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> Section V. Ex amples of Diversit y

> Chapter 10. Microbial Communities Oceanography > A . Water Column Microbial Domains in the : , Volume 2, a quarterlyOceanography 20, Number S The journal of A Lesson from the By Edward F. DeLong

New Views of Sea Microbes (, Archaea, and Eukarya), it is “fishing expeditions” pioneered by Norm Microbial thrives in virtually every only recently that a clear picture of “who Pace and Stephen Giovannoni, and later imaginable in the ocean, from the where” in the ocean has emerged. others, in the early 1990s, proved to be scalding temperatures found at hydro- From the standpoint of oceanogra- reasonably productive enterprises. A ociety. C thermal vents, to frigid environments in phy, why should we care about microbial strong motivation for these microbial and under polar sea ice, to high-pressure Oceanography S opyright 2007 by The in the ocean’s deepest trenches. Our understanding of microbial life in many of these ocean habitats, especially in , has advanced remarkably Our understanding of microbial life in over the past 30 years or so. The recogni- many of these ocean habitats, especially tion of the ubiquity and distribution of in plankton, has advanced remarkably P ociety. Allreserved. rights photoautotrophic such ociety. S as Synechococcus and Prochlorococcus over the past 30 years or so. end all correspondence to:Oceanography S [email protected] Th e or (Johnson and Sieburth, 1979; Waterbury et al., 1979; Chisholm et al., 1988), the of pressure-requiring piezo- ermission is granted to in teaching copy this and research. for use article R philic bacteria (Yayanos et al., 1979), the diversity or microbial taxonomic dis- surveys was the general recognition that discovery of Pelagibacter (Giovannoni tributions and ? As a fledg- microbial activities drive most of the et al., 1990), and recognition of the high ling Assistant Scientist at the Woods major biogeochemical cycles in the sea. abundance of marine phage (Bergh et Hole Oceanographic Institution in Furthermore, it was suspected that many al., 1989) represent just a few recent the late 1980s, I remember my chagrin dominant planktonic microbial groups milestones in microbial oceanography. while pondering critical reviewer com- might be undetected because of their Even at a level as fundamental as the dis- ments on my (failed) grant proposals recalcitrance to cultivation. Given the tribution of life’s three major domains that aimed to survey marine microbial “great plate count anomaly” (e.g., the ociety, P diversity, which read something like this: observation that cultivable planktonic O Box 1931, R O Box Edward F. DeLong (delong@ “DeLong is out on a fishing expedition, microbes accounted for only a small mit.edu) is Professor, Department of without any hypothesis.” At the , percentage of total direct epifluores- ockville, MD 20849-1931, US ockville, Biological Engineering and Department some of us wondered: If you don’t really cence microscopic counts [Staley and epublication, systemmatic , of Civil and Environmental Engineering, know what lives in the ocean, might Konopka, 1985]), it seemed quite pos- Massachusetts Institute of Technology, not a little fishing be a good idea? As it sible that dominant planktonic micro- Cambridge, MA, USA. turned out, the early oceanic microbial bial groups, some responsible for critical A.

