributed among organisms in the environment. Today, we can find similar plasmids in widely distributed locations, indicating that these capabilities have evolved rapidly and the innovations have been geographically distributed very quickly.

The degree to which microbes will evolve new capabilities versus adapting to new conditions using the capabilities already in place remains an open question. But whether they adjust by evolution or adaptation, microbes will continue to affect global environmental conditions in ways that may or may not be favorable from a human point of view.

EXPERIMENTAL TRACTABILITY

Again and again, discoveries made in microbiology have proven to have universal biological significance and microbes have served as model systems for some of the most important discoveries in biology. Many discoveries that were made by investigating microbes have expanded our understanding of evolution in general. A partial list includes:

• ▪ Microbes were the first organisms in which scientists showed that DNA is the hereditary material. (Avery, et al 1944, Hershey and Chase, 1952),
• ▪ Microbes represented the bulk of the data that led to the three domain tree of life (Woese. 1990),
• ▪ Scientists used microbes to demonstrate that evolution proceeds as a result of selection upon pre-existing mutants that arise spontaneously and at random (Luria and Delbruck, 1943), and
• ▪ Observational studies and experiments have demonstrated the importance of host-pathogen arms races as a driver of evolution.

Just as microbes served as highly flexible model systems for molecular biology experimentation, microbes and microbial consortia can also be used for experiments on evolution. Microbes in experimental systems and in real-world situations like infectious disease offer the opportunity to test and observe microbial evolution in action.

With larger organisms, until the advent of molecular biology, biologists were often forced to make inferences about evolution from observation. Genetic study has vastly enriched our understanding of the mechanisms of evolution, but the ability to carry out evolutionary experiments is still limited in long-lived organisms with large and complex genomes. By contrast, experimental evolution with microorganisms offers a rich alternative by providing a testable system based on hypothesis, experiment, and outcome. Control over selective pressures represents another advantage. Moreover, in microbiology it is possible to save and see the “mistakes” or evolutionary dead ends in an experiment; we don't necessarily “lose the losers” in an evolutionary microbiology experiment.

MICROBIAL EVOLUTION CAN BE USED TO SOLVE PROBLEMS

Microbial evolution can be harnessed as a tool to generate new biological entities capable of generating fuels from sunlight, producing bio-based materials, remediating pollution, and recycling wastes. These approaches show great promise for contributing to a transition to sustainable human societies. ‘Directed’ evolution—forward engineering—allows us to circumvent our profound ignorance of how a DNA sequence encodes function and represents a highly effective approach to generating useful new biological molecules and metabolic pathways and to modifying whole organisms for human needs. Although humans have been ‘breeding’ microbes for many years, with the genetic tools now available we can exert more control over the process, for example, by controlling mutation rates and directing mutations to specific genes. We can also extract detailed information on the changes that occur during evolution.

There are many fundamental questions to which we have dangerously incomplete answers.

Only with a more detailed and systematic understanding of microbial evolution can the manipulation of microbial communities be applied to more complex problems. Whether the goal is to use microbial communities to produce desired materials, remedy environmental damage, or mitigate climate change, a comprehensive and predictive understanding of how microbial communities respond to change and stress is critical. There are many fundamental questions to which we have dangerously incomplete answers. For example, we need to know whether microbes increase their rate of genetic change in response to stress. Do they mutate more often or more quickly? Do they exchange plasmids more often? Are they more susceptible to horizontal gene transfer via phages or other mechanisms? If the answer to those questions is yes, that suggests that microbes may be able to evolve more quickly when environmental conditions are in flux. Understanding such responses to stress will be helpful in efforts to drive microbial evolution to meet practical goals.

Even in the absence of perfect understanding of the ‘rules’ of microbial evolution, it has proven possible to modify some microbial systems to achieve a favorable outcome. For example:

• ▪ The ability of microbial communities to remediate oil spills can be enhanced by providing nutrients and co-metabolites like acetate that help microbes break down chlorinated solvents.
• ▪ Land use practices like the timing and amount of fertilization or cultivation of riparian strips can be adjusted to minimize denitrification and leaching, thereby avoiding adverse consequences of nutrient export into coastal and freshwater systems.

Many capabilities seem to be latent in microbial communities, and it seems that every new estimate of microbial diversity soon proves too low. This diversity is amplified by the microbial capacity for gene transfer both within and between species, an ability that allows future populations to share genes, mobilize a direct response to selective pressures and rapidly adapt to both natural and human caused global environmental shifts. Due to their large population sizes and large ranges, microbes represent an enormous natural reservoir of genotypic diversity that is available to undergo natural selection.

