Environmental Diversity of Bacteria and Archaea

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					Syst. Biol. 50(4):470–478, 2001

                          Environmental Diversity of Bacteria and Archaea

                                    EDWARD F. D ELONG 1 AND NORMAN R. PACE2
                 Monterey Bay Aquarium Research Institute, 7700 Sandholdt Rd., Moss Landing, California 95039, USA;
            Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309-0347 , USA

         Abstract.—The microbial way of life spans at least 3.8 billion years of evolution. Microbial organisms
         are pervasive, ubiquitous, and essential components of all ecosystems. The geochemical composition
         of Earth’s biosphere has been molded largely by microbial activities. Yet, despite the predominance of
         microbes during the course of life’s history, general principles and theory of microbial evolution and
         ecology are not well developed. Until recently, investigators had no idea how accurately cultivated
         microorganisms represented overall microbial diversity. The development of molecular phylogenetics
         has recently enabled characterizatio n of naturally occurring microbial biota without cultivation. Free
         from the biases of culture-based studies, molecular phylogenetic surveys have revealed a vast array
         of new microbial groups. Many of these new microbes are widespread and abundant among contem-
         porary microbiota and fall within novel divisions that branch deep within the tree of life. The breadth

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         and extent of extant microbial diversity has become much clearer. A remaining challenge for micro-
         bial biologists is to better characterize the biological properties of these newly described microbial
         taxa. This more comprehensive picture will provide much better perspective on the natural history,
         ecology, and evolution of extant microbial life. [Archaea; bacteria; biodiversity; evolution; microbial;

   Historically, microbial biology has devel-                      virtually unknown and undescribed. Few
oped along research lines largely indepen-                         conspicuous morphological features can
dent of other biological disciplines, mainly                       be used to systematically differentiate mi-
for technical reasons. Simply put, the natu-                       croorganisms or infer their evolutionary
ral microbial world, unlike that of visible or-                    relationships at higher taxonomic levels.
ganisms, cannot be observed in great detail                        Additionally, the microbial fossil record is
by direct methods. The independent devel-                          neither extensive nor informative enough
opment of microbial biology encouraged the                         to provide much insight into ancestral mi-
birth of some elds, a prime example being                          crobial life. Until the mid-1960s, micro-
molecular biology. Meanwhile, however, ap-                         biologists had to be content with sim-
preciation of ecology and evolution in mi-                         ply distinguishing “prokaryotes” (which do
crobial systems has lagged far behind par-                         not possess membrane-bound nuclei) from
allel developments in mainstream biology.                          eukaryotes (which have true nuclear or-
Microbial biologists simply lacked most of                         ganelles). This situation changed dramati-
the classical tools, concepts, and theory avail-                   cally when Zuckerandl and Pauling (1965)
able to paleontologists, systematists, ecolo-                      pointed out that molecules can serve as doc-
gists, and evolutionary biologists. Advances                       uments for evolutionary history. Evolution-
in molecular phylogenetics, macromolecu-                           ary relationships could now be deduced
lar sequencing techniques, and emerging ge-                        from sequence differences observed between
nomic technologies, however, are changing                          homologous macromolecules. For the rst
the playing eld dramatically for microbial                         time, universal comparisons of homologous
biologists (Pace, 1997). Now, serious inroads                      macromolecular features from virtually all
are being made in microbial ecology and                            (known) cellular lifeforms became a practical
evolution, paved largely by the applica-                           reality.
tion of comparative molecular phylogenetic                            Carl Woese was the rst to fully ex-
methods.                                                           ploit the power of molecular phylogenetics
                                                                   for inferring evolutionary relationships be-
                                                                   tween “kingdoms” (deeply related groups),
    T HE T HREE D OMAINS AND M ICROBIAL                            as he and his colleagues sought to create
                 EVOLUTION                                         a uni ed picture of evolutionary relation-
  Until recently, higher-order evolutionary                        ship among prokaryotes in the 1970s (Woese
relationships among microorganisms were                            and Fox, 1977). With remarkable insight, he