124 Oceanography Vol. 20, No. 2 biogeochemical cycling processes, likely of marine microbial life and led to the to most other life forms. The existence remained unknown. This presumption discovery of a new microbial taxo- of novel archaeal types was first hinted turned out to be more or less correct. nomic group in the sea, the planktonic at during cultivation-independent A fundamental advance that acceler- Crenarchaea. The pattern of initial ribosomal RNA surveys in open-ocean ated relatively unbiased microbial cen- discovery using these techniques, and and coastal marine waters. Initial work sus taking was the invention by Norm subsequent in-depth biological and eco- used the polymerase chain reaction Pace and collaborators of cultivation- logical characterization, is now a recur- (PCR) to amplify ribosomal RNA independent, molecular-phylogenetic survey approaches (Olsen et al., 1986). This strategy uses common molecular sequences found in every (e.g., ribo- ...it is only recently that a clear picture of somal RNA sequence) that can serve “who lives where” in the ocean has emerged. as a sort barcode to identify and track microbes by “reading” DNA sequences extracted directly from the environ- ment, without the need for cultivation. ring theme in marine microbial . from mixed microbial populations. In Such cultivation-independent surveys While this story represents only one 1992, Jed Fuhrman of the University taught us that large amounts of micro- example, it illustrates how characteriza- of Southern California first reported bial diversity found in natural habitats tion of dominant microbial inhabitants the existence of a new type of archaeal had totally slipped beneath the radar of in the sea can lead to new insights into ribosomal RNA sequence from deep- cultivation-based approaches. Indeed, global biogeochemical processes. As water planktonic microbes in the Pacific some of the most abundant microbial well, the important interplay and syn- Ocean (Fuhrman et al., 1992). At about groups on our planet have been discov- ergy between cultivation-dependent and the same time, I independently discov- ered using such molecular-based surveys, cultivation-independent approaches for ered and reported on the distribution and had not been evident from cul- characterizing marine microbes in the and abundance of two different coastal ture-based studies. Characterizing these wild is quite evident (see also the article archaeal groups, one related to the deep- is critical for gaining a by Giovannoni et al., this issue). water archaea (planktonic Crenarchaea) deeper and truer understanding of native and another, new group (planktonic microbial inhabitants and their funda- Oceanic Archaea? Euryarchaea) that were phylogenetic mental environmental activities (see The Archaea are a curious phyloge- neighbors to and methano- below). Together, both cultivation-based netic (formerly ) gens (DeLong, 1992). I was also able to and cultivation-independent approaches, comprised of an odd assortment of demonstrate quantitatively that marine which now extend to environmental cultured microbes that fall into three archaea contribute significantly to genomic sequencing surveys, are yielding major groupings: extreme halophiles, marine microbial plankton . significant contributions to our under- , and extreme thermo- The surprise then was that any standing of microbial taxa and activities philes and (Woese, archaea could be found in cold, aero- in the deep blue sea. 1987). Why such an odd assortment of bic habitats of coastal and open-ocean The impact of cultivation- -loving, or anaerobic, or heat-lov- waters—and, to top it off, they were independent surveys of microbes in the ing microbes should form such a coher- abundant. No cultivated, character- environment has been well reviewed ent phylogenetic grouping is still not ized archaea were known to grow at the (Rappé and Giovannoni, 2003). The that well understood. The dogma until combined , temperature, and following story is one tale of how this 1992 was that archaea inhabit mainly concentration found in tem- approach altered our understanding “extreme” environments, inhospitable perate oceanic waters, shallow or deep.

Oceanography June 2007 125 of the total cell counts from water depths of 80–3000 m. At the Hawaii Ocean Time-series (HOT) sta- tion ALOHA, Dave Karl and collabora- tors showed that Crenarchaea comprised as much as 30% of the total microbial counts in deep waters below the euphotic zone (Karner et al., 2001). In aggregate, these and other data suggest that pelagic Crenarchaea comprise a significant pro- portion of overall planktonic microbial biomass throughout the world’s ocean.