MICROBIAL EVOLUTION AND CLIMATE CHANGE ARE INTERTWINED

We are currently struggling to understand how human actions affect global processes, but it is worth remembering that from their beginning, microorganisms have had a profound effect on conditions on Earth. Through the evolution of oxygenic photosynthesis and continuous cycling of carbon, nitrogen and other elements, microbes created and continue to sustain the conditions we enjoy today. This is true at all scales and in all environments, on land, in the ocean, and under the Earth's surface. Microbes directly modulate the amount of bioavailable nitrogen, carbon, phosphorous, sulfur, and many important metals. The ability to transform nitrogen gas from the atmosphere into a form that organisms use to make critical biomolecules like DNA and proteins (the process of nitrogen fixation), evolved in microbes very early in the history of life and microbial nitrogen fixation continues to serve as a fundamental link in the nitrogen cycle. Microbial chemical cycling also plays a role in the strength of the planet's greenhouse effect. Denitrifiers and nitrifiers can generate nitric oxide and nitrous oxide, for example, which are powerful greenhouse gases that have 280-320 times more potential for warming than carbon dioxide. Also, phytoplankton produce dimethylsulfide and dimethylsulfoniopropio-nate, and the cycling of these compounds produces sulfur gasses that impact cloud formation, and, hence, the water cycle and the global albedo (reflectivity). Archaeal methane production is the dominant natural source of methane, a gas that is over 20 times more powerful a greenhouse gas than carbon dioxide.

Human activities are changing the environment in which microbes evolve, creating innumerable new evolutionary pressures.

Human activities are changing the environment in which microbes evolve, creating innumerable new evolutionary pressures. The feedback loop between microbes and the environment now includes another driver. How microbes will react to this new set of variables is critically important, but difficult to predict. The time scale of human-produced changes affects whether they will evoke evolutionary changes or simply adaptation, for example, in microbial community population structure. Over the long term, there is no doubt that if microbial ecosystems are disturbed, their “evolutionary trajectories” will be affected in ways that we cannot predict.

Many of the human-induced changes to the microbial biosphere are felt in the oceans, which represent the largest biome on Earth. Ocean ecosystems are on the receiving end of a great deal of runoff and of other less direct human influences. Chemical runoff from agriculture and industry, including pesticides, herbicides, and metals, stress ocean ecosystems and may increase the availability of metal co-factors like tungsten and molybdenum, which are required for particular proteins and may have been limiting certain biogeochemical processes over time. Increases in temperature, dissolved carbon dioxide, and changes in the water cycle can also be expected in the oceans as the climate continues to change.

Any of these human-caused impacts could have effects on microbial evolution; in combination, the accumulation of stressors may dramatically change the selective pressures faced by microbes in the sea and on land. What will be the impact of all these changes on the microbial communities that are major drivers of global biogeochemical cycles? Are these cycles resilient to change? If so, to what extent does evolution play a role in this resilience? Resilience to change depends on the magnitude of the environmental perturbation, the temporal and spatial extent of the perturbation, and the time required for recovery. After small perturbations, communities recover quickly and internal changes in the community are sufficient for recovery. For more severe perturbations, recovery likely requires dispersal from undisturbed ecosystems, and in the case of severe, widespread, prolonged perturbation, recovery is likely to involve evolutionary change.

EVOLUTIONARY MECHANISMS AT WORK IN MICROBIAL POPULATIONS

Fundamental understanding of evolution in microorganisms lags behind that of multicellular organisms. The tools are becoming available to study the vast expanse of time in Earth's history when evolution was entirely microbial and that saw the evolution of the fundamental pathways shared by all life forms. More effort and resources should be directed to fundamental microbiology research to help us understand the mechanisms through which microbes evolve, especially in response to different environmental changes and challenges. Directing funding solely to applied problems does not fill in these basic gaps in our knowledge in an effective manner. Basic research on microbial evolution, however, has the potential to contribute across sectors and address applied problems in many fields, thereby leading to new approaches to treating disease, raising agricultural productivity, monitoring and addressing climate change, and producing clean energy.

The tools are becoming available to study the vast expanse of time in Earth's history when evolution was entirely microbial and that saw the evolution of the fundamental pathways shared by all life forms.

Only by understanding evolutionary mechanisms can we hope to understand and predict how the evolutionary trajectory of genes, populations, or communities will respond to environmental changes like the impact of introduced organisms (including genetically modified organisms, organisms used in biofuels, and exotic species) in the environment. Also, since all animals and plants have an associated microbial community, we need research that will enable us to understand how those organisms' microbiomes affect their ability to invade new territories or resist invaders, and the impact of extinctions on microbial diversity.