2001            DELONG AND PACE—ENVIRONMENTAL DIVERS ITY OF MICROBES                          471

selected perhaps the single most optimal            genealogy of the organisms is extrapolated
macromolecule for establishing deep re-             from the evolutionary trajectory of single
lationships, 16S ribosomal RNA (rRNA).              genes. Depending on the gene used, its par-
Woese realized that the optimal macro-              ticular history (including lateral transfer),
molecule for constructing global phylogenies        and the magnitude of evolutionary distances
should have a universal distribution, high          considered, these extrapolations will vary in
conservation, some moderate variability, and        accuracy. Despite all of the uncertainties and
minimal lateral genetic transfer. Initially, cat-   artifacts associated with single-gene phylo-
alogues of rRNA oligonucleotides were used          genies, the major features of Woese’s rRNA
to infer relationships among disparate phylo-       tree—and its lessons—have overriding rel-
genetic groups. Later, advanced nucleic acid        evance for contemporary biology. Current
sequencing techniques allowed direct acqui-         genomic studies indicate that lateral gene
sition and comparison of rRNA sequences.            transfer may have played a greater role in
One of the rst and most dramatic results of         the evolution of major lineages than was pre-
Woese’s studies was the discovery of a new          viously appreciated (Doolittle, 1999). How-
microbial kingdom, the Archaea (then called         ever, rRNA, and other core genes involved

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archaebacteria; Woese et al., 1978). These          in information transfer, and therefore appear
anucleate microbes are as evolutionarily dis-       not to have been as extensively laterally ex-
tant from common bacteria, as they are from         changed, provide the most coherent frame-
eukaryotes. The representatives of archaea          work for understanding and interrelating the
known in the early 1980s were a fairly bizarre      main evolutionary branches on the extant
collection of microbes: sulfur-respiring ther-      tree of life (Ochman et al., 2000).
mophiles, extreme halophiles, and obligately
anaerobic methanogens. The evolutionary               CULTIVATION-I NDEPENDENT S URVEY OF
distinctiveness of the Archaea eventually                EXTANT M ICROBIAL D IVERSITY
showed that all known life can be orga-                Until recently, assessment of naturally
nized into three major Domains: Eucarya (all        occurring microbial diversity was an impos-
eukaryotes), Archaea, and Bacteria (Woese           sible undertaking, simply for lack of appro-
et al., 1990). The fruit of Woese’s efforts, a      priate methods. The problem of accurately
universal tree that outlines the evolutionary       describing naturally occurring microbial
relationships of all life, is shown in Figure 1.    assemblages has been extensively discussed
Analyses of most macromolecules involved            and reviewed (Staley and Konopka, 1985;
in nucleic acid-based information processing        Pace et al., 1986; Amman, 1995; Pace, 1997)
(e.g., DNA replication, transcription, transla-     and is largely intertwined with the historical
tion) yield this three-domain topology. Phy-        development of microbiology. The crux of
logenetic analyses of metabolic and regula-         the problem is this: Pure culture techniques,
tory genes, on the other hand, do not result        despite their tremendous utility, are inad-
in similar consistent topologies (Brown and         equate for describing naturally occurring
Doolittle, 1997; Doolittle, 1999).                  microbial assemblages. Microorganisms
   The three-domain tree is simple in form but      commonly recovered by standard micro-
profound in its implications. A casual glance       biological procedures are not generally
at the universal rRNA tree (Fig. 1) shows           representative of the assemblages from
that the lion’s share of phylogenetic diver-        which they originate. For a vast number of
sity resides in the microbial world, whereas        microbial species, appropriate media and
macroscopic organisms occupy small, ter-            conditions for growth are simply not well-
minal nodes on the tree of life. Consider-          developed, available, or practically feasible.
ing the 3.8 billion years over which micro-         In the past, microbial biologists depended
bial life has evolved, this is perhaps not so       nearly exclusively on the isolation and cul-
surprising. Another brief look at the tree in       tivation of individual microbial strains from
Figure 1 reveals the endosymbiotic origins          the environment. Although useful, however,
of several organelles—chloroplasts within           pure culture isolation obliterates the natural
the cyanobacterial cluster (near Synechococ-        assemblage, and the predominant microbes
cus), and mitochondria within the alpha Pro-        are frequently, even generally, invisible to
teobacteria lineage (near Agrobacterium).           the approach.
   A major point to recognize when con-                In the early 1980s, developments in
sidering molecular phylogenies is that the          comparative molecular phylogenetics were
472                                        S YSTEMATIC BIOLOGY                                            VOL. 50

