encyclopedia of biodiversity || bacterial biodiversity

BACTERIALBIODIVERSITY

Erko StackebrandtDSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH

I. Recognizing Gaps in KnowledgeII. Unraveling Phylogenetic Diversity

III. Diversity Extends to the Strain LevelIV. Phylogenetic Diversity and Distribution of Pheno-

typic TraitsV. The Diversity of Symbiotic Prokaryotes

VI. The Diversity of the Uncultured Free-LivingOrganisms

VII. Conclusions

GLOSSARY

domains The highest taxonomic rank defined to clas-sify organisms into Archaea, Bacteria, and Eucarya,which differ from each other in fundamental geno-mic and phenetic properties.

endosymbiont Specialized form of symbiosis in whichone partner thrives within cells, lumen, or tissuesof the host organism; most obligate endosymbiontsbelong to the group of uncultured organisms.

epigenetic level Study of molecules which are theproduct of gene expression.

homology Denoting common ancestry. Structures,processes, sequences, behaviors, etc. are said to behomologous if there is evidence that they are deriva-tions from a common ancestral structure. In molecu-

Encyclopedia of Biodiversity, Volume 1Copyright 2001 by Academic Press. All rights of reproduction in any form reserved. 325

lar biology the term indicates a significant degree ofsimilarity between DNA or proteins.

phylogeny Natural relatedness among life forms; thescience of ordering the genealogy of organisms intoa family tree.

prokaryotes Life forms that are members of the do-mains Archaea and Bacteria as opposed to Eucarya,comprising organisms with a cell nucleus.

rDNA Genes coding for rRNA that play a fundamentalrole in the translation process; the most thoroughlystudied molecule in prokaryotic cells; used in com-parative phylogenetic studies.

uncultured prokaryotes Organisms, the presence ofwhich has been detected by molecular methods inthe environment but they have not been culturedunder artificial laboratory conditions.

BACTERIAL DIVERSITY comprises the total variabilityof prokaryotic life on Earth, covering all genomic, phe-netic, phylogenetic, and ecological variations from thelevel of an individual strain to the community thatevolved over a time span of probably more than 3.5billion years. Determination of the extent of diversityis mainly restriced to cultured prokaryotes because thevast majority of strains are not yet accessible for subse-quent research. Strains involved in obligate symbiotic

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and parasitic relationships with their eukaryotic hostsand free-living strains for which appropriate cultureconditions have not been developed can at best be de-tected by molecular techniques. This article summarizesresults of recent approaches that have broadened thebacteriologist’s view about the immense richness of pro-karyotic diversity.

I. RECOGNIZING GAPSIN KNOWLEDGE

Due to progress in methodologies and concepts, facetsof biodiversity have no been covered equally in differentareas in bacteriology, but progress must be viewed asa logical consequence of available technologies. Intro-duction of groundbreaking methods is usually followedby a period of increased knowledge in those fields forwhich the methods were developed. Numerous exam-ples exist in microbiology, such as the elucidation of(i) the ultrastructure of the prokaryotic cell, followingthe development of the electron microscope; (ii) meta-bolic and biochemical pathways, following the intro-duction of the isotope label technique and enzymology;(iii) anaerobic organisms, following the developmentof appropriate anaerobic cultivation technology; and(iv) cell constituents, following the introduction of theamino acid analyzer, gas chromatography, high-perfor-mance liquid chromatography, and thin layer chroma-tography. Recently, the introduction of high-resolutiongel electrophoresis led to one- and two-dimensionalfingerprinting methods for proteins, ribonucleic acids(RNAs), and deoxyribonucleic acids (DNA); applica-tion of restriction enzymes, cloning strategies, and poly-merase chain reaction (PCR) technology led to im-proved sequence analysis of genes and genomes; andcloning of PCR fragments of environmental DNA, insitu hybridization, and the development of gradient gelelectrophoresis revealed a larger spectrum of prokary-otic diversity than previously known. The latter insightshave been used to develop strategies in which DNA,isolated from environmental samples, is expressed toyield a spectrum of novel enzymes not detected instrains available from biological resource centers. As aresult, taxonomists learned that the vast majority ofprokaryotic species are still undetected, and physiolo-gists, biochemists, and geneticists can expect to findnovel lines of descent containing organisms that expressfundamental deviations from currently known bio-chemical pathways. New models will help to betterunderstand the structure and function of living matter

and the role of prokaryotes in maintaining the bio-sphere.

The number of validly described species of animals,plants, and lower eukaryotes is approximately 400 timeslarger than the number of prokaryotic species(1,600,000:4,000). This fraction of bacterial and arch-aeal species is surprisingly low considering that pro-karyotic species evolved eons ago, exploring and occu-pying any niche that has been investigated for thepresence of prokaryotic organisms. Insects, on the otherhand, which comprise more than 1 million species,evolved late in evolution—less than 600 million yearsago during the Cambium. Certainly, differences in thespecies definition that exist between the biological spe-cies of higher evolved eukaryotes and the pragmaticallydefined prokaryotic species contribute to the tremen-dous discrepancy, but it has been shown convincinglythat the vast majority of prokaryotic strains which arepart of the free-living microbial community species havenot been cultured. Endosymbiotic prokaryotes whichare not free living but firmly associated with eukaryoticcells are another source of uncultured organisms. Thelimitation of recognizing the richness of microbial or-ganisms is most likely due to the use of a restrictedspectrum of enrichment media which selects for a verynarrow spectrum of organisms that compete best underartificial laboratory conditions.

