Molecular Approaches to Marine Microbial Ecology and the Marine Nitrogen Cycle

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 biogeochemical 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 processes 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 oxide, 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

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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 microbial 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 standard 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

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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 oxygen 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, microbiologists 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 evolutionary 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 breakthrough was the capability to determine the sequence of the genes of microbes, including genes retrieved directly from the environment without cultivation of the host organisms. This came about initially by painstaking manual sequencing of genes involved in protein manufacture, the ribosomal RNA genes, in cultivated microbes and led to the discovery that these gene sequences could be used to infer 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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evolutionary relationships among microbes (Woese & Fox 1977, Woese 1987). The introduction of the PCR (Mullis & Faloona 1987) and automated DNA sequencing (Smith et al. 1986) made this process immensely easier, faster, and cheaper. PCR made it possible to extend this approach to natural systems and uncultivated microbes. Thus, since the early 1990s, the sequence database, where the genetic information of countless microbes is archived, has grown rapidly, as has the catalog of recognized microbial diversity.

16S Ribosomal RNA Gene Sequences: Diversity and Evolutionary Relationships The ribosomal RNA genes were the important breakthrough genes because they encode a process that is universal to all organisms, the manufacture of proteins (Figure 1, see color insert). Essentially the same process is performed by ribosomes in every kind of organism from bacteria to humans, and the physical constraints on the design of the ribosome are so strong that random mutation cannot change the sequence very much and still allow function. Thus it is possible to find nucleotide sequences in ribosomal genes that are conserved across all organisms, and then to use those sequences in PCR amplification experiments to find, and to “pluck out,” the homologous genes in unknown and uncultivated organisms. The significance of the 16S rRNA revolution and its current status have been eloquently reviewed by some of the people who first applied the approach to uncultivated microorganisms (Pace 1997, DeLong & Pace 2001, Rappe & Giovannoni 2003). In the phylogenetic trees commonly used to represent evolutionary relationships (see, e.g., Pace 1997), the genetic distance or divergence between two extant organisms, represented by the ends of the branches, is proportional to the length of the branches connecting them. The branch points represent common ancestors from whom extant organisms inherited their characteristics. Thus the common ancestor of green plants was shown to have diverged from the single-celled green algae, whose photosynthetic abilities were inherited from the cyanobacteria, and the anoxygenic photosynthetic bacteria were shown to be ancestral to the oxygenic phototrophs, whose activity makes today’s world of air-breathing animals possible. Every time a new sequence is obtained, either from cultivated organisms or Access provided by Princeton University Library on 01/23/20. For personal use only. directly from the environment, it can be integrated into this tree and its lineage Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org determined. The ability to pluck genes out of nature, without first cultivating the organisms to whose genomes they belong, provides a glimpse of the world outside the culture flask. It is not a purely unbiased look, however, because the application of PCR to discover unknown genes or organisms requires that the target possess enough similarity to known sequences to be amplified with PCR primers that are designed on the basis of known genes. So to some extent, discovery with PCR is limited to finding what you look for. In addition, different sequences amplify with different efficiencies with the same PCR primers, and PCR therefore is considered to impart important but unquantified bias to the amplified population of genes, compared to 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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the microbial assemblage from which it was derived. Nevertheless, the range of diversity within 16S rRNA genes that can be amplified with universal primers is tremendous and far exceeds that found in the culture collection. One of the first discoveries after amplifying DNA extracted from natural samples was that many of the 16 rRNA genes retrieved were not closely related to anything in the culture collection. Thus there is a huge, diverse microbial world, some of whose members are apparently abundant and ubiquitous, have never been cultivated, and whose metabolism has never been characterized. A striking example of this phenomenon arose early in the molecular era. One of the first 16S rRNA clone libraries from seawater was obtained from Sargasso Sea seawater (Giovannoni et al. 1990) and contained many sequences unrelated to sequences from cultivated organisms. These clones are referred to by their original identity in the phylogenetic trees produced by Giovannoni et al. (1990), e.g., SAR11 and SAR07. The group of organisms denoted by rRNA sequences as SAR11 was well represented in this clone library. Sequencing of clone libraries from marine environments far and wide consistently retrieved sequences from this group and it comprised, on average, 25% of marine bacterial 16S rRNA clone libraries (reviewed by Giovannoni & Rappe 2000). Yet there was no cultivated organism whose 16S rRNA sequence was known that appeared to be closely related to the SAR11 group. Because 16S rRNA genes do not encode a physiological function that is important in biogeochemistry, this sequence alone does not provide information on the basic metabolism of the cell. A priori, it was not possible to predict whether SAR11 wasaphotosynthetic organism or a heterotroph or perhaps a novel symbiont. By placing the 16S rRNA sequence in the phylogenetic tree, its closest relatives could be identified, and if some of these were derived from cultivated organisms whose metabolism was known, then we might suggest that SAR11 shared some common metabolic characteristics. This level of inference is sometimes dangerous because of the immense diversity of bacteria and their capability for horizontal gene transfer and rapid mutation. In the case of SAR11, it was not very useful because the 16S rRNA sequence of SAR11 was not very similar to any cultivated strains, making physiological inference impossible. Understandably, SAR11 became the target of a focused cultivation effort. If Access provided by Princeton University Library on 01/23/20. For personal use only. this organism or group of organisms comprised a significant portion of the marine Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org microbial community, it was important to figure out what its metabolism was and what its impact on ocean biogeochemical processes might be. Using a dilution technique with culture medium containing very low levels of organic carbon, SAR11 was finally obtained in pure culture (Rappe et al. 2002). It appears to be an oligotrophic heterotroph, living on low levels of organic carbon in seawater. It is unable to tolerate high substrate levels. The fact that this is an expected result— that it is the kind of organism one would expect to flourish in the oligotrophic Sargasso Sea and much of the low-nutrient world ocean—does not detract from its significance. This body of work is a classic story in the modern era of microbial ecology, one that includes many firsts and sets a high standard for molecular 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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ecological sleuthing. Using the complementary DNA sequence, Morris et al. (2002) were able to detect individual cells of SAR11 in seawater and showed that they comprised up to 50% of the microbial population in surface waters and up to 25% in subsurface waters. Thus the prevalence of SAR11 type 16S rRNA genes in clone libraries was not an artifact of PCR bias or clone library construction; SAR11 really is abundant in nature. Whether all of its close relatives, as identified by 16S rRNA, share its physiology is unknown. A similar story is in the process of unfolding concerning a quite different kind of marine microbe. This one is an Archaeon, a member of the third major domain of life. The first two domains are commonly known as Bacteria and Eukaryotes, the latter containing all the organisms familiar to most people. Prior to the advent of molecular methods for microbial identification, the Archaea were considered to be a weird branch of Bacteria, ancient and characterized by extreme metabolisms and the ability to tolerate extreme environments (high salinity, high temperature, completely anoxic). Very early in the molecular revolution, Carl Woese (Woese et al. 1978) demonstrated that the Archaea actually composed a third major domain, as fundamentally different from Bacteria as Bacteria are from Eukaryotes. Archaea do contain many extremophiles—many are highly thermophilic—and as a group possess the most extreme and diverse metabolic capabilities. So when it was reported that Archaeal 16S rRNA sequences were common in clone libraries retrieved from normal seawater, both in temperate (Furhman et al. 1992, Massana et al. 1997) and in Antarctic (DeLong et al. 1994) waters, another major reevalu- ation was necessary. This group of organisms has, as of this writing, still not been cultivated, but using probes derived from the cloned sequences, (DeLong et al. 1999, Karner et al. 2001) it has been shown that these Archaea are prevalent and abundant over vast volumes of the deep ocean. Because the deep sea comprises a huge and physically rather constant environment, it cannot really be said to be extreme, and this appears to be an example of convergent evolution—Archaea adapted to exploit the same environment so long thought to be dominated by Bac- teria. But the nature of their metabolism and their role in marine biogeochemistry remain unknown. These examples serve to illustrate some of the profound changes in our compre- hension of microbes that the molecular revolution has wrought. In the century or Access provided by Princeton University Library on 01/23/20. For personal use only. so of traditional microbial ecology, we did not succeed in cultivating the microbes Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org that are most abundant (and therefore likely to be very important) in marine and terrestrial environments. It is entirely possible that the culture collection contains mainly weeds, i.e., strains that grow well under conditions that are convenient for the scientist, but may not be important under natural conditions. Therefore, it is possible that the vast uncultivated microbial world carries out biogeochemical transformations in novel and unknown ways, limited only by thermodynamics. In addition to insights about the culture collection and the motivation to attempt novel cultivation methods, the ability to use sequence information to identify and partially describe unknown organisms without culturing them is another important aspect of the molecular revolution. Sequencing of 16S rRNA genes from clone 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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libraries derived from DNA extracted directly from environmental samples has led to the discovery of several major phyla that have no cultivated representatives (Pace 1997). At the beginning of the 16S rRNA era, microbiologists recognized approximately 12 major phyla on the basis of cultivated strains (Woese 1987). The current tree of life contains approximately 40 Bacterial phyla identiﬁed on the basis of their 16S rRNA sequences, and approximately one third of those groups have no cultivated members (DeLong & Pace 2001). These groups represent deeply branching divisions in the phylogenetic tree, and on the basis of rRNA sequence, suggest the existence of groups as different from any known group as cyanobacteria are from Escherichia coli.Asimilar story has unfolded for the Archaea—this domain too contains a far greater diversity than previously recognized, and many distinct and divergent groups contain no cultivated representatives. At present, the metabolic characteristics of the uncultivated groups are unknown, and the need for this knowledge is a major motivation for the new ﬁeld of research in environmental genomics (see below). Even the Eukarya, previously thought to contain most of the biological diversity on Earth, have not been immune to this revolution. New lineages of Eukarya, some uncultivated, have been discovered, especially in the ocean. But the main revision in terms of the Eukarya is the recognition that metazoan life contains only a small fraction of the total diversity on the planet; on the basis of their genes, the multicellular Eukarya represent a thin slice of life. Clearly, the major mechanisms of evolution must be understood in the context of prokaryotic life.