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  FIGURE 1. Universal phylogenetic tree based on small-subunit rRNA sequences. “Universal” unrooted phyloge-
netic tree showing representative taxa was inferred by maximum likelihood analysis of 1,620 homologous positions
of sequences of small subunit rRNAs from the indicated organisms or environmental clones. Numbers indicate
percentage of bootstrap resamplings that support indicated branches in maximum likelihood (before slash) or max-
imum parsimony (after slash) analyses for only those groups that attained >60% support with at least one of the
two methods. Analyses of duplicated protein genes have placed the root of the tree on the branch at the base of
the bacterial line (Iwabe et al., 1989). Evolutionary distance (sequence changes) between the species shown is read
along line segments. The scale bar corresponds to 0.1 changes per nucleotide. pSL50, pSL4, pSL22, pSL12, pJP27,
pJP78, and marine SBAR5 represent rRNA sequences obtained directly from environmental samples. [Figure from
Barns et al., 1996; reproduced with permission.]
2001           DELONG AND PACE—ENVIRONMENTAL DIVERS ITY OF MICROBES                             473

instrumental in removing the roadblocks that             e`
                                                  1995; B´ ja et al., 2000). Additionally, the great
prevented accurate description of natural         concordance between many independent
microbial diversity (Stahl et al., 1984; Pace     studies conducted in similar habitats tends
et al., 1986). The cultivation-independent ap-    to support the conclusions about micro-
proach involves recovery of phylogeneti-          bial diversity and distribution provided by
cally informative gene sequences from nu-         cultivation-independent approaches.
cleic acids extracted directly from naturally        Cultivation-independent approaches such
occurring microbial biomass. Phylogeneti-         as those described above have invigorated
cally informative gene sequences extracted        the eld of microbial ecology. Phylogenetic
from mixed microbial populations are iso-         comparison of rRNA genes retrieved di-
lated as DNA clones that are then sorted and      rectly from the environment has fast be-
sequenced. Molecular phylogenetic compar-         come the standard for surveying natural mi-
isons provide phylogenetic identi cation of       crobial diversity (Amann et al., 1995; Pace,
individual population constituents. Small         1997). The approach has led to the discov-
subunit rRNA genes are the most com-              ery of many novel microbial taxa, ranging
monly used phylogenetic markers to date,          from new species, to new phyla, even new

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because of their ubiquity and conserved           “kingdoms”! And these newly recognized
nature.                                           microbes are not minor players in the envi-
   The above-described methods also pro-          ronment: They often represent the major taxa
vide phylogenetically based markers that can      present in both terrestrial and marine ecosys-
be used to tag speci c phylogenetic types of      tems. Cultivation-independent surveys have
organisms for identi cation. Small subunit        greatly expanded the known phylogenetic
rRNAs, the targets for these nucleic acid–        variety of known microbial species (Pace,
based hybridization probes, have proven ex-       1997). These studies suggest that the pheno-
traordinarily useful for such “molecular tag-     typic properties of many of the most abun-
ging” approaches. Relatively high amounts         dant microbes inhabiting Earth remain to be
of intracellular rRNA provide abundant tar-       determined.
get for phylogenetic identi cation of indi-
vidual cells by using uor-labeled oligonu-
cleotide probes (DeLong et al., 1989; Amann           Cultivation-Independent Surveys of the
et al., 1995). Individual microbial cells can                    Domain Bacteria
now be stained with color-coded probes               In 1987, Carl Woese published a bench-
and identi ed with uorescence microscopic         mark paper in microbial biology, the rst
techniques. Nested suites of probes speci c       comprehensive synthesis of bacterial evolu-
for different taxonomic levels (e.g., domains,    tion placed in the context of all lifeforms
genera, species) can now be designed, allow-      (Woese, 1987). In his treatise, Woese outlined
ing hierarchical taxonomic dissection of com-     the evolutionary relationships of the major
plex, naturally occurring microbial assem-        bacterial phyla, as inferred from rRNA se-
blages (Amann et al., 1995).                      quence data. The 12 major bacterial divisions
   Cultivation-independent molecular phy-         identi ed still represent most of the taxa that
logenetic approaches, like any other method-      can be readily cultivated and characterized
ologies, have their own speci c pitfalls and      by using cultivation methods. Nevertheless,
biases (for a recent review and detailed dis-     recent cultivation-independent molecular
cussion, see Wintzingerode et al., 1997). Po-     surveys have revealed that the bacterial
tential biases include differential cell lysis,   domain consists of many more divisions,
preferential DNA recovery from speci c cell       having few or no cultured representatives.
types, and several analytical complications       Figure 2 compares the view of bacterial
introduced by using the polymerase chain          phylogenetic representation in 1987 with
reaction (Reysenbach et al., 1992; Suzuki         the current view derived from cultivation-
and Giovannoni, 1996; Wang and Wang,              independent studies. The current tree of
1996; Wintzingerode et al., 1997; Poltz and       bacteria now contains >40 phylogenetically
Cavanaugh, 1998). Nonetheless, results from       well-resolved bacterial divisions (Pace, 1997;
culture-independent studies using different       Hugenholtz et al., 1998). The newly dis-
techniques, with each expected to produce         covered groups within the domain Bacte-
different biases, tend to yield similar re-       ria, most with no cultivated representatives,
sults (Schmidt et al., 1991; Mullins et al.,      demonstrate that the microbial species in our
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  FIGURE 2. Diagrammatic representation of currently known major groupings in the bacterial domain, inferred
from rRNA sequences. Division-level groupings of two or more rRNA sequences are depicted as wedges. Divisions
that have cultivated representatives are shown in black; divisions represented only by rRNA sequences retrieved
from environmental samples are shown in white. The small tree in the upper right is an outline of known bacterial
divisions in 1987, as compiled by Woese (1987).