Biology and Ecology of Planktonic Marine Crenarchaea Creative application of biochemical, geochemical, and genomic techniques have provided considerable data on the planktonic Crenarchaea. These stud- Figure 1. Epifluorescence micrograph ofCenarchaeum symbiosum, a symbiotic marine crenarchaeon (green cells) closely related to planktonic archaea. The green fluorescence is derived from fluorescein- ies, combined with isolation in pure labeled, rRNA-targeted probes used for in situ nucleic hybridization to identify archaeal cells. culture of a marine crenarchaeon (see below), now provide some specific clues regarding the biogeochemical and eco- logical importance of this abundant Following on the heels of these first oce- planktonic Crenarchaea were found to marine microbial group. anic sightings, archaeal groups began contribute as much as 20% to the total analyses of cold marine sedi- cropping up in many unexpected habi- microbial rRNA in late-winter Antarctic ments by Jaap Damste’s group at the tats. The next steps were to quantify the coastal waters at -1.8°C (DeLong et Netherlands Institute for Sea Research distribution and abundance of these al., 1994). Surveys in temperate waters (NIOZ) revealed a first for this envi- unusual microbes and to begin to under- off the coast of California also showed ronment—high levels of tetraether lip- stand their biological properties and that the planktonic Crenarchaea tended ids, that were previously found only in ecological significance. to be most abundant in waters below thermophilic Crenarchaea (Hoefs et al., the euphotic zone (Massana et al., 1997). Stuart Wakeham at the Skidaway Marine Archaeal Abundance, 1997). This trend has generally held Institute of Marine Science and my Distribution, and Variability in a global sense in both coastal and group at the University of California, Once it was clear that archaea were rea- open-ocean settings. Santa Barbara, then showed that marine sonably abundant players in microbial Ribosomal RNA-targeted fluorescence plankton samples with high numbers plankton, scientists initiated studies probes (DeLong et al., 1989) have been of planktonic Crenarchaea also con- that employed radiolabeled, archaea- used to provide estimates of archaeal tained high levels of the same tetraether specific oligonucleotide probes to quan- cell numbers in the water column. Off (DeLong et al., 1998). In addi- tify total, extractable archaeal rRNA in the California coast, for example, plank- tion, Preston et al. (1996) showed that marine plankton (Figure 1). Surprisingly, tonic Crenarchaea represent > 20% symbiosum, a crenarchaeal

126 Oceanography Vol. 20, No. 2 symbiont of marine , contained A the very same tetraether lipids. These EURYARCHAEA could be used to infer the lipid’s detailed “Marine Archaeal Group II” chemical structure. Collectively, these ermoplasmatales data establish that the sources of abun- Figure 2. Phylogeny, lipids, and vertical distribu- dant marine tetraether lipids in the tion of planktonic marine plankton are indeed derived from plank- Crenarchaea. (A) A phylo- tonic Crenarchaea (Figure 2). ermococcales genetic tree showing the Archaeoglobales general affiliations of two A real surprise came when Ann different planktonic archaeal pJP33 Pearson, during her graduate work groups. (B) General struc- ermop with Tim Eglinton at Woods Hole pSL12 tures of abundant tetraether lipids found in Crenarchaea. Oceanographic Institution, provided ro (C) Distribution of plank- radioisotopic data suggesting pelagic teale tonic Crenarchaea and s Euryarchaea in the North Crenarchaea may be chemoautotro- “Marine Archaeal Group I” Pacific Subtropical Gyre, as phic (Pearson et al., 2001). She puri- CRENARCHAEA detected in large DNA-insert fied large amounts of marine archaeal genomic libraries (data from DeLong et al., 2006). lipids from deep-sea surficial derived from the deep-water plank- HO B I O O tonic Crenarchaea. Natural 14C isotope O O OH analyses of archaeal lipids suggested that HO deep-water archaea were not consum- II O O O ing much organic derived from O OH surface primary . Rather, deep-water Crenarchaea appeared to be using dissolved inorganic as their main carbon source. Pearson’s analyses C 10 are now supported by a variety of inde- pendent studies. For example, when Cornelia Wuchter, a graduate student 13 100

at NIOZ, added C-labeled bicarbon- ) ate to a sample from the North Sea, then incubated the in the dark, the pth (m heavy-isotope label from CO was nearly De 2 1000 exclusively incorporated into crenar- chaeal lipids (Wuchter et al., 2003). % Crenarchaea 4000 % Euryarchaea The observation of CO2-fixing Crenarchaea of course immediately 0.25 0.3 led to the question: exactly what are 0.05 0.1 0.15 0.2 0.35 % rRNA-containing fragments these ubiquitous CO2-fixing marine Crenarchaea using as their source? Early clues came from the dis- covery of an monooxygenase in Crenarchaea that encodes a key