Inferring Metabolic Function from 16S rRNA Gene Sequences Ribosomal RNA sequencing opened the treasure chest of microbial diversity. Be- cause of the relatively stable nature of rRNA (rRNA genes rarely, if ever, are transferred horizontally between unrelated organisms), these sequences provide the new phylogeny of microbial relationships. But these genes do not encode enzymes and thus are not directly related to the interface between the organism and its environment, i.e., they may not reflect directly the selective pressures that shape the physiology of the organism. The sequence of rRNA genes alone does not provide direct information about the metabolic capabilities of the cell from Access provided by Princeton University Library on 01/23/20. For personal use only. which they are derived. Only a few groups exhibit such high coherence between Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org phylogeny and physiology that the rRNA sequence is uniquely identified with a metabolic functionality. For these groups, it is sufficient, at least initially, to detect them using unique 16S rRNA sequences, and on the basis of 16S rRNA sequence alone, to infer the presence of their associated metabolism. There are several examples of monophyletic groups that have important roles in biogeochemistry. The cyanobacteria are one diverse but deeply branching phy- lum in the 16S rRNA tree of life; all members of the cyanobacteria are single- celled photosynthetic organisms. Two cyanobacterial genera, Synechococcus and Prochlorococcus, constitute the most abundant photosynthetic microbes on Earth. They were discovered in the ocean as recently as 1979 (Waterbury et al. 1979) 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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and 1988 (Chisholm et al. 1988), using microscopy and flow cytometry, respectively. Both genera of picoplanktonic cyanobacteria contain diverse ecotypes with widespread geographic occurrence (Rocap et al. 2002). Prochlorococcus occurs ubiquitously in surface waters between 40◦N and 40◦S. Cultured strains verify that temperature is probably the critical factor in limiting its poleward distribution (Partensky et al. 1999). Synechococcus occurs more widely, although its abundance decreases with decreasing temperature below 14◦C (Li 1998). At their maximum concentrations, Synechococcus and Prochlococcus can attain cell densities of several hundred thousand cells per ml of surface seawater, and Prochlorococcus is generally tenfold more abundant than Synechococcus (Partensky et al. 1999). These densities make these two tiny prokaryotes the most abundant photosynthetic organisms on Earth, and their contribution to global photosynthetic carbon fixation may exceed 50% of the total marine contribution (Maranon et al. 2001). Prochlorococ- cus is the smallest photosynthetic cell known and probably represents the minimal size and minimal genome required for photosynthetic life. Complete genomes for both Prochlorococus and Synechococcus have been sequenced and are beginning to shed light on niche differentiation and regulatory structure for these important microbes (Palenik et al. 2003, Rocap et al. 2003). Sequence analysis of the ribosomal genes, intergenic spacer regions, RNA polymerase, and photosynthetic genes all suggest that both Synechococcus and Prochlorococcus contain a great deal of intra-generic genetic diversity, and the significance of this diversity in terms of ecosystem function is currently under investigation. Even though all members of the two genera are assumed to be photosynthetic, there is already documented evidence of their differential metabolic capabilities regarding nitrogen uptake and motility, and the vastly different genomic compositions imply many additional inter-generic differences will be found. It was relatively recently that the deep subsurface of Earth was recognized to contain viable microbial communities. The deep subsurface is characterized by absence of light and oxygen, presence of high heat and pressure, abundance of re- duced substrates, but scarcity of organic carbon. Thus it is not surprising that this environment is characterized by microbial assemblages with metabolic characteristics similar to those found in the limited and extreme environments of Earth’s surface, such as anaerobic muds, hot springs, and hydrothermal vents. Cultivation Access provided by Princeton University Library on 01/23/20. For personal use only. of microbes from such environments is very challenging, and study of their genes is Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org about the only way to identify them and their metabolic capabilities. Based largely on the composition of the clone library that resulted from using PCR to amplify 16S rRNA genes from bulk DNA extracts, Chapelle et al. (2002) concluded that the deep subsurface community retrieved from hydrothermal waters circulating through deep igneous rocks was dominated by hydrogen-utilizing methanogenic bacteria (Table 1). The capability for methanogensis among cultivated organisms is restricted to subgroups of the Archaea; the similarity of the retrieved 16S rRNA sequences to those of known methanogens allowed the conclusion that organisms with similar metabolism dominated this subsurface community. In the absence of light and organic compounds for food, the community derives its energy from the oxidation of hydrogen by CO2 to produce methane and water. 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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TABLE 1 Biogeochemical transformations investigated in recent molecular ecological studies Process Net reaction

Methanogenesis (hydrogen utilizing) 4 H2 + CO2 → CH4 + 2H2O Autotrophic aerobic nitriﬁcation + → − + + + A. Ammonia oxidation NH3 1.5 O2 NO2 H2O H − + → − B. Nitrite oxidation 2 NO2 O2 2NO3

Autotrophic aerobic methanotrophy CH4 + 2O2 → CO2 + 2H2O − + + +→ + Denitriﬁcation 4 NO3 5(CH2O) 4H 2N2 2 CO2 + 7H2O + Nitrogen ﬁxation 2 N2 + 3CH2O + 2H2O + 4H → CO2 + + 4NH4 + = → = + Sulfate reduction 2H2 SO4 S 4H2O + + − → + Anaerobic ammonia oxidation (anammox) NH4 NO2 N2 2H2O = + → − + − + Anaerobic methane oxidation coupled to SO4 CH4 HCO3 HS 2H2O sulfate reduction

Aerobic heterotrophy CH2O + O2 → CO2 + H2O

The autotrophic aerobic nitrifying bacteria (oxidation of ammonium to nitrite and of nitrite to nitrate), the autotrophic aerobic methanotrophs (oxidation of methane to CO2), and a few other groups are distinguishable on the basis of 16S rRNA with high degrees of certainty. But for many biogeochemical functions (e.g., Table 1), such as denitriﬁcation (respiratory reduction of nitrate to nitrogen gas), general aerobic heterotrophy (oxidation of organic carbon compounds to support growth at the expense of oxygen), and hydrocarbon degradation (usually oxidative degradation of hydrocarbons, both natural and anthropogenically produced), the capability for the function is present in many organisms that are not closely related on the basis of rRNA sequences. Conversely, organisms that have very similar 16S rRNA sequences can have very different metabolic capabilities. This is due to the ability of bacteria to respond to selective forces by

Access provided by Princeton University Library on 01/23/20. For personal use only. acquiring or losing gene function by horizontal gene transfer and the capacity for Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org rapid evolution over many short microbial generations. For processes that involve many interregulated genes, such as nitrogen fixation and photosynthesis, the ability to perform those processes is not easily lost or acquired, and thus the functional groups are relatively coherent with the groups identified by 16S rRNA phylogenetic analysis. The sulfate-reducing bacteria (SRB) are an interesting example. This is a highly constrained metabolism, in that it confers important environmental and physiological capacities on the organism to tolerate anoxia and high levels of sulfide. In the 16S rRNA tree of life, four major phyla contain sulfate-reducing groups, three in the Bacterial domain and one in the Archaeal domain (Castro et al. 2000). The 16S rRNA phylogenies of the four groups each show rather tight clustering, so that 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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all members of the cluster are SRB, even though each cluster falls within a larger group of Bacteria or Archaea whose members share some features in common (e.g., spore formation) but are not SRB. Thus, 16S rRNA genes from uncultivated organisms that fall within these clusters can be assumed to represent SRBs, depending on the clarity of cluster definition. Interestingly, the key functional genes involved in the sulfate reduction pathway appear to have moved laterally among the groups, even between Bacteria and Archaeal lineages (Klein et al. 2001, Friedrich 2002) (see below for functional genes). Twonovel metabolisms have recently been discovered and the microbial agents responsible for them have been identified using 16S rRNA sequences, even though the organisms have not yet been brought into pure culture. In the case of anaerobic ammonium oxidation, a cell enrichment from a bioreactor in which the process was occurring was found to be comprised of >80% of a single cell type (Jetten et al. 1998). Its rRNA sequence identified it as a member of the Planctomycetales, a Bacterial group characterized by unique cellular structures. Such structures were detected by electron microscopy in the enrichment culture, and sequences similar to the 16S rRNA sequence derived from the original enrichment have now been reported in many bioreactor samples (Toh et al. 2002). Detection of sequences very similar to this unique sequence in the oxygen minimum zones of the Black Sea (Kuypers et al. 2003) and the Gulfo Duce (South America) (Dalsgaard et al. 2003) is taken as evidence that similar bacteria are responsible for anaerobic ammonia oxidation in these natural environments as well. Similarly, the anaerobic oxidation of methane has been shown to be associated with clusters of cells that contain two main 16S rRNA gene types (Boetius et al. 2000). One is closely related to known SRB and the other to known methanogens. The two organisms are thought to grow in very close association so that the hydrogen concentration in the immediate vicinity of the methanogenic cells is main- tained at very low levels owing to its consumption by SRB. In this way, the typical methanogenic reaction to produce methane from CO2 and H2 essentially runs in reverse and methane is oxidized anaerobically (Table 1). Interestingly, both of these newly recognized anaerobic oxidation processes are thought to depend on consortia, which means that cultivation of the individual members and elucidation of the metabolic pathways involved by classical approaches would be extremely Access provided by Princeton University Library on 01/23/20. For personal use only. difficult. Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org The monophyletic groups described above tend to be associated with metabolisms that represent major genomic commitments and obligate the cell to complex highly regulated biochemistry, e.g., methanogensis, photosynthesis, aerobic nitrification. In contrast, for highly inducible processes that involve few genes and are potentially less essential for the cell, such as denitrification and degradation of individual hydrocarbon substrates, mutation, and gene transfer are more likely to lead to variation in gene sequence and gene content, thus loosening the link between 16S rRNA phylogeny and metabolic function. The necessity for specific electron donors and regulatory interactions generally precludes direct substitution of single genes among strains, but transfer of partial operons could account for 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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the cassette-like nature of gene organization in denitriﬁcation, for example, and implies the relative ease of horizontal gene transfer for this process. A focus on the genes, known as functional genes, which encode the essential enzymes themselves, is necessary to link the microbes with the biogeochemical transformation of interest.