culture collections provide only a skewed                 “extremophiles”. One of the two main lin-
and incomplete picture of extant microbial                eages of Archaea, the Crenarchaeota, was
diversity.                                                especially notorious for growth at high
                                                          temperatures. All cultivated Crenarchaeota
                                                          originate from extremely hot environments,
      Cultivation-Independent Surveys of the              including hydrothermal vents and geother-
                 Domain Archaea                           mal springs. Many are hyperthermophiles,
  Archaea, although prokaryotic in cellular               requiring temperatures greater than 80± C for
ultrastructure, are evolutionarily quite dis-             optimal growth.
tant from their microbial cousins, Bacteria. At              It was surprising, then, when culture-
the time of their discovery, known and cul-               independent surveys revealed an abundance
tured Archaea (then archaebacteria) seemed                of archaea in many diverse habitats. The
to be an odd, perhaps rare, collection of or-             discovery of widespread diversity of ar-
ganisms, united mainly by the evolution-                  chaea in “nonextreme” habitats is one of
ary heritage indicated by rRNA genes and                  the particularly striking ndings of culture-
a few other molecular features. The known                 independent surveys. Previously, there was
habitats of Archaea were hypersaline brines               no reason to believe that Archaea contributed
(extremely halophilic archaea), geothermal                signi cantly to the ecology of aerobic ma-
environments (hyperthermophilic archaea),                 rine or terrestrial habitats. Yet now it is
and strictly anoxic habitats (methanogens).               apparent that two major groups of Archaea
In common parlance, cultivated Archaea                    are common and very abundant compo-
were, without exception, considered to be                 nents of marine plankton and elsewhere
2001              DELONG AND PACE—ENVIRONMENTAL DIVERS ITY OF MICROBES                                       475

(DeLong, 1998a, b; DeLong et al., 1999a).                 cell densities of 1 £105/ml, at depths rang-
Shallow and deep marine waters at po-                     ing from 100 m to the bottom of the abyss
lar, temperate, and tropical latitudes; the               (DeLong et al., 1999a). At these cell densities,
guts of abyssal sea cucumbers; and ma-                    archaea represent »20% or more of ALL
rine sediments all show evidence of these                 microbial cells in the oceans, a habitat where
cold-adapted cousins of hyperthermophilic                 10 years ago they were not thought to exist!
archaea (DeLong, 1998a, b; see Group 1a,                     Do these abundant archaeal cells, detected
Fig. 3). Archaea are now known to be com-                 by rRNA gene surveys, actually represent ac-
monly present in the marine environment at                tive, authochthonous community members?

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   FIGURE 3. Diagrammatic representation of major groupings in the archaeal domain, inferred from rRNA se-
quences. Major archaeal divisions are depicted as wedges. Divisions that have cultivated representatives are shown
in black; divisions represented only by rRNA sequences retrieved from environmental samples are shown in white.
The predominant habitats of the uncultivated archaeal groups are indicated in italics.
476                                  S YSTEMATIC BIOLOGY                                    VOL. 50