In particular, we need to understand processes that drive evolutionary diversification. Generating sequence data sets from a wide diversity of organisms for many highly conserved genes beyond rRNA will reveal a great deal about early evolutionary events. Distinguishing evolution from adaptation can be challenging at the microbial level. Microbial communities can respond to a change in the environment in different ways, including community level changes (e.g., ecological replacement, in which one organism replaces another), physiological adaptations (phenotypic change in response to the environment), and evolution through natural selection, but the relative importance of these different responses in various environments or under various circumstances is not known.

LINKING FIELD AND LABORATORY MICROBIOLOGY TO LEARN MORE ABOUT HOW MICROBIAL EVOLUTION WORKS

When making his case for how natural selection shapes evolution, Darwin pointed to the large phenotypic changes that could be achieved in relatively few generations of artificial selection. The same strategy applied to microbes, experimental evolution, has allowed us to observe how rapidly microbes and their biological components (proteins, DNA, RNA) adapt in the face of defined selection pressures and, with the tools now available to sequence genes and even whole genomes, even delineate the precise mechanisms by which adaptation occurs. These studies will continue to yield insights into evolution at a molecular level, and may also provide insight into how microbes have responded in the past and will respond in the future to specific changes in their natural environments. For example, experimental evolution studies have already predicted specific mutations conferring antibiotic resistance. Experimental evolution helps us to understand mechanisms of evolution and to measure and constrain evolutionarily relevant parameters.

It is now time to tackle questions of evolution in mixed communities that better reflect the environments in which microorganisms actually evolve.

At the same time, microbial ecologists increasingly can investigate the population structure of real microbial communities in the field. But they traditionally have not addressed the question of evolution in the complex communities they study. Conversely, evolutionary microbiologists in the laboratory have focused on the mechanisms of evolution in experimental microcosms, but haven't expanded their findings to complex natural systems. Both communities will gain greatly by collaborating. Micro-biologists need to bring these two paradigms together by linking comparative studies of microbial communities in the environment with experimental work in the lab. We need to study systems from the “top down”, by observation, and “bottom up”, through experimentation. Some of the priorities for these collaborative studies include:

• ▪ Linking population structure with concepts of microbial fitness based on what has been learned in experimental systems,
• ▪ Seeking environmental confirmation of the lessons about fitness that microbial evolutionists are learning in the lab,
• ▪ Taking biome mapping to the next level and interpreting the population structures that we see based on what has been learned in past decades in experimental evolution.

LEARNING FROM NATURAL MICROCOSMS

Most of what we know about microbial evolution has come from the study of pure cultures of organisms like E. coli and Salmonella. It is now time to tackle questions of evolution in mixed communities that better reflect the environments in which micro-organisms actually evolve. Researchers need to employ tractable, simple systems with small groups of microorganisms in which the pattern and consequences of genetic and phenotypic changes can be manipulated and tracked. Some possibilities include communities from simple extreme environments, like acid mine drainage, or invertebrate gut communities.

The living cells harbored in deeply buried marine sediments (>1 meter to several kilometers in depth), often referred to as the “deep biosphere”, represent another possible natural microcosm of both population-level adaptation and cellular evolution. Studies of sediment cores below one meter have detected population-level enrichment of a very specific population of microbes that are not observed in any other habitat – it is a distinct biome.

The deep biosphere may also contain a sort of “microbial Galapagos” – evolutionary islands where isolation and selective pressures have resulted in speciation. If ocean water can be considered a gel to microorganisms, the deep sediments can be considered a solid. Because these sediments are impermeable, the microbial habitat is marked by increasing isolation and individual cells in the deep biosphere represent time capsules of evolution. Some of these microbes have been effectively isolated for tens of millions of years.

LINKING MICROBES TO CLIMATE

Global climate change will have profound impacts on all ecosystems, but we have little understanding of what those impacts will be, especially at the microbial level. The effect of temperature changes on microbial systems is poorly understood, so we cannot predict, for example, the impacts of warming in northern latitudes on biogeochemical cycles or the effect of warmer oceans on primary productivity. Global climate change will also alter the ranges and geographical distribution of insects and other disease vectors and, consequently, the distribution patterns of diseases, with unknown impacts on agriculture, human populations, and ecosystems.

Global climate change will have profound impacts on all ecosystems, but we have little understanding of what those impacts will be, especially at the microbial level.