The discussion of diversity issues and the increasingawareness of the importance of microorganisms inmaintaining the biosphere is embedded in discussionof the implementation of the articles of the Conventionon Biological Diversity (CBD). The driving force behindthe CBD has been the recognition of the possibility ofa significant reduction or loss of biological diversity atsource by human activities, and the preamble admits ageneral lack of information and knowledge regardingbiological diversity. Nowhere is the lack of knowledgemore acute than for microbial diversity (prokaryotes,fungi, yeasts, and heterotrophic and autotrophic pro-tists) and there is a widespread appreciation amongmicrobiologists that cultured microorganisms representa very small, not necessarily ecologically important,fraction of natural microbial diversity.

Considering the total number of nucleotides per ge-nome, far more recognizable diversity can be seen atthe molecular level than at the epigenetic level. Com-pared to the total number of described species, thenumber of organisms analyzed in genome sequencingprojects is still small (about 2.5%), but this portion willincrease rapidly and genomic screening and data miningwill develop into dominating biological disciplines inthe future. However, taxonomists will not lose interest

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in the more traditional properties of a cell because ofthe demanding process of describing species and gen-era. The polyphasic approach to classification requiresinformation about morphology, ultrastructure, metabo-lism, chemical structure of cell constituents, and geno-mic features. Thus, from more than 100 years of bacte-rial classification, an enormous wealth of phenotypicand epigenetic data have accumulated and are of benefitto bacterial taxonomy, which many scientists regard asthe mother of biological sciences. However, taxono-mists are concerned that this spectrum of diversity willhave to be sacrificed for an approach that considersmolecular/genomic data more appropriate for delineat-ing taxonomic ranks. Indeed, this cut will happensooner or later, but it does not mean that the avalancheof genomic data will bury all activities directed towardthe elucidation of the phenotype.

II. UNRAVELING PHYLOGENETICDIVERSITY

Whereas the past century unraveled the diversity ofepigenetic properties, i.e., those characters that are theresult of gene expression, the next century will provea tremendous wealth of information on genomic prop-erties. The terms genomics and reverse genetics havebeen coined for the strategy of obtaining value-addedinformation from sequences of genomes and genes. Datamining and meganetworking programs will replace thesimple search for similarities between a few homolo-gous genes, and it can be expected that the importanceof phenotypic traits will be reintroduced as a conse-quence of the desire to understand the horizontal andvertical flow of genes and their regulation and ex-pression.

The combination of information of a (small) portionof the genome with phenotypic properties is nowhereexemplified better than in modern classification strategyof prokaryotes. The first component, the backbone ofthe system, is provided by the primary structure ofhomologous molecules which have accompanied theorganisms since early evolution and they can be rankedaccording to their evolutionary history. The numberand nature of sequence differences among proteins andgenes coding for rRNA and proteins allow the recogni-tion of pairs or groups of organisms which evolvedfrom a common ancestor, and the order in which lin-eages have emerged in time facilitates decisions aboutthe grouping of organisms.

The main problem that has emerged in phylogenetic

studies during the past 10 years is the question ofwhether to place emphasis on the genealogical relation-ships derived from comparative analyses of a singlehomologous gene, a gene cluster, or even large partsof the genome. It has been demonstrated convincinglythat the historic fate of ribosomal (r)DNA genes doesnot necessarily represent the fate of other genes becausethe phylogenetic branching patterns of different genesmay show significant deviations. Analysis of the pres-ence and position of genes in completely sequencedprokaryotic and eukaryotic genomes and horizontalgene transfer among genomes with a high plasticity hasplayed an enormous role in designing and shaping earlyevolved organisms, and the chimerical genomic struc-ture of descendants of early evolving organisms hasbeen proven beyond doubt. The 16S rDNA is the mostwidely analyzed molecule in phylogenetic studies be-cause of its alternating change of degrees of sequenceconservatism that allows the recognition of most distantrelationships and moderate and close relationships. Theavailability of a database comprising more than 10,000sequences of prokaryotic strains makes it easy to eitherunambiguously affiliate a new isolate to one of the 4000species or to postulate the finding of a new taxon incase no highly similar match is obtained with the isolateand a recognized species. Analysis of rDNA is the goldstandard for analyzing phylogenetic relatedness;Schleifer and Ludwig (1989) provided excellent evi-dence that the rDNA data are closely matched by resultsof comparative analyses of other conservative moleculesresponsible for central cell functions. Second, the phy-logenetic branching patterns provide a scenario inwhich clusters of related organisms also share a highportion of epigenetic properties. The resolution powerof the 16S rDNA is restricted, however, because neithervery early nor very recent evolutionary events are wellresolved. Branching patterns are influenced not only bythe treeing algorithms applied for inferring phyloge-netic relationships but also by the size of the database.A phylogenetic tree is a dynamic construct which will tosome extend change its topology with any new sequenceadded. Consequently, it is difficult for the scientist tojudge whether the phylogenetic tree generated is a truereflection of the evolutionary history of the analyzedmolecule. If the tree is generated for the purpose ofmaking conclusions on the fate of genomic and epige-netic characteristics of the organisms concerned, con-clusions should be underpinned by the analysis of addi-tional evolutionary marker.