Oceanography June 2007 127 used by nitrifiers in the first isolates grew exclusively on ammonia in the water column (Mincer et al., step of ammonia oxidation (Treusch et for energy, producing as the end 2007). Future studies promise to further al., 2005). This new archaeal ammonia product. The archaeal nitrifiers were elucidate the ecophysiological properties monooxygenase gene was subsequently also shown to use CO2 as their carbon of the ubiquitous marine crenarchaeal detected in many marine environments source, and they did not appear capable nitrifiers and to better quantify and as well (Wuchter et al., 2006; Mincer of growing on organic matter. These constrain and CO2 fixation et al., 2007). Genomic analyses subse- data, combined with earlier informa- rates, as well as further characterize their quently revealed many other genes asso- tion on crenarchaeal distributions and ecological interactions. ciated with nitrification and CO2 fixation abundance, indicate that planktonic in marine Crenarchaea (Hallam et al., Crenarchaea are indeed critical players in Conclusion 2006a, 2006b). A definitive demonstra- the ocean’s cycle, and they exert The discovery and ecological character- tion that marine Crenarchaea are indeed a large influence on oceanic nitrification ization of the planktonic marine archaea nitrifiers was achieved by Dave Stahl’s in the sea (Francis et al., 2007). represent but one example in a positive group at the University of Washington, New observations on the biology, and accelerating trend in marine micro- in collaboration with John Waterbury ecology, and activity of planktonic bial ecology. The general story shows the at the Woods Hole Oceanographic Crenarchaea continue to accumulate. utility of cultivation-independent, DNA- Institution (Konneke et al., 2005). In These include detailed quantitative anal- based survey approaches for identifying cultures designed to isolate nitrifying yses modeling in situ carbon sources of and tracking microbes in the environ- bacteria (bacteria that oxidize ammonia planktonic Crenarchaea (Ingalls et al., ment. The interplay between nucleic- obtain energy and utilize CO2 as their 2006), microautoradiography studies to acid-based approaches and geochemi- carbon source), Stahl’s group unexpect- track substrate assimilation into differ- cal and biomarker studies that leverage edly found ammonia-oxidizing cultures ent cell types (Herndl et al., 2005), and stable- and radio-isotopic tracers is also that did not appear to be typical bacte- observations of Crenarchaea in anoxic central to the tale. As well, the important rial nitrifiers—in fact, these nitrifying zones of the Black Sea (Coolen et al., synergy between cultivation-dependent microbes did not even belong to the 2007) and the Arabian Sea (Damste et and cultivation-independent micro- bial surveys is also clear. Other similar parables include the discovery, ecologi- cal characterization, and isolation of the ubiquitous bacterioplankter Pelagibacter ...newly evolving strategies for characterizing (see Giovannoni article, this issue), the microbes in situ and in the lab...will advance recognition of anaerobic ammonia- our knowledge of microbial life and activity oxidizing bacteria in marine oxygen- minimum zones (see Ward et al., this in the global . issue), and the discovery of anaerobic, -oxidizing archaea at methane seeps (Hinrichs et al., 1999). It is clear that combining these and other newly domain Bacteria (Konneke et al., 2005). al., 2002). The potential interactions evolving strategies for characterizing After some major microbe sleuthing, between Crenarchaea and other bac- microbes in situ and in the lab, including Stahl’s group discovered that the new terioplankton have also recently been nanoscale DNA sequencing, proteomics, nitrifiers were, in fact, the same type of suggested by the similar distributions of and single-cell stable isotope analyses, Crenarchaea that are so abundant in bacterial nitrite oxidizers belonging to will advance our knowledge of microbial marine plankton. These crenarchaeal the Nitrospina and to Crenarchaea life and activity in the global oceans.

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