Other research priorities with respect to microbes and climate include:

• ▪ Determining how decomposers will respond to the increases in productivity and carbon dioxide consumption that are anticipated as average temperatures climb,
• ▪ Exploring the effects of falling pH on productivity in the oceans,
• ▪ Distinguishing between cause and effect with respect to microbes and climate to determine whether microbes are causing certain changes or “buffering” the system from faster change, and,
• ▪ Exploring the genomics and population structure of microbial systems to determine whether or not there are genetic “backup systems”. In other words, if one species that performs some key function is lost, are there other microbes to perform that function in its stead?

UNDERTAKING LARGE ENVIRONMENTAL STUDIES

To understand how microbes will evolve in response to human impacts like carbon emissions and nutrient run-off, microbiology needs a baseline of information about current genetic diversity and the natural history of populations and communities in different habitats. Large-scale space and time surveys of both living and non-living factors in a range of different communities are needed to fill these gaps in our knowledge base.

PREDICTING PHENOTYPE FROM GENOTYPE

Darwin was able to observe that different beak shapes among the Galapagos finches had adaptive significance—a critical piece of evidence linking fitness and natural selection to evolution over time. With the advent of molecular biology, linking characteristics like beak shape (phenotype) to genetic variation (genotype) became feasible, which fueled rapid progress in understanding the mechanisms of evolution. The genotype-phenotype connection is by no means solved for macroorganisms, but the challenge is vastly greater in microorganisms. The function of most microbial genes is unknown—even in well-characterized organisms—and metagenomic studies reveal more and more genetic sequences that encode unknown proteins of unknown function. Thus much of the incredible wealth of genomic sequence data available today is untapped. When a protein's function is unknown, it is difficult to discern the adaptive or ecological significance of variations in that protein within a population.

…much of the incredible wealth of genomic sequence data available today is untapped.

Solving the genotype-phenotype question is critical. We need to become better able to make predictions about the structure and function of proteins from DNA sequence data. Progress in this area will not only improve our understanding of microbial evolution. Historically, study of molecular pathways in model organisms like E. coli and yeast has contributed enormously to our understanding of plant, animal, and human physiology and disease. Because so many key metabolic and regulatory pathways are evolutionarily conserved across life's kingdoms, the same will be true of tackling the genotype-phenotype challenge at the microbial level.

Bioinformatics is a crucial tool for identifying patterns of genetic variation and recognizing genes that are under positive selection. Computational biology can vastly expand our ability to detect these genes and infer their biochemical, physiological, and evolutionary significance.

DETERMINING THE RATES AND CONSTRAINTS ON MICROBIAL EVOLUTION

How quickly can microbes evolve? And what constrains that rate? Researchers need to determine the temporal scales over which different kinds of evolutionary change occur in microbial populations and identify the factors that affect the rate of evolution. We also need tools, methods and conceptual frameworks to measure such evolutionary parameters as:

• ▪ mutation rate,
• ▪ recombination rate,
• ▪ environmental growth rates, and
• ▪ effective population sizes.

UNDERSTANDING THE EXTENT AND SIGNIFICANCE OF HORIZONTAL GENE TRANSFER

Horizontal gene transfer is a—perhaps the—major source of genetic variation in microorganisms but our understanding of the ‘rules’ governing which genes are shared among which organisms, and under what conditions, is rudimentary. We need to be able to quantify the relative contribution of horizontal gene transfer from related and unrelated organisms in a given strain and to determine the relative contributions of different methods of gene exchange (transduction, transformation, conjugation) to its genome. Research is needed to explore how the importance of these mechanisms changes between organisms and environments and what implications these mechanisms have for the rates and types of evolutionary changes that occur in genomes and populations. Are there underlying principles governing the structure of pan-genomes, and do these affect organisms' evolutionary history and potential?

EDUCATION AND TRAINING

Deeper understanding of microbial evolution will have implications across biology because so many of life's most basic systems and processes evolved first in microbes. Therefore, educational outreach is absolutely critical. Microbiologists have an obligation not only to train the next generation of students to be multidisciplinary but also to recognize, communicate, and incorporate into education the connections between microbiology and the rest of biology and other sciences. The general public and policy makers also need to be informed of work in microbial evolution, and especially of the many potential practical outcomes.

Microbiologists have an obligation not only to train the next generation of students to be multidisciplinary but also to recognize, communicate, and incorporate into education the connections between microbiology and the rest of biology and other sciences.

Students and researchers need access to more opportunities for interdisciplinary training and research, for example, integrating evolutionary microbiology and microbial physiology to facilitate a better understanding of the adaptive significance of genomic variations between different organisms.