18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV 10.1146/annurev.earth.33.092203.122514 Annu. Rev. Earth Planet. Sci. 2005. 33:301–33 doi: 10.1146/annurev.earth.33.092203.122514 Copyright c 2005 by Annual Reviews. All rights reserved First published online as a Review in Advance on December 15, 2004 MOLECULAR APPROACHES TO MARINE MICROBIAL ECOLOGY AND THE MARINE NITROGEN CYCLE Bess B. Ward Department of Geosciences, Princeton University, Princeton, New Jersey 08544; email: [email protected] KeyWords 16S rRNA, diversity, functional genes, gene expression, genomics ■ Abstract Microbes are recognized as important components of the Earth system, playing key roles in controlling the composition of the atmosphere and surface waters, forming the basis of the marine food web, and the cycling of chemicals in the ocean. A revolution in microbial ecology has occurred in the past 15–20 years with the advent of rapid methods for discovering and sequencing the genes of uncultivated microbes from natural environments. Initially based on sequences from the 16S rRNA gene, this revolution made it possible to identify microorganisms without first cultivating them, to discover and characterize the immense previously unsuspected diversity of the microbial world, and to reconstruct the evolutionary relationships among microbes. Subsequent focus on functional genes, those that encode enzymes that catalyze bio- geochemical transformations, and current work on larger DNA fragments and entire genomes make it possible to link microbial diversity to ecosystem function. These approaches have yielded insights into the regulation of microbial activity and proof of the microbial role in biogeochemical processes previously unknown. Questions raised by the molecular revolution, which are now the focus of microbial ecology research, include the significance of microbial diversity and redundancy to biogeochemical pro- cesses and ecosystem function. Access provided by Princeton University Library on 01/23/20. For personal use only. Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org INTRODUCTION With the rise of the environmental geosciences has come an increased appreciation of the importance of microbes to many processes in low temperature geochemistry throughout Earth history. Microbes are responsible for the origin and maintenance of multicellular life on the planet through the production of oxygen, and they par- ticipate in weathering and long-term geological processes through the production of CO2 in respiration. It is also clear that microbes play important roles in the production and consumption of greenhouse gases such as methane and nitrous ox- ide, although the magnitudes and regulation of the fluxes are still quite uncertain. The fertility of soils and the quality of natural waters have long been recognized 0084-6597/05/0519-0301$20.00 301 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV 302 WARD as dependent on microbial processes. Because microbes have the capacity to both remediate and worsen environmental damage caused by humans, discovering and understanding their activities is ever more essential. The main goal of modern mi- crobial ecology in the context of Earth sciences is to discover the roles microbes play in biogeochemistry and to understand the regulation of microbial activity and the factors that control biogeochemical transformations. Research in microbial biogeochemistry has changed dramatically in recent years owing to the integra- tion of molecular biological methods into biogeochemical investigations. These new methods not only provide answers to longstanding questions, but they create the ability to ask new questions and entirely change our perceptions of natural systems. The technical advances that made this revolution in microbial ecology possible are central to the kinds of questions now being addressed, but a primer in molecular methods is beyond the scope of this review. The reader is referred to some stan- dard methods manuals for details on polymerase chain reaction (PCR) (Bartlett & Stirling 2003), cloning, automated DNA sequencing (Graham & Hill 2001), and phylogenetic analysis (Hillis et al. 1996). A BRIEF HISTORY OF MICROBIAL ECOLOGY IN BIOGEOCHEMISTRY The great classic contributions toward understanding the importance of microbes in the natural world were made when nineteenth century microbiologists first learned to cultivate microorganisms involved in disease and food spoilage (reviewed for the general audience by De Kruif 1996 and Postgate 1992). These advances in identifying the organisms responsible for specific processes are exemplified by the application of Koch’s postulates to the identification of pathogens. Once these pathogens were identified and characterized in pure culture, the solutions to most of humankind’s major epidemic illnesses were obtained within the space of a few years. In a sense, this golden age of microbiology is only now dawning in environmental microbiology. The past few years have seen the identification of new organisms and their direct linkage to important biogeochemical processes, Access provided by Princeton University Library on 01/23/20. For personal use only. and the discovery of a microbial role in processes previously thought to be abiotic Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org or even impossible. For example, molecular methods proved uniquely powerful in identifying the agents responsible for anaerobic ammonia oxidation and anaerobic methane oxidation. Both of these processes had been suggested on the basis of isotope and chemical evidence, but known cultivated nitrifiers and methanotrophs, respectively, were not capable of oxidation in the absence of oxygen. In analogy with recognizing the microbial basis of disease, microbiologists pursued the microbes that might be responsible for the spoilage of food and the transformations of materials associated with the fertility of soils. Once in culture, the biochemical transformations involved in, for example, fermentation, nitrogen fixation, denitrification, photosynthesis, etc., could be elucidated and their inter- mediates, substrates, and cofactors identified. Thus the cultivation of microbes, 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV BIOGEOCHEMISTRY AND MOLECULAR ECOLOGY 303 and the careful study of their growth and physiology in simple controlled systems, has been essential to understanding their activity in nature. The power of a pure culture for understanding biogeochemical transformations is that every aspect of the system can be defined. The fate of every carbon atom, the source of every oxy- gen atom, the stoichiometry of product formation and substrate utilization, and the pH or oxygen levels required to allow growth can all be known exactly. Ironically, cultivation as a tool of microbial ecology has also limited our vision of the real world. It was implicitly assumed that microbes in culture represented those in nature, and we relied on cultivated model organisms to extrapolate to processes in nature. The processes that occurred under specified conditions in laboratory culture were assumed to occur under similar conditions in nature and to be performed by organisms similar to those in culture. The converse also slipped into convention; if a process could not be shown to occur in culture, it was easy to assume that the process could not occur in nature. The need to reconcile observed processes in nature with the potential of microbes in culture led to creative cultivation methods and to the use of methods not requiring cultivation to establish the role of microbes in certain processes. Following the lead of taxonomists working with macroorganisms, microbiol- ogists attempted a classification of microbes based on their observable features. These features included some aspects of their biochemistry (utilization of specific substrates, production of particular end products, requirements for oxygen) and thus classification was not constrained entirely to the very limited morphological repertoire of microbes (i.e., round, comma-, sausage-, or spiral-shaped). Still, the classification scheme remained simply that: a system of classifying rather than a system of understanding or of inferring evolutionary relationships. The issues of diversity, the importance and constraints of cultivation, and evolu- tionary relationships among microbes were all revolutionized by the introduction of molecular methods and the insights they provided into microbial ecology. THE MOLECULAR BREAKTHROUGH The single most important breakthrough of modern microbial ecology was the abil- Access provided by Princeton University Library on 01/23/20. For personal use only. ity to read the information in microbial genes, and it accomplished at least three Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org major advances: (a)itallowed the simple unambiguous identification of microbes without cultivation; (b)itmade possible the discovery and partial characterization of microbes that have never been cultivated, and thus provided a much broader view of the natural microbial world; and (c)itprovided a basis for evolutionary inference and for discovery of evolutionary relationships among microbes that did not possess distinguishing characteristics or had not been cultivated. That break- through was the capability to determine the sequence of the genes of microbes, including genes retrieved directly