Increasingly, genes with a resolution higher than 16SrDNA are sequenced for determining intrageneric andintraspecies relatedness (e.g., genes hsp65 and hsp70


coding for heat shock proteins and gyraseB). Tradition-ally, and still required in the polyphasic approach toclassification, DNA–DNA hybridization studies are per-formed at the level of highly related species and atthe interspecies level, but the laborious experimentalburden and lack of a cumulative database excludewide application.

III. DIVERSITY EXTENDS TO THESTRAIN LEVEL

In the past, selected physiological reactions and com-puter-assisted techniques were applied to elucidate in-traspecific relationships. This goal can also be achievedby generating patterns from DNA, RNA, and proteinsthat represent a strain-specific fingerprint or a bar code-type profile. Spontaneous mutations occur at an averagerate of about 10�7 per gene and generation in prokary-otes but vary to a great extent in genetic loci. The extentto which strains of a species differ from each other alsodepends on the relative evolutionary time during whichstrains evolved from each other. Because prokaryoticspecies are man-made constructs established to facili-tate taxonomy, strains included in a species may varysignificantly from each other in phylogenetic depth andhence in the extent to which macromolecules diverged.The complexity of the patterns obtained depends onthe size, length, and degree of conservatism of the mac-romolecule of choice and the tools used to cleave, am-plify, hybridize, and separate these markers. Separationof DNA fragments by pulsed field electrophoresis, prob-ing of defined genes with labeled probes, visualizationof bands via computerized, laser-analyzed densitometerscanning, and the use of fully automated, reproducibletechniques have improved the resolution and the moni-toring part of the analyzes significantly. Pattern identi-fication is rapid, discriminating, and applicable to anyspecies for which DNA, RNA, and whole cell proteinscan be isolated. Providing the potential for discrimina-tion, isolates and reference strains that exhibit a highdegree of pattern similarity can be considered related.These techniques complement traditional typing meth-ods used mainly in the clinical environment, such asserotyping, biotyping, and phage typing. The decisionof whether a strain with a unique pattern actually be-longs to a described species or should be described asa new species requires more quantitative methods atthe genomic level that allow one to measure the degreeof relatedness.

IV. PHYLOGENETIC DIVERSITYAND DISTRIBUTION OF

PHENOTYPIC TRAITS

The phylogenetic and phenotypic separation of the do-main Archaea from the domains Bacteria and Eucarya(Fig. 1) is the most exciting result since the introductionof comparative sequence analysis by Woese and Fox inthe mid-1970s. The presence of two prokaryotic do-mains in which members are defined by clearly differentgenomic and phenotypic properties has changed funda-mentally the hypothesis on the dichotomy of life formsand revolutionized ideas about the evolution of theeukaryotic cell. Domains were introduced by Woese,Kandler, and Wheelis to denote that these primary linesof descent constitute higher entities than the traditionaleukaryotic kingdoms. The most significant epigeneticdifferences among members of Archaea and Bacteriaare the compositions of the cell wall and fatty acid andthe modification pattern of tRNA.

Figure 1 schematically depicts the tripartition of thedomain Archaea that guided the description of threekingdoms, the Euryarchaeota, the Crenarchaeota, andthe Korarchaeota, for some uncultured organisms. Nokingdoms have been described for the rich phylogeneticstructure of the domain Bacteria, outlined in Fig. 2,because of the significantly large number of organismsand lineages involved, which are not always well sepa-rated from each other. The order at which these lineagesevolved is of low statistical significance but the phyloge-netic composition of organisms within these lineagescan be recovered by analyses of other genes, such asthose coding for 23 rRNA, 5S rRNA, ribosomal proteins,ATPase, elongation factors, and heat shock proteinHSP70. In some lineages certain characteristics are in-deed of phylogenetic significance, such as morphologyand/or ultrastructural features (Thermotogales, Plancto-mycetales, Verrucomicrobiales, Spirochaetales, and Myxo-bacteriales), chemotaxonomic properties such as cellwall composition (Thermotogales/Deinococcus, clos-tridia, and Actinobacteria) or lack thereof (Planctomyce-tales), and physiology, i.e., the composition of thephotosynthetic apparatus (Chloroflexus, Chlorobiales,and cyanobacteria). Most lineages, however, have awide variation of morphological, chemical, ultrastruc-tural, and biochemical diversity, some traits of whichmay have been acquired in the course of their evolutionby horizontal gene transfer, whereas others may haveevolved as a response to occupying new environmentalniches (e.g., autotrophic and chemolitothrophic forms).Traits formerly believed to be of monophyletic origin—


FIGURE 1 Schematic illustration of the main lines of 16S rDNA sequences showing the triparti-tion of the domains Bacteria, Archaea, and Eucarya. The circle indicates the approximate positionof the root of the tree, making the domains Archaea and Eucarya phylogenetic neighbors. Thescale bar indicates 10 estimated changes per nucleotide position.

and hence of taxonomic value—lost their significancein classification when their polyphyletic origin wasdemonstrated or when these properties were found tobe of little genomic stability. In general, this is truefor morphology, spore formation, the relationship tooxygen, the presence of a photosynthetic apparatus,gliding motility, and many other characters. In modernclassification, these properties are no longer used as thesole basis for the description of higher taxa, such asfamilies, orders, and classes; today, the rational for ahigher taxon is primarily the distinct phylogeneticgrouping of its members, whereas their phenotypic de-scription may be rather broad.