Apparently yes, according to several lines of      rine plankton and sediments, respectively.
evidence. Fluorescence in situ hybridization       Many other yet uncultivated lineages con-
microscopy of planktonic archaea shows             tinue to be discovered, as ongoing culture-
intact (and sometimes dividing) cells that         independent surveys probe unexplored mi-
contain appreciable amounts of rRNA,               crobial habitats.
cells that reach an abundance maximum
(30% of the total planktonic microbial cell                         CONCLUSION
population) at speci c depths in the water            Despite the impact of culture-independent
column (DeLong et al., 1999a); these data are      rRNA gene surveys on microbial ecology,
suggestive of an active archaeal population.       phylogenetic information provided by a sin-
In Antarctica, surface-water archaea peak          gle gene (e.g., rRNA) is usually insuf cient
in relative abundance in late winter (up           for inferring the physiology or ecological sig-
to 20% of the total planktonic microbial           ni cance of organisms known only by rRNA
population), decrease to undetectable num          sequence. To a large extent, the lack of alter-
bers during the austral summer (Murray             natives to cultivation has severely limited the
et al., 1998), and again increase with the onset   abilities of microbial biologists to character-

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of the subsequent austral winter. These data       ize naturally occurring microbes. The unex-
are again suggestive of an active and dy-          pected phylogenetic breadth and diversity of
namic population, one that in Antarctic wa-        many of the newly discovered microbial lin-
ters has a distinct, seasonal cycle. Biomarkers    eages imply novel evolutionary innovations,
of planktonic archaeal metabolism (specif-         new phenotypic properties, and unantic-
ically, archaea-unique tetraether lipids) are      ipated ecological roles. New approaches
also detectable and accumulate in planktonic       for more comprehensive biological charac-
and sedimentary marine habitats, a distinct        terization of these novel microbial taxa are
biogeochemical in uence of the cold-water          desperately needed to fully appreciate the
archaea on their surrounding environment           signi cance of extant microbial life on Earth.
(Hoefs et al., 1997; DeLong et al., 1998).            Recent developments in genome science
Finally, a speci c symbiotic association           now offer substantial promise for fur-
between a marine sponge and a strain of            ther characterizing uncultivated microbial
cold-water archaea indicates that these mi-        species (Shizuya et al., 1992; Stein et al.,
croorganisms have radiated into many dif-          1996; Schleper et al., 1998; Vergin et al., 1998;
ferent habitats, including associations with                               ea
                                                   DeLong et al., 1999b; B´ j` et al., 2000; Rondon
metazoan hosts (Preston et al., 1996). All the     et al., 2000). Using bacterial arti cal chro-
above data strongly suggest that the newly         mosomes (BACs), one can retrieve genome
detected marine archaea are active, dynamic,       fragments larger than 100 kb from uncul-
and likely to have marked impacts and inter-       tivated microbial species. BAC clones pre-
actions with surrounding habitats and biota.       pared from the chromosomal DNA of un-
   Since their initial detection in marine         cultured microbes now provide the raw data
plankton, evidence for a widespread distri-        necessary to dissect the genomes, and re-
bution of new types of archaea has been fur-       construct the biochemical pathways, of nat-
ther extended in forest and agricultural soils,    urally occurring microbes. Coupled with the
deep subsurface environments, freshwater           high-throughput approaches developed for
lake sediments, deep sea sediments, and in         standard genome projects, large amounts of
association with certain metazoan species          previously unavailable data are now becom-
(Pace, 1997; Delong, 1998a, b). Several of         ing readily accessible. Soon, comparative in-
the new archaeal groups are now very com-          formation on the genome structure, content,
monly encountered in culture-independent           and organization will become available from
ecological surveys (Fig. 3). Distant relatives     some of the most evolutionarily divergent
of thermophilic Crenarchaeota (Group C1a,          lifeforms on our planet. This information
C1b, C1c; Group C2, C3, C4) are found in           promises to expand our understanding of the
many low-temperature terrestrial and ma-           evolution and ecology of microbes remark-
rine habitats. Two recently discovered ar-         ably. These empirical data should help cat-
chaeal types fall within the other main            alyze new syntheses that include models and
branch of archaea, the Euryarchaeota. These        theory to help explain the observed ecolog-
uncultured groups (Group E2 and E3 in              ical patterns and evolutionary processes of
Fig. 3) are found predominantly in ma-             microbial life on Earth.
2001               DELONG AND PACE—ENVIRONMENTAL DIVERS ITY OF MICROBES                                            477

               ACKNOWLEDGMENTS                                 MURRAY, A. E., C. M. PRES TON, R. MASSANA, L. T.
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