Focus on the Process: Functional Genes Microbial ecologists are beginning to investigate the diversity and function of microbes involved in biogeochemical cycles based on information from their functional genes. Basic biochemistry to purify and characterize the enzyme of interest is usually required initially because it is necessary to know the sequence of at least one functional gene before it is possible to develop molecular probes for unknown members of a functional group. Thus, this approach is not fully culture independent because the initial gene sequence is usually derived from a cultivated microbe (just as the initial database for the development of universal rRNA primers was developed from the culture collection). Nevertheless, using probes and primers developed on the basis of a few initial sequences, it has been possible to detect many functional genes in the environment, and once again to detect organisms whose sequences are different from any cultured organisms. The functional genes that have received the most attention are those that encode specific enzymes that are unambiguously linked to a specific process of importance in the environment, such as the examples in Table 2. Even in the case of monophyletic groups, the use of functional genes increases the resolution of diversity and function studies because the rate of mutation and divergence of functional genes generally exceeds that of ribosomal genes. Thus the diversity of a functional group can be described with greater resolution on the basis of functional enzyme encoding genes than using 16S rRNA genes of the same group (Rotthauwe et al. 1997, O’Mullan & Ward 2005). Probes developed from the sequences of functional genes have been used to detect similar genes in the environment, without having to enrich for and cultivate organisms involved in the process of interest. Perhaps the most deeply investigated at present are the genes involved in the anaerobic respiratory pathways of denitri- Access provided by Princeton University Library on 01/23/20. For personal use only. fication and sulfate reduction. In both cases, it is the second step in the multistep Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org pathway that is the focus of most molecular studies. Nitrite reductase (both the Fe and the Cu forms, encoded by nirS and nirK, respectively) and nitrous oxide reductase (nosZ) are unique to the denitrification pathway. For sulfate reduction, the enzyme sulfite reductase is thought to be unique to the pathway (although it occurs in some organisms that begin the pathway with reduction of sulfite, rather than sulfate). The following examples are dominated by the N cycle (Figure 2, see color insert), but similar progress has been made in other biogeochemical processes, such as the genes in the sulfate reduction pathway (Friedrich 2002, Klein et al. 2001, Perez-Jiminez et al. 2001), methane oxidation (Murrell & Radajewski 2000), 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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TABLE 2 Functional genes that are used to study biogeochemical processes (with examples of their application in the marine environment where possible) Enzyme Gene Pathway References

Nitrite reductase nirK, nirS Denitrification Braker et al. 2000, 2001; Jayakumar et al. 2004; Prieme et al. 2002 Nitrous oxide nosZ Denitrification Scala & Kerkhof 1999, 2000 reductase Ammonia mono- amoA Ammonia Kowalchuk & Stephen 2001, Francis oxygenase oxidation et al. 2003, O’Mullan & Ward 2004 Nitrogenase nifH Nitrogen fixation Zehr et al. 2001, 2003; Jenkins et al. 2004 Dissimilatory dsrAB Sulfate reduction Perez-Jiminez et al. 2001, Dhillon sulfite reductase et al. 2003, Fukuba et al. 2003 Methane mono- pmmo Methane oxidation Costello & Lidstrom 1999, Murrell & oxygenase Radajewski 2000 Ribulose rbcL Photosynthetic Paul et al. 2000a, Wawrik et al. 2003 bis-phosphate carbon fixation carboxylase Catechol 2,3 xylE Aromatic Mesarch et al. 2000, Siciliano et al. di-oxygenase hydrocarbon 2003 degradation

methanogenesis (Garcia et al. 2000), and hydrocarbon degradation (Junca & Pieper 2004, Meyer et al. 1999).

DENITRIFICATION In denitrification, oxides of nitrogen, including nitrate, nitrite, and nitric and nitrous oxides, are used in respiration in place of oxygen by facultative anaerobic bacteria. The end product of complete denitrification is N2 gas (Figure 2), and this process represents the most important loss of fixed nitrogen from the environment (Figure 3, see color insert). It is essential in water treatment Access provided by Princeton University Library on 01/23/20. For personal use only. because it removes the oxidized nitrogenous nutrients from waste water before Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org it is returned to the environment, where excess N loading leads to eutrophication and undesirable alteration of natural communities. The same process in agriculture leads to loss of applied N fertilizer, an added biological and economic cost. In the ocean, denitrification in a few regions is responsible for system-wide N limitation. Because of its importance in natural and constructed ecosystems, the factors that control denitrification have been well studied and its regulation in the environment is of some significance. These are questions that can be addressed explicitly and in mechanistic ways using molecular genetic information. The study of denitrification raises many issues that are common in biogeochemical systems. Denitrification is performed by a diverse range of different kinds of 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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microbes (whose 16S rRNA sequences place them widely across all three domains of life), including representatives of many phyla that appear to share little else. Therefore, it is necessary to focus on a gene directly involved in denitrification (Figure 2) to investigate the diversity of denitrifying bacteria in the environment, and to link their distributions and activity to the process itself. Nitrite reductase is the first committed step in the denitrification pathway because it is at this point that the nitrogen leaves the dissolved solute pool and becomes a gas, a major change affecting N availability for other microbes. There are two basic kinds of nitrite reductase enzymes, one with Cu in the active site and one that contains only Fe. Aside from the fact that the mechanism of reduction is different between the two, there appears to be little of physiological significance to distinguish them, and indeed geneticists have shown that in some cases, one enzyme is functional in the genetic background of organisms that normally express the other (Glockner et al. 1993). nirS, which encodes the Fe-type enzyme, occurs predominantly in the Proteobacteria, one of the major divisions in the Bacterial domain. nirK, which encodes the Cu-type enzyme, is more widely distributed, and has been reported in Proteobacteria, many other Bacterial groups, Archaea, and even in fungi (Zumft 1997). Using PCR primers to retrieve nirS genes from various marine and terrestrial environments, it has been shown that nirS is ubiquitous and that a surprisingly large variety of nirS genes can be found even in a relatively small sample. Braker et al. (2000) retrieved several groups of nirS genes from marine sediments, including some that were essentially identical with the most commonly cultivated marine denitrifier, and used a terminal restriction pattern analysis to compare their distribution (Braker et al. 2001). In a study that relied more heavily on detailed sequence analysis (C.A. Francis & B.B. Ward, in preparation), approximately 600 nirS fragments were sequenced from clone libraries based on 1.5 g of sediment from each of five sites in Chesapeake Bay. This combined dataset appears to en- compass the full range of nirS diversity, including the cultivated organisms and sequences obtained from other environments. Many of the sequences, while ob- viously homologous with known nirS sequences, are not very closely related to sequences from cultivated denitrifiers. If the different nirS genes belong to different kinds of microbes, and assuming the sequences represent potentially active Access provided by Princeton University Library on 01/23/20. For personal use only. genes, there is an immense diversity of microbial life involved in denitrification, Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org a huge guild of apparently redundant microbial capacity. At present, it has not been ascertained (nor is it a simple matter to do so) whether the different genes are expressed or regulated under different conditions. It could be argued that the sequence variability we find is not functional, but rather represents random neutral mutation that does not affect the enzyme function and therefore does not represent truly functional diversity. One counter to this argument is the large degree of sequence diversity that occurs in patterns not likely to be random or neutral, and the detection of similar sequence groups in ecologically similar but geographi- cally distant environments. For example, in the first report of nirS sequences from a marine water column (all previous reports were from sediment environments), 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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many novel nirS sequences were described from shallow coastal Arabian Sea waters (Jayakumar et al. 2004). Nevertheless, some of the Arabian Sea sequences showed high similarity to nirS sequences derived from cultures and from marine sediment environments in coastal North America and in English stream sediments. If it were not functionally significant why would this diversity persist in coherent evolutionary patterns? The PCR approach has been less effective for nirK, probably because it occurs more widely among different groups and is itself more diverse. Although several sets of PCR primers have been published and applied to both marine and terrestrial environments, fewer total sequences have been obtained. Whether this reflects the limited occurrence of the Cu variant of the enzyme or a limitation of the sequence database for primer development is unknown. In a terrestrial environment, nirS could not be detected in forest soils, but extensive diversity in nirS genes was found in marsh sediments (Prieme et al. 2002). nirK was detected in both forest and marsh, but in overall less diversity, which suggests that the total nirK assemblage is not adequately sampled by any single set of PCR primers. In addition to metabolic diversity, the possession of nirK versus nirS imposes slightly different trace metal requirements on the organisms. Almost all organisms require iron, and bacteria can be limited by iron availability (Tortell et al. 1996). The requirement for copper as a cofactor for redox enzymes is less common than the requirement for iron, and copper is in fact toxic to many organisms. Denitrifiers, however, can be limited by copper availability in culture (Granger & Ward 2003) and it is hypothesized that copper availability may control the rate of denitrification or the nature of its terminal products in some environments. nosZ is the gene that encodes the enzyme nitrous oxide reductase, which catalyzes the last step in complete denitrification to N2 (Figure 2). It is a copper- containing enzyme that is absent in fungal and Archaeal denitrifiers, which generally stop at the production of N2O. Scala & Kerkhof (2000) used the diversity of nosZ gene fragments to investigate patchiness in the diversity and distribution of denitrifying bacteria in marine sediments. They found that the greatest variation occurred between the most distant samples (at 1-km scales), but was also large between seasons at the same site. In terrestrial systems, different groups of denitrifiers, represented by nosZ genes, occurred in forest and meadow soils, Access provided by Princeton University Library on 01/23/20. For personal use only. and community composition was implicated in the patterns of denitrification rates Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org observed (Rich et al. 2003). Thus the apparent redundancy represented by many different versions of the same gene may in fact represent ecological resilience to change in environmental factors that act selectively on different members of the same functional group, i.e., denitrifiers.