A good example of the inability of phenotypic prop-erties to serve as phylogenetic markers is provided bymembers of the class Proteobacteria, which includes themajority of Gram-negative bacteria. This class is highlydiverse with respect to physiological and morphological

properties. Morphological diversity ranges from simplespherical forms to the highly complex fruiting bodiesof myxobacteria. Physiologies include chemolithoauto-trophy, photosynthesis, fermentation, anaerobic respi-ration, and nitrogen fixation. To reliably affiliate a newisolate to a described genus by these properties is im-probable; the chance of doing so is increased by thepresence of unique biochemical properties, such as ni-trate or ammonium oxidation (Nitrobacter in the � sub-class and Nitrosomonas and relatives predominantly inthe � subclass, respectively) and sulfate reduction (De-sulfovibrio and relatives; � subclass). Sulfur and sulfateoxidation as well as anaerobic photosynthesis are poorphylogenetic markers because members of Thiobacillusare found in the �, �, and � subclasses and the sulfurlessphotosynthetic organisms occupy different sublines ofdescent within the � and � subclasses.

As a consequence of the recognized discrepancies


FIGURE 2 Relative order of the main lines of descent within the domain Bacteria based on16S rDNA sequences. The horizontal portion of triangles indicates the phylogenetic depth ofthat lineage, whereas the size of triangles is not indicative of species numbers. Lineagesfrom which symbionts evolved are indicated by open arrowheads. Lineages which encompasssequences of as yet uncultured free-living bacteria are indicated by solid arrowheads. Thescale bar indicates 10 estimated changes per nucleotide position.

between the phylogeny of prokaryotic taxa and theirprevious taxonomic treatment, the recent phylogeny-oriented classification system of genera and higher taxawas developed to primarily match the 16S rDNA data.Newer textbooks have adopted the modern approach,in which, by providing the phylogenetic framework,the origin and evolution of certain phenotypic traitsmay be better understood than expressed by traditionalsuperficial lumping.

V. THE DIVERSITY OF SYMBIOTICPROKARYOTES

Endosymbiotic associations, recognized more than acentury ago, initially concentrated mainly on the elu-cidation of the origin of chloroplasts. Later, the im-portance of eukaryote–prokaryote relationships wasrecognized for associations between plants and nitro-


gen-fixing bacteria, e.g., legumes and Rhizobium spe-cies, monocots and Azospirillum species, Casuarina sp.and Frankia sp., ferns and Anabena sp., and the prokary-otic origin of plant mitochondria. However, most endo-symbiotic relationships between microorganisms andtheir eukaryotic hosts were mainly descriptive and thetaxonomic affiliation of the vast majority of unculturedmicroorganisms remained virtually unknown. In con-trast, microbial partners participating in nonobligatesymbiotic relationships were identified long before themolecular era; there are numerous associations for theectosymbiotic microbiota of the rumen, intestine, gut,and skin.

A. Identification of SymbiontsMost endosymbiotic bacteria are defined as ‘‘as yetuncultured’’ organisms, many of which do not existas a pure culture within the host’s tissue. Hence,genes coding for rRNA cannot be isolated selectivelybut must be identified within clone libraries consistingof PCR-amplified rDNA using prokaryote-specific PCRprimers and total DNA extracted from the plant,animal, or even the prokaryotic cell. If the hostcontains a single symbiotic partner only, the clonelibrary will consist exclusively of the one uniquerDNA insert; if the association is more complex, theclone library will contain phylogenetically differentinserts. Authentification of the symbiont and verifica-tion of the location of the putative endosymbiontwithin the host’s tissue is performed by fluorescencein situ hybridization techniques as elegantly developedby Stahl, Amann, and coworkers. In most cases the16S rDNA sequence will provide sufficient uniquenucleotide stretches to allow generation of symbiont-specific oligonucleotide probes.

Not until 1982 was Prochloron didemni, the endosym-biont of the ascidian Lissoclinum patellum, identifiedby molecular techniques. Ten years later, a wealth ofinformation was available on the molecular phylogenyof symbionts and endosymbionts from a broad spec-trum of eukaryotic hosts, ranging from protozoa tovertebrates. The availability of a large database, con-sisting of thousands of 16S rDNA sequences of free-living prokaryotic species, facilitates the search for thephylogenetic affiliation of the more than 500 16S rDNAsequences available for host-associated bacteria. Funda-mental questions about the identity of the prokaryoticpartner, the evolution of symbiotic relationships, andthe mechanisms of symbiont transmission can nowbe addressed.

B. The Evolutionary Origin of SymbiontsMost endosymbionts investigated to date originate fromancestors within the domain Bacteria, in which theyare found in a few main lines of descent (open arrow-heads in Fig. 2). The ability to thrive in certain anaero-bic protozoa of the genera Metopus, Plagiopyla, andTrimyema appears to be widespread among methano-genic Archaea (kingdom Euryarchaeota). No endosym-bionts have been described to share a common ancestrywith those prokaryotes which define the most deeplybranching lineages, such as the Crenarchaeota, domainArchaea, and Aquificales, Thermotogales, and otherbranches comprising thermophilic and phototrophicorganisms of the domain Bacteria. Because symbiontsand nonsymbionts share more than 80% 16S rDNAsequence similarity it can be concluded that the inva-sion of the eukaryotic host by prokaryotic cells musthave occurred less than 2 Gy ago. Figure 3 shows recentevolutionary events by plotting geological time against16S rDNA similarity values determined for the originof organisms defined by key physiological types (i.e.,oxygen-generating photosynthesis by cyanobacteria,fermentative metabolism in facultative anaerobic bacte-ria, and origin of respiration chain in aerobic bacteria).Thus, as derived from 16S rDNA similarity values, theorigin of endosymbionts correlates with the origin ofthe eukaryotic cell and endosymbiosis has occurredrepeatedly in (perhaps all) eukaryotic lineage.

In order to study the history of symbiotic associa-tions, the phylogenetic trees of hosts and their symbi-onts should be compared. Few data are available, withthe most convincing study being that on the endosymbi-

FIGURE 3 Correlation plot of geological time (as deduced from thefossil record and oxygen content) and 16S rDNA similarity values.Only the past 2 billion years are shown. The origin of aerobic bacteriaand symbiotic bacteria correlates with the increased content of oxygenin the atmosphere and the eukaryotic cell, respectively (modifiedfrom Stackebrandt 1995).


onts of aphids. The topology of the symbiont Buchneraaphidicola tree is completely concordant with host phy-logeny based on morphology. The fossil and biogeo-graphic time points for the aphid phylogeny have beenused by Wilson and Bauman to calibrate the 16S rDNAof the closely related endosymbionts (�8% similarity).The value of 1% fixed substitutions per 25–50 millionyears determined for the symbionts of aphids is similarto the value of 1% per 50 million years determined onthe basis of a broader range of nonobligate symbioticrelationships (e.g., Rhizobium/legumes, Photobac-terium/fish, and enterobacteria/mammals) and to thevalue of 1% per 60 million years for the past 500 millionyears (Fig. 3).

The host’s advantage of the association has beenunraveled in a few cases, such as the removal of hydro-gen produced from hydrogenosomes of ciliates by arch-aeal methanogenic endosymbionts, provision of nutri-tional carbon to the host bivalves by sulfur-oxidizinggill symbionts, or essential amino acids to aphids bytheir endosymbionts. Application of the PCR tech-niques has allowed the elucidation of the transmissionroute of symbionts in ovaries, testis, and gill tissue oftropical lunicid bivalves and deep sea bivalves.

C. Phylogenetic Affiliationof Endosymbionts

The majority of endosymbionts cluster phylogeneticallymost closely with Gram-negative free-living bacteria. Itcan therefore be deduced that Gram-negative bacteriaare the most successful candidates for forming symbio-sis, including obligate endosymbiotic associations. Afew nonproteobacterial symbioses have been described,such as those found between wood-eating cockroachesand termites and spirochetes and Gram-positive host-associated bacteria, such as the microparasite Pasteuriapenetrans (in root-knot nematodes), Epulopiscium (afish symbiont), and Frankia (nitrogen fixing on Casuar-ina and relatives). The association between Sphingobac-terium comitans, a member of the Bacteroides/Flavobac-terium phylum, and the myxobacterium Chondromycescrocatus is an example of a prokaryote–prokaryote sym-biosis. Endosymbionts of Archaea are members of thekingdom Euryarchaeota and they are, in contrast tothose of Bacteria, organisms containing a Gram-positivecell wall. These anaerobic symbionts, originating fromancestors of the families Methanosarcinaceae, Metha-nomicrobiaceae, and Methanocorpusculaceae, have beenidentified in several genera of termites and protozoa.The basis of the symbiotic interaction appears to be

hydrogen transfer from host to endosymbiont, whichcan use the gas for methanogenesis.

1. Symbionts of the ProteobacteriaProteobacteria embrace organisms known to have closeassociations with eukaryotic hosts as pathogens, parthe-nogenesis bacteria, incompatibility bacteria, symbionts,and organelles (such as the mitochondria of plantswhich evolved from � proteobacterial ancestors). Endo-symbionts and nonobligatory associates of the samehost may belong to different phylogenetic groups, indi-cating that the same host is susceptible to more thana single invasion process and that not all symbioticrelationships result in obligate endosymbiosis.

a. Symbionts of the � Proteobacteria

The � subclass of Proteobacteria contains a widespectrum of organisms that are closely associated witheukaryotic cells. Prime examples are members of thenitrogen-fixing genera Rhizobium, Sinorhizobium,Bradyrhizobium, Mesorhizobium, and Azorhizobium.Pathogens include Afipia, Brucella, Bartonella, Rickettt-sia, Ehrlichia, Orienta, and Anaplasma. A highly relatedcluster of host-associated organisms, including symbi-onts of insects, cytoplasmatically inherited bacteriasuch as the parthenogenesis bacteria (PB), and the cyto-plasmatic incompatibility bacteria (CIB) is related toWolbachia pipientis. Other members of this group arethe PB and CIB of Culex and Drosophila. Because of thedegree of 16S rDNA similarity among cultured strains,these symbionts must be considered members of thesame species. These relationships demonstrate that thecommon ancestor of Wolbachia and relatives invadeda broad spectrum of insect hosts which, as shown bythe high values of up to 99% 16S rDNA similarity, musthave occurred recently in evolution.

b. Symbionts of the � Proteobacteria

Members of the � subclass of Proteobacteria encompassa wide range of mainly pathogenic plant- and animal-associated bacteria, such as members of Burkholderia,Azoarcus, Ralstonia, Bordetella, Kingella, Eikenella, andNeisseria. Also included in this subclass is the kineto-plast of Crithidia. The diversity of endosymbionts, how-ever, is rare and restricted to the endosymbionts of themealy bugs, which are moderately related members ofthe genera Ralstonia and Burkholderia.