NITRIFICATION Nitrification is sometimes considered to be the opposite of denitrification because it is an oxidative rather than reductive process involving nitrate and nitrite (Figures 2 and 3). Conventional nitrification involves two groups of organisms, the ammonia-oxidizing bacteria (AOB), which oxidize ammonia to nitrite via hydroxylamine, and the nitrite-oxidizing bacteria (NOB), which oxidize 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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nitrite to nitrate. Both groups are predominantly or obligately autotrophic, using the reducing power of their nitrogenous substrates to fix CO2 via the Calvin-Benson cycle. This is a notoriously poor way to make a living because the oxidations of ammonia and nitrite are relatively low energy yielding, and the requirement to fix CO2 adds to the metabolic cost, compared to utilization of preformed organic matter. The capability to exploit these energy sources presumably constitutes an ecological advantage, however, that only a few species have evolved to exploit. Nitrification does not affect the budget of fixed nitrogen in the environment, but does change its form and thus its availability to other organisms. By consuming ammonia, which being positively charged tends to stick to soil particles, and releasing it as nitrate, whose negative charge tends to partition it into the water phase, nitrification decreases the efficiency of fertilizer additions. It also supplies the substrate for denitrification, and the linkage of these two processes is responsible for the net loss of fixed N in soil and aquatic environments. The question of most obvious economic significance for nitrification is its essential role in sewage treatment and water quality. The nitrate produced by nitrifiers is a favored substrate for water plants and algae, thus a prime cause of eutrophication of coastal waters. Denitrification removes the nitrate and thus lowers the net N load. Thus the diversity, interactions, and regulation of these groups are important questions in both natural and human-made systems (Figure 3). The problems faced in the study of nitrification are quite different from the challenges of denitrification. Current evidence is that the aerobic autotrophic nitrifiers—conventional nitrifiers—occur in relatively low abundance in natural environments (Ward 2002b). Cultivated strains, both AOB and NOB, grow very slowly (generation times on the order of a day), so isolation, cultivation, and in vitro experimentation with nitrifiers is a slow, time-consuming process. Prior to the introduction of molecular methods in microbial ecology, nitrification was a difficult process to study, primarily owing to the slow growth of the organisms in culture and the necessity to use stable isotopes (rather than radioisotopes) to make direct measurements of transformation rates in natural samples. The nitrifiers have avery restricted phylogeny (i.e., the ability to nitrify is found in only a few groups within the Bacterial domain), especially the AOB, and their 16S rRNA phylogeny is essentially coherent with that derived from the key functional gene, ammonia Access provided by Princeton University Library on 01/23/20. For personal use only. monooxygenase (amoA) (Purkhold et al. 2000, 2003) (Figure 2). This has made Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org the nitrifiers very attractive and amenable to detection and study by molecular methods, and much has been learned recently of their diversity and ecology. Most attention has focused on the AOB, and much more on the Betaproteobac- terial group than on the other major group of AOB, the Gammaproteobacteria. The process of ammonia oxidation was slow to yield to classical biochemical investigations of its mechanism due to the interdependent electron transfers involved in the oxidation of ammonia to hydroxylamine and then to nitrite. However, the initial purification and partial sequencing of the ammonia monooxygenase protein (McTavish et al. 1993) led rapidly to the development of DNA probes for PCR amplification. Most of the many recent surveys of the diversity of AOB in the 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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environment, including investigation of the response of the assemblage to changes in ammonium levels or to land use changes, have used exclusively 16S rRNA sequences. In the few cases that attempted to link information from both 16S rRNA and amoA genes, it appears that the 16S rRNA and amoA sequences were derived from the same population. The ecology of nitrifier diversity was recently reviewed (Kowalchuk & Stephen 2001); different groups appear to dominate different major environments (e.g., freshwater versus seawater versus soil), but in most environments, the local assemblages are quite diverse and may contain representatives of many phylogenetically defined clusters. Essentially, the entire range of amoA diversity was encompassed in a set of clone libraries from Chesapeake Bay sediments, except that very few sequences closely related to cultivated strains were found (Francis et al. 2003). Given the limited ecological niche of nitrifiers, there would appear to be relatively few environmental parameters that could define microniches to sustain high species diversity over small spatial scales. Therefore, small-scale temporal variability on small spatial scales in parameters such as ammonium and oxygen concentration are invoked to explain the persistence of multiple species assemblages in environments that appear to be relatively constant. Small-scale variability was invoked to explain the cooccurrence of intermingled clusters of different AOB species (defined by 16S rRNA sequence) in bioreactor biofilms (Gieseke et al. 2001). It may also be responsible for the substantial variability in amoA sequences over the space of ten vertical meters in a relatively well-mixed marine water column (O’Mullan & Ward 2005). Casciotti et al. (2003) directly addressed the question of how the sequence of the functional gene amoA might affect the product formed. They showed that the isotopic enrichment factor, ε,of15Ninammonia and nitrite in the oxidation reaction varied systematically in relation to the gene/protein sequence—organisms whose sequences clustered together in the amoA phylogenetic tree had similar ε’s and ε differed by more than twofold among phylogenetically defined clusters (Figure 4, see color insert). This ε difference could be diagnostic for identifying which organisms are responsible for particular transformations and pinpointing the source of particular compounds in the environment. It was concluded that the N2O found in the surface ocean was probably due to nitrification by marine strains of Nitrosomonas, which have quite different ε than the model terrestrial Access provided by Princeton University Library on 01/23/20. For personal use only. AOB, Nitrosomonas europaea (Casciotti et al. 2003). In the case of AOB, the Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org 16S rRNA and amoA gene phylogenies have parallel topology, so that variation in ε for ammonium oxidation is correlated with both. Interestingly, variation in ε for sulfite oxidation was not correlated with16s rRNA gene phylogeny in SRB (Detmers et al. 2001), probably owing to lateral gene transfer of the functional gene dsr across distant phylogenetic boundaries (Klein et al. 2001).

NITROGEN FIXATION The functional gene for which the greatest number of partial gene sequences has been obtained from the environment is nifH, which encodes the second major component of the nitrogen ﬁxation enzyme complex, nitrogenase (Figure 2). Because nitrogen is the element most likely to limit primary production 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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in both terrestrial and aquatic systems, the study of nitrogen fixation has always been a high priority in microbial ecology. Measurement of nitrogen fixation rates is difficult technically and is usually accomplished by the proxy measurement of acetylene reduction (the enzyme nitrogenase reduces acetylene in parallel with dinitrogen, and its product, ethylene, is easily quantified by gas chromatography). On the ecosystem scale, nitrogen fixation is implicated by N:P ratios in biomass that are higher than can be explained easily by the availability of inorganic N nutrients such as nitrate. Based on conventional rate measurements and cultivation of microbes, it appears that the capability for nitrogen fixation is more limited than might be expected from the prevalence of nitrogen limitation in nature. Thus important questions about nitrogen fixation include, How widespread across the microbial world is the capability for nitrogen fixation? What environmental factors regulate the expression of N fixation genes, the activity of the N fixation enzymes, and thus the rate of fixation? These questions have been addressed powerfully through the study of genes involved in N fixation. Using PCR, as described above, to obtain nifH sequences from many different environments, it has been shown that the genetic capability for N fixation is common in many environments, even those where inorganic N is abundant. For example, diverse nifH genes apparently derived from most major groups (including cyanobacteria, aerobic heterotrophic proteobacteria, and anaerobic types typical of anoxic sediments) have been reported from eutrophic estuaries (Zehr et al. 2003), and the number of different groups represented is greater than in the oligotrophic ocean, where N limitation is expected to be a major selective factor. The capability to fix nitrogen requires several genes and is highly regulated, thus implying a serious genomic and physiological commitment to the maintenance of the capability. It therefore seems likely that complex regulation and variable environmental conditions ensure that the capability is not readily lost. The ocean is N limited over vast expanses of surface water, and it has long been a mystery why so few obvious N-fixing microbes have adapted to existence there. The major N-fixing organism is a single genus of nonheterocystous filamentous cyanobacteria, Trichodesmium.Itwas suspected that additional N-fixing organisms were present but had remained undetected because it was too difficult to measure the process directly or to figure out which microscopic cells might be doing it. Access provided by Princeton University Library on 01/23/20. For personal use only. This mystery was elucidated by showing that the N-fixation genes were not only Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org present but expressed in unicellular cyanobacteria from the equatorial surface 15 15 ocean (Zehr et al. 2001). Conversion of N2 to N-biomass by these cells was also demonstrated, showing definitively that organisms in addition to Trichodesmium contribute to the flux of new nitrogen into surface waters in N-limited oceans.

PHOTOSYNTHESIS The most abundant enzyme on Earth is ribulose bis phosphate carboxylase (RubisCO), the initial enzyme in the Calvin-Benson cycle that is responsible for most photosynthetic carbon ﬁxation on Earth today. Marine phytoplankton contribute about half of the annual oxygen production for Earth’s atmosphere, and in so doing, constitute a massive sink for CO2 and a possible route 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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to long-term carbon sequestration. Because different phytoplankton have different nutritional requirements and capabilities (including N fixation), the overall rate of photosynthesis depends on which microscopic plants are involved in CO2 fixation in the ocean. The genes (rbcL) that encode the large subunits of RubisCO are highly conserved throughout evolution and exhibit phylogenetic patterns that make it possible to identify the kind of organism from which the gene was derived without directly culturing that organism. This approach has been powerfully exploited to explore the composition of the photosynthetic assemblage of ocean waters (Paul et al. 2000b) and to assess its response to changing environmental conditions (Paul et al. 2000a). As for nirS and nifH above, the genetic exploration has yielded sequences that undoubtedly encode rbcL genes but do not belong to organisms represented in the culture collection. This group also showed that dis- tinctly different subsets of rbcL genes, associated with different phytoplankton groups such as green algae, prasinophytes, prymnesiophytes, and cyanobacteria, were distributed and expressed in different patterns throughout the water column and in relation to distance from the Mississippi River plume in the Gulf of Mexico (Wawrik et al. 2002). This level of functional resolution is difficult to attain by classical cultivation or microscopic approaches, and the specific identification of which groups are active (denoted by gene expression) is powerful new information that could not be obtained in other ways. These genetic approaches are also amenable to high throughput assays (see below) that can exploit the growing sequence database.

HowMany Species Are There, or Does Microbial Diversity Matter to Ecosystem Function? Both nosZ and nirS/nirK databases mentioned above imply that denitrifiers com- prise a huge and extremely diverse group of species. Although the questions of how to define a species and how many species exist are of philosophical and sci- entific importance (D. Ward 1998, Hughes et al. 2001, Curtis et al. 2002, B.Ward 2002a), they have very practical implications as well. To compare the diversity or species composition of two different samples, it is necessary to know how well

Access provided by Princeton University Library on 01/23/20. For personal use only. the attempts to measure diversity have described the community from which the

Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org samples are taken. For example, if the community of denitrifiers is comprised of 1000 species or (because it is difficult to apply the standard species definition to microbes) operational taxonomic units (OTUs), and the subsample from each of two sites detects ten species, then there is little basis for comparison because nei- ther site has been adequately sampled. Attempts to compute how many species of denitrifiers there are can be based on diversity in the nirS gene. Using a sequence identify difference of as much as 15% to define an OTU for the nirS gene yielded estimates on the order of thousands in Chesapeake Bay alone (G.A. Jackson, in preparation). In the case of denitrifiers, it appears that even 1000 gene sequences for individual functional genes may not be adequate to describe the community of a single site. This level of diversity within the functional guild denitrifiers was 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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not suspected before the advent of gene sequencing. These observations of apparently great sequence diversity and large functional redundancy have been made repeatedly for many of the functional genes in Table 2 as their databases grow and receive intense scrutiny. Although substantial sequence diversity has been observed at the functional gene level in AOB, it appears that the nitrifiers are less diverse overall than the denitrifiers. This conclusion is obvious at the culture level and at the level of 16S rRNA, and it has implications for ecological studies. “Species” or OTU abundance curves derived from amoA sequences tend to converge on the truncated Gaussian curve, which is expected to describe species that are lognormally distributed (Curtis et al. 2002; G.A. Jackson, in preparation). This implies that for amoA,afew hundred sequences are sufficient to sample the most abundant types from any particular environment. By contrast, even several hundred sequences are not sufficient to describe the composition of the denitrifer assemblage based on nirS or nirK sequences. The implication for ecological work is that much deeper sequencing is needed, at least for some initial studies, to determine the limits of functional diversity. By analogy, only in the simplest systems is there any chance that a normal clone library will sample the essence of the community on the basis of its 16S rRNA genes. The significance of functional diversity and its relationship to ecosystem function is one of the paradigmatic questions of modern microbial ecology. If these thousands of gene sequences represent biologically distinct organisms, then biogeochemistry depends on the integrated activity of many partially redundant microbes and the regulation by environmental factors and response to environmental change may be extremely complex.

BIOGEOCHEMISTRY FROM GENES

The most successful molecular studies so far have provided huge amounts of new information about the diversity and distribution of functional genes and the microbial guilds associated with them. Quantiﬁcation of the number of organisms (proportional to the number of genes of a particular type) is the next step, and

Access provided by Princeton University Library on 01/23/20. For personal use only. several approaches to quantiﬁcation have been made. Early attempts used quan-

Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org titative hybridization with 16S rRNA genes. In this method, the intensity of a radioactive or fluorescent signal is proportional to the amount of complementary (matching) sequence from a sample that binds to a known sequence (Sahm et al. 1999, MacGregor et al. 2001). Within the limits of its selective bias, PCR can also produce quantitative results. An example of one approach, competitive PCR, is the estimation of the abundance of functional genes associated with nitrification in waste water treatment plants (Dionisi et al. 2002). Very sensitive detection limits (10 to 100 gene copies) were demonstrated using competitive PCR to detect the gene encoding the ring-breaking step in BTEX compounds in soils (Mesarch et al. 2000). The most recent application of quantitative PCR is the real time PCR (RT-PCR) approach in which the accumulation of amplified product is monitored 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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throughout the reaction, which by comparison with calibrated standards, allows the determination of the initial number of copies of the target gene that were present before amplification [e.g., aromatic oxygenase genes (Baldwin et al. 2003), nifH genes (Short et al. 2004)]. This approach is also useful for quantification of mes- senger RNA (see below). Another way to associate activity specifically with genes is the stable isotope pairing method, in which a substrate (e.g., 13CO) is incubated with a complex sample. After incubation, the rRNA from the whole assemblage is extracted and the heavier molecules, which have incorporated the label, are sepa- rated by ultracentrifugation. PCR can then be used to identify which components have incorporated the label, and can provide at least comparative quantification of incorporation rates and comparison of active members of different communities (Whitby et al. 2001, Manefield et al. 2002).

Gene Expression Detecting the number of functional genes present in a sample provides information on the genetic capacity for the function encoded in those genes but does not neces- sarily provide information on whether the organisms that possess those genes are active at the time. Gene expression is under the control of regulatory mechanisms in the cell that respond to environmental cues; some genes are expressed constitutively, i.e., whenever the cell is active, but most genes are expressed at variable levels depending on the activity level or particular physiological needs of the cell. Quantification of gene expression, as well as gene abundance, should provide information on which particular organisms are active under which environmental conditions. For example, most denitrifying bacteria are facultative and can persist utilizing oxygen instead of nitrate for respiration when sediments become aerated after strong mixing. In that case, nirS or nirK gene abundance alone would give an overestimate of the active population size. Some of the most impressive quantification results have been obtained in studies of the eukaryotic phytoplankton communities of surface seawater. The gene that encodes RubisCO is very common and abundant in surface waters, thus providing the best signal-to-noise ratio for quantitative approaches. Wawrik et al. (2003) quantified mRNA levels for three different forms of rbcL genes, which represent Access provided by Princeton University Library on 01/23/20. For personal use only. three different taxonomic groups of phytoplankton. The distributions of mRNA Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org levels as a function of depth in the water column were distinct from the depth distributions of cell number (quantified by flow cytometry for cells containing one RubisCO form) and photosynthetic pigments (quantified by HPLC; different pigments are associated with different kinds of phytoplankton). This implies that the different phytoplankton groups were differentially affected by environmental variables, such as the inflow of water from the Mississippi River plume, light, and 14 nutrient levels. Carbon fixation rates (as measured by CO2 incorporation) and mRNA levels in the various groups were not highly correlated. The measured C fixation rate represents the overall photosynthetic production of the entire assemblage, which contains members that were not detected by the mRNA analysis. The 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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power of the mRNA analysis, however, is the ability to detect patterns of activity within different components of the assemblage. For genes that are inducible and whose expression responds directly to environmental conditions, it is unlikely that gene expression should relate directly to rates of processes catalyzed by the encoded enzyme. Typically, the level of mRNA over the time course of induction of gene expression and enzyme activity shows very low or zero levels of constitutive expression, a large peak in mRNA level as the gene expression responds to a specific induction (e.g., disappearance of oxygen in the presence of nitrate in the case of denitrification), followed by much lower levels of mRNA as the process continues after initial induction under favorable conditions. This pattern implies that high levels of mRNA signals will only be observed during transition phases and that relatively low mRNA levels will be associated with the highest rates of corresponding enzyme activity, i.e., after initial induction of gene expression, enzyme level is high but mRNA level is low. The relationship between mRNA levels and enzyme activity no doubt varies with the gene/enzyme system and may be more straightforward for genes that are constitutively expressed. It will rarely if ever be the case that quantification of mRNA levels will allow direct quantification of the corresponding enzymatic reaction. Therefore, if the goal of biogeochemistry is to determine the rates of transformations in the environment, then molecular biology at present does not contribute directly. If the goal is, however, to understand those rates and the factors both biotic and abiotic that may regulate them, then molecular biology is probably the only direct approach to the problem. The exploration of functional gene diversity represents the second step of the molecular revolution in biogeochemistry. A major remaining challenge is to link the two pieces of information, i.e., to link diversity and function in the environment. At the molecular level alone, it is difficult to know whether functional and ribosomal genes, independently retrieved from the same sample, represent the same organisms. The uncertainty arises partly owing to the shear size and com- plexity of microbial communities. The total number of bacteria per gram of soil or sediments is often on the order of 109 cells, whereas 106 cells can be found in a milliliter of surface sea or lake water. The number of species or OTUs in that same sample is variously estimated to range from a few hundred to many thousand Access provided by Princeton University Library on 01/23/20. For personal use only. (Hughes et al. 2001, Curtis et al. 2002). Thus two clone libraries of 100 16S rRNA Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org genes and 100 nirS genes each, for example, could contain gene fragments from completely unrelated members of the community, even at the scale of 1 g or 1 ml of sample. If the gene sequences in the clone libraries are distinctive enough, i.e., uniquely characteristic of particular organisms, then it is possible that they are indeed derived from the same cells or related cells. For the monophyletic group, the Betaproteobacterial AOB, this may be possible. Caffrey et al. (2003) reported that both16S rRNA and amoA sequences obtained from estuarine mud samples were most closely related to the same cultivated Nitrosomonas marina strain, and deduced that organisms of this kind dominated the clone library and possibly the environment. O’Mullan & Ward (2005) analyzed the degree of sequence variation 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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in 16S rRNA and amoA sequences obtained with AOB-specific primers and concluded that they were likely derived from the same AOB population in Monterey Bay seawater. However, this kind of analysis is not possible for most genes and most groups, and still does not physically link the two genes. In the case of the phytoplankton discussed above, it was assumed that the rbcL gene sequences contained sufficient phylogenetic information, i.e., it was not necessary to have both 18S/16S rRNA information and the rbcL gene information to identify the players. As discussed above, the link between community composition and overall rate of C fixation was shown to be variable and implied differential responses of various members of the assemblage. Wawrik et al. (2002) used quantitative RT-PCR to evaluate the abundance of mRNA from rbcL genes of diatoms. In a culture of Phaeodactylum tricornutum, mRNA levels varied on a diel basis, being highest in the morning. RubisCO enzyme activity and C fixation rates, however, were highest in the afternoon, illustrating that different kinds of information are deduced from gene expression and enzyme activity measurements. Quantitative (Q)RT-PCR is a powerful method for quantification of specific mRNA molecules, but the method requires the use of specific primers. The degenerate primers that are so powerful for detecting related, but not identical, genes in the diversity studies described above are not always directly adaptable for QRT-PCR. Thus the target group must be carefully defined before comparisons can be made between activity, diversity, and gene expression assays.