c. Symbionts of the � Proteobacteria

By far the higher number of endosymbionts of insectsand vertebrates are members of the � subclass of Proteo-


bacteria. In addition, this taxon contains a wide spec-trum of animal and human pathogens and nonobligatesymbionts such as members of Enterobacteriaceae, Legi-onellaceae, Pasteurellaceae, Vibrionaceae, Pseudomonas(sensu stricto), and Acinetobacter. Many endosymbiontscluster according to the phylogenetic rank of theirhosts, which may be indicative of coevolution events:(i) The primary endosymbionts of giant ants, aphids,tse-tse, and the sweet potato white fly form a phyloge-netically coherent cluster within the radiation of entero-bacteria; (ii) the symbionts of fish with light organs,such as the deep-sea anglerfish and the flashlight fish,cluster with different members of Vibrio; (iii) two mod-erately related subcluster consist of the gill symbiontsof bivalves. These organisms are remotely related tomethylotrophic bacteria. The latter symbionts are sul-furoxidizing organisms which provide their hosts withnutritional carbon. Nitrogen-fixing symbiosis are notrestricted to plants but the gland of an invertebrateshipworm contains large numbers of � proteobacteriawhich possess the ability to digest cellulose and fix ni-trogen.

VI. THE DIVERSITY OFTHE UNCULTURED

FREE-LIVING ORGANISMS

For more than a century, assessment of prokaryoticspecies has been evaluated by the culturing approach.The number of different growth media is unknown, butall aimed to recover the largest possible diversity oforganisms. However, it is not the enrichment and isola-tion but rather the lack of cheap and reliable molecularidentification methods that still slow microbiologists intheir attempts to classify strains to the species level andto describe new prokaryotic species. The number ofnovel strains that has been eliminated during the isola-tion process, or which were only included in a biasedsearch for specific properties before they disappearedin nonpublic resource collections, cannot be counted.It is unknown how many strains have been investigatedin parallel. Although this problem may one day be over-come by more facilitated species definitions and theavailability of a global network of biological informa-tion, microbiologists are currently confronted with theproblem that there is a remarkable difference in thenumber and morphology of organisms in natural sam-ples with enrichment cultures and isolated colonies.Staley and Konopka introduced the phrase ‘‘great plateanomaly’’ to indicate that only a small fraction of pro-

karyotic species observed under the microscope willgrow under artificial laboratory conditions.

A. The Vast Majority of ProkaryoticSpecies Have Not Been Cultured

Pace and colleagues first suggested that rRNA sequencescould be used to characterize natural communitieswithout the need to culture. The delay in publishingthe first studies by the research groups of Giovannoniand Ward was largely due to the need to develop robustand simple technologies whereby 16S rRNA sequencescould be recovered from complex mixtures of environ-mental nucleic acids and then individually sequenced.Since then, there have been numerous 16S rRNA- and16S rDNA-based studies in which sequences were ana-lyzed to explore microbial diversity in different environ-ments. Most of these studies differ in details of method-ologies, such as isolation of nucleic acids, PCRamplification conditions, and source of cloning vectorsand ligation enzymes, but in one aspect the outcomeof all of these studies was similar: The vast majority ofthe more than 1200 environmental partial 16S rDNAsequences, deposited in public databases, were not iden-tical with and often not even similar to the homologoussequences of described species accessible in the exten-sive databases of cultured bacteria (Fig. 2, solid arrow-heads). Also, the sequences were rarely identical tosequences obtained from strains that were isolated inparallel to the molecular work from the same environ-ment (Fig. 4). As judged from the low degree of se-quence similarity, one could even conclude that manysequences are indicative of the presence of higher taxa.This finding reinforced the previously mentioned ideathat the vast majority of species have not yet been iso-lated.

Additional molecular techniques have been devel-oped for understanding the composition of microbialcommunities that reach beyond the mere assessment ofphylogenetic relationships of clones and strains. Amongothers, methods comprise (i) the application of gradientelectrophoresis of PCR-amplified 16S rDNA sequencesto facilitate recognition of changes of populations intime and space, (ii) the development of biologicalprobes to detect the presence of genes encoding meta-bolic enzymes or to identify bacterial species directlyin an environmental sample, (iii) the development ofbiosensors to determined microprofiles of inorganiccompounds, (iv) flow cytometry and cell sorting toenumerate and separate groups of organisms accordingto size and taxon specificity, (v) subtractive hybridiza-


tion to facilitate comparative analysis of environmentalsamples, and (vi) extension of the database to includegenes other than 16S rDNA (e.g., nif genes).

The environments discussed in the following sec-tions were selected because they provide the largestdatabase of phylogenetic information on unculturedorganisms. Many other environments have been investi-gated, but the overall picture of prokaryotic diversityis less focused; these include, fresh water, paddy fields,marsh oil, marine plants, bioremediation sites, bioleachreactors, or the multistructured associations betweenprokaryotes and eukaryotic cells.