Genomics With the advent of mass sequencing facilities (and their decreased level of use after the completion of the human genome sequencing projects), it has become surprisingly routine to obtain the sequence of entire microbial genomes. Most Bacteria and Archaea have one chromosome per cell and the total size of the genome varies from slightly over 1 MB (megabase, 1 × 106 base pairs) to several MB. Almost 200 microbial genomes have been completed (The Institute for Genomic Research Comprehensive Microbial Resource, http://www.tigr.org/tigr- scripts/CMR2/CMRGenomes.spl), and the early analysis of these data provides

Access provided by Princeton University Library on 01/23/20. For personal use only. some fascinating and surprising insights. Even when two organisms of the same

Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org genus were sequenced, for example, two strains of the picoplanktonic phototrophs Prochlorococcus,itwas found that approximately one third of their total genomic complement was entirely different from each other (Rocap et al. 2003). Approxi- mately one third of the total contained recognizable genes related to known genes, and the last third of the sequences could be recognized as probable genes but of so far unidentiﬁed function. The degree of unknown or unrecognized sequence decreases with every subsequent sequencing project, but every genome appears to contain unique sequences. Even among the similar organisms, the arrangement of genes within the chromosome varies greatly. This suggests the occurrence of evolutionary events in which genes or operons were lost entirely, leading to loss of function and thus to niche specialization (Rocap et al. 2003). 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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Temperature is one of the most important variables because all biological processes and organisms typically survive over a limited temperature range. As of this writing, 16 Archaeal genomes have been completed, and they include organisms with temperature optima ranging from 0◦Cto100◦C. The genomes of two cold-adapted Archaea were compared with those of the other, higher-temperature Archaea to discover the mechanism of temperature adaptation (Saunders et al. 2003). Genome-wide differences that may be associated with adaptation to cold were found, including trends in amino acid and transfer (t)RNA composition and differences in protein structure and composition. The opposite analysis, to identify the features that allow life at extreme high temperatures, has obvious economic as well as ecological implications. Decoding the genomes of cultivated organisms continues to yield both expected and surprising results. For example, as expected, the genome of the terrestrial AOB, Nitrosomonas europaea, contains all the genes required for an obligately chemolithoautotrophic life using energy only from ammonia oxidation and carbon only from CO2 ﬁxation (Chain et al. 2003). However, the genes for a complete tricarboxylic acid cycle were also present—absence of one or more TCA cycle genes had been suspected as the reason that AOB are incapable of heterotrophic growth on preformed organic carbon. Either one or more of these genes are non- functional or there must be some other factor that constrains N. europaea to this lifestyle. Even the complete genome does not contain all the information needed to understand the life of the cell. The next stage in exploration is proteomics, or the study of the interacting function of the proteins produced by the cell. Nevertheless, it is clear that genomics of cultivated organisms, although currently providing a torrent of new information, will not be sufﬁcient to understand the genetics of natural populations given the unculturability and immense diversity of microbes in nature.

Environmental Genomics A more direct linkage between function and phylogeny in natural assemblages is possible using metagenomic approaches. A very powerful method involves the

Access provided by Princeton University Library on 01/23/20. For personal use only. use of bacterial artiﬁcial chromosomes (BAC), which can be used to clone very

Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org large DNA fragments. DNA extracted from environmental samples is fragmented and cloned without prior screening or amplification with specific primers. Thus, although BAC libraries may have their own biases and limitations, they avoid the bias of PCR in which something must be known of the target sequence to find it in the first place. These large pieces of DNA in BAC libraries can then be screened for targets similar to known genes, either by amplification with gene-specific primers or by hybridization with gene probes. The first efforts screened for the 16S rRNA genes themselves and then investigated nearby functional genes. BACs can be used to amplify very large fragments of DNA, up to 200 kbp. An average prokaryotic gene is approximately 1000 bp, so such a fragment might contain 200 genes, all derived in one linear piece from the same organism. Therefore, if a ribosomal gene 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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and a functional gene are found on the same BAC, they were both derived from the same organism. The opposite approach can also be used—screen for sequences with homology to known functional genes to investigate their genetic background. When a recognizable gene is found, the host DNA fragment can be sequenced to find out what kind of genes are nearby. If one of those nearby genes is a 16S rRNA gene, then the phylogeny of the organism from which the gene was obtained can be deduced directly. Regardless of the 16S linkage, sequencing the vicinity of a known functional gene can shed light on the operonal organization of the genome from which the functional gene was derived. Beja et al. (2000) were the first to apply this approach to a BAC library derived from environmental samples. One of their initial goals had been to learn more about bacteria whose 16S rRNA sequences were commonly retrieved from seawater but whose identify and metabolic characteristics were unknown. Beja et al. (2001) screened the BAC library using primers specific for rRNA fragments of the uncultivated organisms, based on sequences that had been obtained previously from 16S rRNA clone libraries. In a BAC library of 6240 clones, two were found to contain 16S rRNA fragments closely related to SAR86 sequences. SAR86 was another group whose 16S rRNA sequences were commonly retrieved from seawater but which had no cultivated close relatives (Mullins et al. 1995). After further sequencing of the DNA in the same BAC, Beja et al. (2001) discovered a gene with high homology to the rhodopsin gene of Archaea, which encodes a protein used in light sensing and proton transport. This new proteorhodopsin gene, however, was linked to the 16S rRNA gene of an uncultivated Bacterium rather than an Archaeon. Beja et al. (2001) went further to show that proteorhodopsin is widespread in marine bacteria, and this suggests that a photo-assisted metabolism may be prevalent and important in the energetics of marine systems. Liles et al. (2003) screened a BAC library of 24,400 clones from soil and found 28 16S genes. They compared the phylogenetic affiliations of these genes with 16S rRNA genes retrieved by direct PCR and cloning using universal 16S rRNA primers. Most of the groups shown by previous 16S clone libraries to be common in soils were detected in the BAC library, but the relative proportion of representation of some groups differed significantly. It is likely that both approaches confer bias on the results; direct PCR is much more efficient at retrieving target genes but yields Access provided by Princeton University Library on 01/23/20. For personal use only. no ancillary information on the other characteristics of the organisms from which Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org the genes are derived. BAC clones, in contrast, provide access to genes that are physically linked to the 16S rRNA genes. This study (Liles et al. 2003) focused on the Acidobacterium division, which is important in acid soil environments. Although 16S rRNA gene sequences associated with this group are often obtained with high representation in clone libraries, most subgroups identified by sequence analysis have no cultured representatives. Thus by sequencing the same BAC insert from which an Acidobacterium 16S rRNA gene was obtained, genes in 20 additional operons were characterized. By homology with genes of known function, genes associated with cell cycling and cell division, substrate metabolism, amino acid uptake, and DNA repair, among others, were identified. Although very 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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labor intensive, this approach is a powerful one for revealing the physiology and metabolism of uncultivated organisms. Subsequent mass sequencing efforts of BAC libraries have yielded tantalizing clues as to the genomic make up of natural samples using the approach described above to screen for genes with known homologies as a starting point. In this effort, the ends of the DNA fragments that are directly linked to the BAC vector were sequenced as a screening method to look for interesting genes for further investigation. Clearly, this kind of approach requires a huge sequencing capacity for exploration. A recent example of the mass sequencing approach targeted the Sargasso Sea again (Venter et al. 2004) and more than 1 Gbp of total DNA that had been extracted from samples of up to 200 liters of Sargasso Sea water were cloned and sequenced. Approximately 25% of the 1.66 million sequences obtained could be assembled into contiguous sequences of more than one gene, i.e., possibly linked in a chromosome. This demonstrates that even massive random sequencing has a long way to go before genes can be recognized at the organism level and before organisms can be assembled from their genes alone. Over a million genes were recognized as protein coding units, and less than 500,000 could be assigned putative identity by homology with known genes. The distribution of the putative known genes was nevertheless intriguing; for example, over 800 sequences of the proteorhodopsin discovered by Beja et al. (2001) were detected in the Sargasso Sea metagenome.

Linking Genes and Processes Linking genetic information and biogeochemical transformation processes is addressed presently through correlation or interpretation of parallel experiments in which gene diversity and or gene expression is measured on the same samples 14 that are subjected to incubation-based rate measurements (such as CO2 uptake for primary production, 15N-based methods for nitrification and nitrogen fixation, N2O accumulation in the presence of acetylene for denitrification, or acetylene reduction for nitrogen fixation). Early hopes that gene expression, as quantified by the amount of mRNA, would yield direct measurement of rate processes were na¨ıve; although correlations between levels of gene expression and rates may oc- Access provided by Princeton University Library on 01/23/20. For personal use only. cur, they are unlikely to be robust (see above for discussion of the usual pattern of Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org induction of gene expression). Despite and because of the revolutionary advances in molecular microbial ecology and biogeochemistry, we are left with the recognition that most current studies present only a snapshot in time and space. Without historical data (history being the past five minutes to two weeks or whatever the relevant timescale of response by the organisms of interest), it is difficult to infer cause and effect. For example, does the presence of large population levels of certain microbes at one point in time imply that the coexisting environmental conditions are favorable for that organism? Or is it more likely that the current conditions are not favorable, and that a crash is about to occur because the conditions of the day before were very favorable 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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buthavenow changed, perhaps even as a result of the organism modifying its environment? The labor-intensive nature of making and analyzing clone libraries has meant that snapshots are often the limit of our view of the environment and has provided the impetus for the development of high-throughput methods. Such methods might be able to detect the instantaneous response to environmental change in terms of gene expression or to describe the species composition or the active membership of a sample in near real time or at least rapidly, making it possible to collect and analyze many samples with high spatial and temporal resolution. Genes expressed in real time could be detected in concert with changing environmental conditions to study regulation of biogeochemical processes in situ. Microarrays are one such high-throughput approach that have been developed and tested in a number of formats and that promise to introduce a higher-density and higher-resolution study of the environment. Microarrays are simply glass slides onto which fragments of genes have been bound in a pattern of many tiny dots. When complementary DNA or RNA from the sample, which has been labeled with a fluorescent tag, hybridizes to matching sequences and binds to the dots, the pattern of fluorescence identifies which genes were present in the sample by their location on the array. The most common kind of array is one in which each dot represents a different gene from the same organism, a genome array. These arrays are used to investigate the response of the organism to some set of or change in environmental conditions. mRNA is collected from the culture under defined conditions, transcribed into DNA, and bound to the array. The genes that were being expressed at the time can thus be identified. For example, this approach has been very powerful in medical applications in identifying the genes involved in various kids of pathologies and cancer. The approach is beginning to be applied to environmental organisms as the genomes for microbes become available. Most of the published examples concern pathogens, but this is a very active area of environmental microbiological research. A second kind of array contains different fragments of DNA or many different versions of the same gene from different organisms. This kind of array is used to identify which kinds of organisms are present in the sample when sample DNA is hybridized, and it could be used to examine gene expression in natural samples when mRNA, transcribed into DNA, is hybridized. The most common application Access provided by Princeton University Library on 01/23/20. For personal use only. of multigene microarrays so far is a diversity survey method, a replacement for Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org clone libraries. One approach (Cho & Tiedje 2001) used fragments of bacterial genomic DNA as the probes on microarrays. The pattern of hybridization when DNA from cultures or complex natural mixtures of genomic DNAs are used as targets can be interpreted to determine the taxonomic identify of the target DNA. This kind of approach might have potential for rapid description of community composition. Loy et al. (2002) developed a microarray based on nested probe sets with various levels of specificity for the 16S rRNA genes of all the major SRB groups. The design ensured that each target would bind with several different probes in a diagnostic pattern. For example, one of the organisms that was detected in a cyanobacterial mat from a solar lake environment was a Desulfonema species. The hybridization 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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pattern included positive signals from the specific probes for that particular group of Desulfonema strains, as well as the more general probe for a cluster of several genera closely related to Desulfonema, and also to the much more general probes to which all sulfate-reducing members of the Deltaproteobacteria should bind. Adamczyk et al. (2003) used an array containing 16S rRNA probes specific for various groups of AOB to assess activity of different AOB types in activated sludge 14 samples. The unknown target sample was incubated with CO2, and the total RNA was extracted. After hybridization with the array, the amount of radioactivity detected in the probe dots was quantified, providing an indication of what kinds of organisms were active in the incubated sample. This creative attempt to link diversity and function suffered from sensitivity limitations but could avoid some bias associated with amplification of target DNA from natural samples. The functional genes associated with particular processes again offer a direct approach to linking diversity and function. Functional gene microarrays contain many versions of different functional genes representing different organisms. Be- cause most functional genes occur in one or only a few copies in the cell (rRNA genes occur in up to 13 copies per cell), this kind of array has better prospects for quantification in terms of cell numbers representing each type of gene. Taroncher- Oldenburg et al. (2003) demonstrated the specificity of microarray containing 70-mer oligonucleotides representing functional genes for amoA, nirS, nirK, and nifH, and showed that different communities of denitrifiers could be detected in Chesapeake Bay sediments on the basis of nirS diversity. A similar microarray containing many different versions of the methane monooxygenase gene was used to characterize methanotrophic communities in landfill samples (Bodrossy et al. 2003, Stralis-Pavese et al. 2004). In a slightly different format (Jenkins et al. 2004, Steward et al. 2004), arrays containing phylogenetically diagnostic patterns of nifH genes were used to investigate the diversity of nitrogen-fixing microbes in Chesapeake Bay. Tiquia et al. (2004) constructed a very large microarray containing many different functional genes and used it to detect organisms involved in several different biogeochemical cycles in soils. At present, the functional gene arrays have been limited to detection of genes and inference about processes, but the hope is that they will be able to detect mRNA and thus to provide direct information about which groups of microbes are active in the sample. Detection Access provided by Princeton University Library on 01/23/20. For personal use only. limits and problems with obtaining good quality RNA from some sample matrices Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org presently limit this application. Methods such as QRT-PCR (above) remain the best way to assess gene expression, and they are not presently high-throughput methods owing to the nature of the probes and the protocols required.

CONCLUSIONS

The introduction of molecular methods and approaches into microbial ecology and the study of microbially mediated processes in environmental biogeochemistry has changed the ﬁeld of endeavor at every level. New technologies are now required for cutting-edge research, and questions not even imagined previously 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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are now routinely addressed. The microbial world is recognized as immensely more diverse than previously thought, and processes only vaguely imagined have been linked to novel organisms and shown to represent important ﬂuxes in the environment. Some of the important questions raised by this revolution include the following: What is the evolutionary and ecological signiﬁcance of microbial diversity? How can studies at the molecular level contribute to quantitative understanding of biogeochemical processes and their environmental regulation? We are enjoying a deluge of new information, much of which remains to be comprehended and linked to the fundamental processes of biogeochemistry.

The Annual Review of Earth and Planetary Science is online at http://earth.annualreviews.org

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The ammonia monooxygenase structural plication in analysing landfill methanotroph gene amoA as a functional marker: molec- communities under different plant covers. ular fine-scale analysis of natural ammonia- Environ. Microbiol. 6:347–63 oxidizing populations. Appl. Environ. Micro- Taroncher-Oldenburg G, Griner EM, Francis biol. 63:4704–12 CA, Ward BB. 2003. Oligonucleotide mi- Sahm K, MacGregor BJ, Jorgensen BB, Stahl croarray for the study of functional gene DA. 1999. Sulphate reduction and verti- diversity in the nitrogen cycle in the environ- cal distribution of sulphate-reducing bacteria ment. Appl. Environ. Microbiol. 69:1159– quantified by rRNA slot-blot hybridization 71 in a coastal marine sediment. Appl. Environ. Tiquia SM, Wu L, Chong SC, Passovets S, Xu Microbiol. 1:65–74 D, et al. 2004. Evaluation of 50-mer oligonu- Saunders NFW, Thomas T, Curmi PMG, cleotide arrays for detecting microbial pop- Mattick JS, Kuczek E, et al. 2003. Mecha- ulations in environmental samples. Biotech- nisms of thermal adaptation revealed from niques 36:664–75 the genomes of the Antarctic Archaea Toh SK, Webb RI, Ashbolt NJ. 2002. En- Methanogenium frigidum and Methanococ- richment of autotrophic anaerobic ammonia- coides burtonii. Genome Res. 13:1580–88 oxidizing consortia from various wastewa- Scala DJ, Kerkhof LJ. 1999. Diversity of ni- ters. Microb. Ecol. 43:154–67 trous oxide reductase (nosZ) genes in con- Tortell PD, Maldonado MT, Price NM. 1996. tinental shelf sediments. Appl. Environ. Mi- The role of heterotrophic bacteria in iron- crobiol. 65:1681–87 limited ocean ecosystems. Nature 383:330– Scala DJ, Kerkhof LJ. 2000. Horizontal hetero- 32 geneity of denitrifying bacterial communi- Venter CJ, Remington K, Heidelberg JG, ties in marine sediments by terminal restric- Halpern AL, Rusch D, et al. 2004. Environ- tion fragment length polymorphism analysis. mental genome shotgun sequencing of the Appl. Environ. Microbiol. 66:1980–86 Sargasso Sea. Science 304:66–74 Short SM, Jenkins BD, Zehr JP. 2004. Spatial Ward BB. 2002a. How many species of and temporal distribution of two diazotrophic prokaryotes are there? Proc. Natl. Acad. Sci. bacteria in Chesapeake Bay. Appl. Environ. USA 99:10234–36 Microbiol. 70:2186–92 Ward BB. 2002b. Nitrification in aquatic sys- Siciliano SD, Germida JJ, Banks K, Greer tems. In Encyclopedia of Environmental Mi- CW. 2003. Changes in microbial community crobiology, ed. DA Capone, pp. 2144–67. composition and function during a polyaro- New York: Wiley matic hydrocarbon phytoremediation field Ward DM. 1998. A natural species concept for trial. Appl. Environ. Microbiol. 69:483– prokaryotes. Curr. Opin. Microbiol. 1:271– 89 77 Access provided by Princeton University Library on 01/23/20. For personal use only. Smith LM, Sanders JZ, Kaiser RJ, Hughes P, Waterbury JB, Watson SW, Guillard RRL, Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org Dood C, et al. 1986. Fluorescence detection Brand LE. 1979. Widespread occurrence of in automated DNA-sequence analysis. Na- a unicellular, marine, planktonic, cyanobac- ture 321:674–79 terium. Nature 277:293–94 Steward GF, Jenkins BD, Ward BB, Zehr Wawrik B, Paul JH, Campbell MJ, Griffin L, JP. 2004. Development and testing of a Houchin L, et al. 2003. Vertical structure DNA macroarray to assess nitrogenase (nifH) of the phytoplankton community associated gene diversity. Appl. Environ. Microbiol. 70: with a coastal plume in the Gulf of Mexico. 1455–65 Mar. Ecol. Progr. Ser. 251:87–101 Stralis-Pavese N, Sessitsch A, Weilharter A, Wawrik B, Paul JH, Tabita FR. 2002. Real- Reichenauer T, Riesing J, et al. 2004. Op- time PCR quantification of rbcL (ribulose- timization of diagnostic microarray for ap- 1,5-bisphosphate carboxylase/oxygenase) 18 Mar 2005 11:48 AR AR233-EA33-10.tex XMLPublishSM(2004/02/24) P1: KUV