1. The Marine Environmenta. The Uncultured Archaea

The use of archaeal-specific 16S rDNA PCR primersand subsequent analysis of clone libraries from DNAof oxygenated coastal surface waters and oligotrophicopen-ocean samples by the groups of DeLong and Fuhr-man revealed the widespread occurrence of two typesof archaeal diversity. Analyses of different marine sites,including surface and subsurface waters of the Pacific,Atlantic, Antarctic coastal waters, offshore slope re-gions, and a deep-sea marine holothurian, have subse-quently confirmed the affiliation of archaeal sequencesto these two groups. Clone sequences of the first groupcontained 16S rDNA genes of Crenarchaeota organismsfound at depths �100 to 4800 m, whereas others werefound at different marine sites at more shallow depths.Organisms from which sequences originated constitutea significant component of approximately 5–14% of themarine picoplancton assemblages and were novel andclosely related to the archaean ‘‘Crenarchaeum symbio-sum,’’ a symbiont of the sponge Axinella mexicana. Ata lower level of relatedness these sequences form aseparate branch at the basis of the kingdom Crenar-chaeota, where they show some distant relatedness toa group of sequences isolated from sediment organismsof the Obsidian Pool, Yellowstone.

A few sequences have been described to belong to thesecond sequence group originating from Euryarchaeotaand are distantly related to the terrestrial species Ther-moplasma acidophilum. Except for a holothurian arch-aeal sequence, they have been retrieved from the sameenvironment from which the crenarchaeotal sequenceswere obtained, but mostly at lower depth (0–100 m).The high sequence similarity of members of this groupfrom geographically separate sites contrasts the findingby Munson and colleagues, who demonstrated the highdegree of phylogenetic diversity between archaeal se-quences retrieved from 16S rDNA clone libraries gener-

ated from material sampled in marsh sediment samplesand adjacent vegetative marshland in the United King-dom. In this study, clone sequences formed about15 different phylogenetic groups, each of which washighly to distantly related to cultivated Euryarchaeotaspecies.

The origin of marine archaea is unclear because norepresentative has been cultured. One may argue thatthese prokaryotes originate from dormant stages of re-leased commensales or symbionts of marine inverte-brates. This view, however, is contradictory to the highcell number of living organisms of this group—up to14% of the total community. Disturbed deep-sea sedi-ments, which have not been investigated, may be an-other possible source for these organisms. In contrast,euryarchaeal clone sequences retrieved from surfacewater material may be coastal and even terrestrial origin,considering that there is a specific relationship of thesesequences to some of the DNA retrieved from coastalsalt marsh.

b. The Uncultured Bacteria

Phylogenetic analysis of rDNA from phytoplanktonof the Sargasso Sea by Giovannoni, Britschgi, andcoworkers revealed that the majority of the mostlyeubacterial sequences were novel to systematists. Someclones represented oxygenic phototrophs and couldbe assigned to the Synechococcus group of the Cyano-bacteriales. Groups of related sequences of mainly the� and � Proteobacteria were identified and definedas SAR (Sargasso) groups, showing distant relatednessto cultured bacteria of Shewanella, Vibrio, and Ocean-ospirillum. Analysis of the phytoplankton from thenorth Pacific near Hawaii by Schmidt and colleaguesled to the unexpected result that the population wasvery similar to that of the Atlantic Ocean. Somesequences were similar to those of common culturedmarine organisms (e.g., Vibrio, Pseudoalteromonas, andChromatium), whereas others represented novel lin-eages which were distantly related to the Fibrobactergroup and Chlorobium.

Following studies extended the range of samplingsites in the Atlantic and Pacific Oceans, the AntarcticSea, and the Mediterranean Sea. Basically, the findingsof the first studies were confirmed in that many ofthe new sequences were highly related, although notidentical, to those defined earlier, irrespective of thelocation. The ecological role of the hitherto unde-scribed organisms remains unresolved, although theirgeneral physiological capacity is probably similar tothat of described species because � Proteobacteria


encompass mainly lithotrophic and oligocarbophilicorganisms, whereas � Proteobacteria and cytophagesexhibit strong hydrolyzing and degrading capacities.The few sequences of Gram-positive bacteria are prob-ably of terrestrial origin because they are mainly foundin coastal regions and in sediments which must beregarded as a deposit mainly for endospore-forming or-ganisms.

B. Hot Spring EnvironmentsResearch in hot spring environments has been stimu-lated by the discovery of Thermus aquaticus as a speciesof high biotechnological value. The community of matsespecially provided an excellent comparison of micro-bial composition as assessed by selective culture tech-niques introduced by Brock and Castenholz. Resultsof the sequence investigations by Ward, Weller, andcolleagues clearly demonstrated that even such a ratherclosed system inhabits a phylogenetically diverse com-munity. Consistent with the findings in the marineenvironment, none of the recovered sequences closelyresembled sequences from cultured taxa isolated fromsimilar environments. Sequences of cultured organismsbelieved to constitute a major component of the matcommunity, such as Synechoccous lividus or Chloroflexusaurantiacus, were not recovered. Analysis of prokaryoticDNA from hot springs located in Yellowstone NationalPark by Pace and coworkers revealed an unexpectedlylarge number of distinct bacterial sequences, indicativeof novel main lines of descent. Archaeal sequences weredetermined to be specifically related to sequences fromthe Crenarchaeota. A few sequences showed high simi-larities with 16S rRNA sequences of cultivated Archaea,(e.g., Desulfurococcus mobilis, Pyrobaculum islandicum,and Thermofilum pendens) but were not identical to any.The archaeal sequences from the hot spring environ-ment were not closely related to the novel archaealsequences retrieved from the marine environment,which appear to possess a similar position intermediateto the Crenarchaeota. Results of these studies indicatedthat the domain Archaea possesses a third line of de-scent, the kingdom Korarchaeota, suggesting that notonly the phylogenetic but also the physiological diver-sity of the Archaea are significantly larger than reflectedby the few cultured representatives.

C. Soil EnvironmentIn contrast to the early extensive work on differentmarine sites and hot springs, determination of micro-

bial biodiversity in soil was delayed until appropriatemethods were developed that circumvented method-ological difficulties such as the isolation of PCR-ableDNA from humic acid-containing soil and semiquanti-tative cell recovery. Despite these shortcomings studiesby Liesack and Triplett clearly demonstrated that soilsamples from the Southern and Northern Hemispherescontain a rich and varied bacterial flora, includingnonthermophilic archaeal members from the kingdomCrenarchaeota.

The first soil sample investigated by sequencing andprobing 16S rRNA genes was located in a subtropical,moderately acidophilic, and forested environment inQueensland, Australia. Unexpectedly, only a few se-quences were obtained from commonly isolated soilorganisms, such as Streptomyces, although members ofthis genus were cultured in large numbers from thesame soil sample. The reasons for the low representationof Gram-positive bacteria in libraries generated withuniversal 16S rDNA primers are not known, but thismay be explained by the cell wall structure of restingand dormant cells which fail to disintegrate under mildenzymatic lysis. By using actinomycete-specific prim-ers, Embley and colleagues were indeed able to dem-onstrate the presence of a rich diversity of thesetaxa in soil. Alternatively, Gram-positive bacteria maybe a minor (numerically) component of the soil flora,with selective isolation exaggerating their numbers.Some Australian soil sequences were closely relatedto those of nitrogen-fixing species of the � Proteobac-teria, but the majority of clone sequences representednovel groups that were only remotely related toknown taxa, e.g., Planctomycetales, Actinobacteria,Verrucomicrobium, Acidiphilium, and Thiobacillus acidi-philius.

Several actinobacterial clone sequences, retrievedfrom rDNA of different soil types and different geo-graphical locations, formed two clusters groupingremotely with members of Rubrobacter, Acidicrobium,and Atopobium. In addition to their presence in Austra-lian forested soil, their occurrence was verified in ahot spring (Australia), geothermally heated soil (NewZealand), paddy fields and soybean fields (Japan),cultivated soil (Mexico), peat bog and garden soil(Germany), grassland soil (The Netherlands), andforest soil (Finland) (Fig. 4). These sequences formedfractions of 1–23% of the respective clone librariesand one of these organisms constituted about 6% ofthe metabolically active part of a Dutch grasslandsoil community as shown by ribosomal RNA analysis.It can be deduced that these uncultured organisms


FIGURE 4 Distribution of 16S rDNA clone sequences, representinguncultured bacteria, within the radiation of 16S rDNA sequences ofsome cultured bacteria. Origin of clone sequences: MC, Australiansoil; PAD, Japanese paddy field; FIE, Japanese soy bean field; NH,north Pacific Ocean. The scale bar indicates 10 estimated changesper nucleotide position

are distributed worldwide and play a physiologicallyimportant role in the soil ecology.

VII. CONCLUSIONS

Despite tremendous progress in the elucidation of pro-karyotic diversity, many pitfalls have been identifiedwhich influence the composition of sequences in a clonelibrary and hence these data can be used neither toquantify nor to qualify the composition of communities.Any estimation of the relation of cultured and as yetnoncultured organisms is nothing more than a guessand not supported by scientific data. Most environmen-tal analyses have revealed a heterogeneous mixture of

deep and shallow branching lineages, very few of whichhave shown close relationships to cultured taxa. Manylineages are very closely related to each other, some ofthis diversity is due to microheterogeneity at the levelof rrn operons within a single cell, whereas others mayrepresent true strain diversity.

Although the contribution of rDNA and rRNA tomicrobial ecology must be considered significant, onemolecule alone cannot nearly cover all facets of micro-bial ecology. Not only must the function of an ecosys-tem be deduced from analyses of genes expressedthrough rRNA, mRNA, and proteins, and not onlyshould the network of broad physiological interactionsbe verified by in vitro reconstitution of isolates butalso all these strategies must include data on thoroughphysical and chemical analysis of the natural sample.Because ecological interactions are performed bystrains and not by species, the 16S rDNA in not theappropriate tool to unravel the diversity of this highphylogenetic level. Strain diversity may thus be oneor more magnitudes higher than mirrored by theanalysis of such an evolutionary conserved gene.Ecological niches must be defined and the difficultyin doing so is increased with the complexity of thesample. Soil samples, for example, are homogenizedin that during the isolation of DNA many individualmicroniches with their individual populations aredestroyed. In order to understand ecological interac-tions, population sizes must be known, strain richnessand strain abundance must be assessed, the biochemi-cal diversity of strains must be recognized by applyingfunctional probes for the detection of the expressionof specific genes, and physical probes must be appliedfor the assessment of the chemical and physical condi-tions of the environment. Modern environmental stud-ies must maintain the isolation component becausebiotechnological exploitation of cosmid libraries is asdesirable as the increase in knowledge of the evolution,phylogeny, and ecology of pure cultures.

See Also the Following ArticlesARCHAEA, ORIGIN OF • BACTERIAL GENETICS •

EUKARYOTES, ORIGIN OF • MICROBIAL BIODIVERSITY,MEASUREMENT OF • THERMOPHILES, ORIGIN OF

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