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mRNA in diatoms and pelagophytes. Appl. Woese CR, Magrum LJ, Fox GE. 1978. Archae- Environ. Microbiol. 68:3771–79 bacteria. J. Mol. Evol. 11:245–52 Whitby CB, Hall G, Pickup R, Saunders JR, Zehr JP, Jenkins BD, Short SM, Steward GF. Ineson P, et al. 2001. C-13 incorporation 2003. Nitrogenase gene diversity and mi- into DNA as a means of identifying the ac- crobial community structure: a cross-system tive components of ammonia-oxidizer popu- comparison. Environ. Microbiol. 5:539–54 lations. Lett. Appl. Microbiol. 32:398–401 Zehr JP, Waterbury JB, Turner PJ, Montoya Woese CR. 1987. Bacterial evolution. Micro- JP, Omoregie E, et al. 2001. Unicellular biol. Rev. 51:221–71 cyanobacteria fix N2 in the subtropical North Woese CR, Fox GE. 1977. Phylogenetic struc- Pacific Ocean. Nature 412:635–38 ture of the prokaryotic domain: the pri- Zumft WG. 1997. Cell biology and molecular mary kingdoms. Proc. Natl. Acad. Sci. USA basis of denitrification. Microbiol. Mol. Biol. 74:5088–90 Rev. 61:533–616 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 HI-RES-EA33-Ward.qxd 3/29/05 10:21 AM Page 1

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Figure 1 Schematic representation of a bacterial cell. The circular chromosome contains the DNA, which encodes both rRNA and functional genes. Several genes, including the 16S rRNA gene, are transcribed to make up the ribosome. The DNA is also transcribed into mRNA, which is translated by the ribosomes to make proteins, including the enzymes that catalyze biogeochemical transformations. Enzymes are shown catalyzing reactions within the cell, transporting materials into and out of the cell, and exchanging substrates (S) and products (P) between the cell and its environment. Information at the DNA level (i.e., gene sequences) is used to identify organisms by phylogenetic analysis and to investigate diversity. The presence of mRNA corresponding to particular functional genes implies active gene expression and is used to identify active organisms and processes. 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 HI-RES-EA33-Ward.qxd 3/29/05 10:21 AM Page 2

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Figure 2 Schematic of the microbial N cycle, showing the major processes and the functional genes that are used to study these processes. narB encodes the assimilato- ry nitrate reductase in cyanobacteria (the similar gene in eukaryotic algae is referred to as NR, and in heterotrophic bacteria as nasA). nirA encodes the siroheme nitrite reductase in plants and algae, which catalyzes the six-electron reduction of nitrite to Access provided by Princeton University Library on 01/23/20. For personal use only. ammonium. amoA encodes the ammonia monooxygenase enzyme that catalyzes the Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org first step in chemoautotrophic ammonia oxidation to hydroxylamine. hao encodes hydroxylamine oxidoreductase, which catalyzes the further oxidation of hydroxylamine to nitrite in ammonia oxidizing bacteria. nirK and nirS encode the copper and heme-type dissimilatory nitrite reductases, respectively, which catalyze the reduction of nitrite to nitric oxide in denitrifying and nitrifying bacteria. norB encodes nitric oxide reductase, which reduces NO to N2O. nosZ encodes nitrous oxide reductase, which catalyzes the final step in denitrification leading to N2. The two arrows labeled aax represent anaerobic ammonia oxidation, for which the enzymes and genes are currently unknown. nifH encodes Component II of nitrogenase, the central enzyme in nitrogen fixation. HI-RES-EA33-Ward.qxd 3/29/05 10:21 AM Page 3

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Figure 3 Nitrogen cycle processes pictured in a sediment/water interface environment. Aerobic processes (conventional nitrification) occur in the water and at the interface. The same nitrogen compounds used in nitrification are exchanged with the sediments (dotted lines imply diffusion), where denitrification and anaerobic ammonia oxidation (red arrows) both lead to the production of N2. The linkage of these processes leads to the loss of fixed nitrogen from the system. Because the same compounds serve as both products and substrates for different organisms, it is difficult to determine which reactions control net nitrogen fluxes. Both isotopic and molecular methods have been crucial in disentangling the transformations. 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 HI-RES-EA33-Ward.qxd 3/29/05 10:21 AM Page 4

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Figure 4 Phylogenetic tree based on a DNA fragment encoding 177 amino acids deduced from sequences of the amoA gene from chemoautotrophic ammonia-oxidizing bacteria. Nodes that are supported by bootstrap values >70% are marked with filled circles and weaker nodes (<70%) with open circles. The scale bar indicates 1% substitution per amino acid site. Isotope effects were measured in cultures of representatives (colored names) of several clusters and indicate the fractionation that occurs in the process of oxidation of ammonia to nitrite (assuming a pseudo one step reaction in which no fractionation is associated with the transitory production of NH2OH). The degree of fractionation parallels the variation in amino acid sequence of the enzyme, suggesting a direct link between genetic diversity and biogeochemical function. With permission from Casciotti et al. (2003). 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 P1: KUV March 23, 2005 16:53 Annual Reviews AR233-FM

Annual Review of Earth and Planetary Sciences Volume 33, 2005

CONTENTS

THE EARLY HISTORY OF ATMOSPHERIC OXYGEN:HOMAGE TO ROBERT M. GARRELS, D.E. Canﬁeld 1 THE NORTH ANATOLIAN FAULT:ANEW LOOK, A.M.C. S¸engor,¬ Okan Tuys¬ uz¬ , Caner Im˙ ren, Mehmet Sakõnc¸, Haluk Eyidogˇan, Naci Gor¬ ur,¬ Xavier Le Pichon, and Claude Rangin 37 ARE THE ALPS COLLAPSING?, Jane Selverstone 113 EARLY CRUSTAL EVOLUTION OF MARS, Francis Nimmo and Ken Tanaka 133 REPRESENTING MODEL UNCERTAINTY IN WEATHER AND CLIMATE PREDICTION, T.N. Palmer, G.J. Shutts, R. Hagedorn, F.J. Doblas-Reyes, T. Jung, and M. Leutbecher 163 REAL-TIME SEISMOLOGY AND EARTHQUAKE DAMAGE MITIGATION, Hiroo Kanamori 195 LAKES BENEATH THE ICE SHEET:THE OCCURRENCE,ANALYSIS, AND FUTURE EXPLORATION OF LAKE VOSTOK AND OTHER ANTARCTIC SUBGLACIAL LAKES, Martin J. Siegert 215 SUBGLACIAL PROCESSES, Garry K.C. Clarke 247 FEATHERED DINOSAURS, Mark A. Norell and Xing Xu 277 MOLECULAR APPROACHES TO MARINE MICROBIAL ECOLOGY AND THE MARINE NITROGEN CYCLE, Bess B. Ward 301 EARTHQUAKE TRIGGERING BY STATIC,DYNAMIC, AND POSTSEISMIC STRESS TRANSFER, Andrew M. Freed 335

Access provided by Princeton University Library on 01/23/20. For personal use only. EVOLUTION OF THE CONTINENTAL LITHOSPHERE, Norman H. Sleep 369 Annu. Rev. Earth Planet. Sci. 2005.33:301-333. Downloaded from www.annualreviews.org EVOLUTION OF FISH-SHAPED REPTILES (REPTILIA:ICHTHYOPTERYGIA) IN THEIR PHYSICAL ENVIRONMENTS AND CONSTRAINTS, Ryosuke Motani 395 THE EDIACARA BIOTA:NEOPROTEROZOIC ORIGIN OF ANIMALS AND THEIR ECOSYSTEMS, Guy M. Narbonne 421 MATHEMATICAL MODELING OF WHOLE-LANDSCAPE EVOLUTION, Garry Willgoose 443 VOLCANIC SEISMOLOGY, Stephen R. McNutt 461

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x CONTENTS

THE INTERIORS OF GIANT PLANETS:MODELS AND OUTSTANDING QUESTIONS, Tristan Guillot 493 THE Hf-W ISOTOPIC SYSTEM AND THE ORIGIN OF THE EARTH AND MOON, Stein B. Jacobsen 531 PLANETARY SEISMOLOGY, Philippe Lognonne« 571 ATMOSPHERIC MOIST CONVECTION, Bjorn Stevens 605 OROGRAPHIC PRECIPITATION, Gerard H. Roe 645

INDEXES Subject Index 673 Cumulative Index of Contributing Authors, Volumes 23Ð33 693 Cumulative Index of Chapter Titles, Volumes 22Ð33 696

ERRATA An online log of corrections to Annual Review of Earth and Planetary Sciences chapters may be found at http://earth.annualreviews.org 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