B I O D I V E R S I T Y 3 ( 2 ) 3

A R T I C L E S

This article is apopularized adaptationof the authors’ paper“The impact oftaxonomic genelibraries on ourknowledge of bacterialdiversity: the exampleof the marineArchaea,” published inRushton B.S., P.Hackney, and C.R.Tyrie (eds), 2001,Biological collectionsand biodiversity,Linnean SocietyOccasional Publica-tions 3. (See review ofthis title in BookReview section, page45.) Special thanks toR. Powell for his workin popularizing theoriginal.

Bacteria and Archaea: Molecular techniquesreveal astonishing diversity

James O. McInerney,Marice Mullarkey,Martina E. Wernecke,and Richard Powell

James O. McInerneyDepartment of BiologyNational University of IrelandMaynooth, County Kildare, IrelandEmail: james.o.mcinerney@may.ie

Marice Mullarkey,Martina E. Wernecke, and Richard PowellDepartment of MicrobiologyNational University of IrelandGalway, Ireland.

Abstract. The last decade has produced a significantadvance in our appreciation of the diversity of prokary-otic organisms (commonly given the generic term “bac-teria”). The need for this improvement was clear as thecurrent list of approximately 5,000 accredited species haslong been known to be a major underestimate of livingprokaryotic species. The primary reasons for this poorcensus were 1) the inability to cultivate the vast majorityof prokaryotic species in the laboratory and 2) a classi-fication system that inherently required laboratory cul-ture. Fortunately, the impact of DNA-based methods hasremarkably improved our knowledge by providing both anew alternative classification system (i.e. phylogeneticclassification), and critically, new experimental strate-gies to identify non-culturable species. The resulting datahave not only highlighted the true breath of prokaryoticdiversity but have also changed some of our previousviews of biological evolution. Phylogenetic analysis ofgene sequences retrieved from both cultured and uncul-tured bacteria has shown that all cellular life can be or-dered into three taxa (termed Domains) - Bacteria,Archaea, and Eucarya. Intriguingly, this has resulted ina major taxonomic promotion for the Archaea, which werepreviously thought to be a series of unusual bacterialspecies. In addition, the use of DNA-based methods toidentify and catalogue non-culturable species has radi-cally improved our knowledge of the diversity foundwithin living prokaryotes. This paper describes our cur-rent view of prokaryotic diversity describing the impactover the last decade of DNA-based methods. It is a popu-lar adaptation of a previously published paper (2001).

INTRODUCING THE HIDDENWORLD OF THE PROKARYOTESOne of the most significant developments in microbiologyhas been the discovery of many new bacterial species thatare so unique that taxonomists have accorded them therank of new phyla and even kingdoms. The collectivescientific name for these organisms is “Prokaryote,”meaning a cell characterized by the lack of a distinctmembrane-bound nucleus. (In contrast, cells whosechromosomes are contained within a membrane-boundnucleus are termed eukaryotes.) Far more commonlyprokaryotes are given the generic term “bacteria.” Theyare found throughout the entire planetary ecosystemincluding niches where eukaryotic species are rare orabsent (e.g. the ocean depths, the planet’s subsurface,thermal and polar environments, and oxygen-freeenvironments). This wide ecological range reflects theirvast metabolic capabilities that allow different prokaryoticspecies to inhabit different environments.

Prokaryotes also occur in great abundance. A recentanalysis suggested that the total number of livingprokaryotic cells is 4 - 6 x 1030 composed of 1.2 x1029 cells in the ocean, 2.6 x 1029 cells in soil, and0.25 - 2.4 x 1030 cells within the Earth’s subsurface(Whitman et al 1998). An alternative way to appreci-ate these figures is that even while accounting for theidea that a prokaryote cell is typically about 10,000-fold smaller in volume than a eukaryotic cell, the to-tal amount of prokaryote biomass is still approxi-mately 10,000 times greater than the amount of hu-man biomass currently living on Earth. Because ofthese large numbers, their metabolic capabilities, andtheir ubiquity, prokaryotes play an essential functionin the planet’s biochemical processes including de-composition in soil, the provision of atmospheric com-ponents, nitrogen fixation, and photosynthesis.

Despite this significance, we have as yet only a verypoor description of living prokaryotic species, andperhaps for obvious reasons, surveys of biodiversityoften overlook bacteria. There are severe technicallimitations among the traditional census-gatheringmethods of microscopy and bacteriology. Most spe-cies are indistinguishable under the microscope, andit has long been observed that only a fraction of thebacteria observed under the microscope can be suc-cessfully cultivated in the laboratory. Compoundingthis, those prokaryotic species that readily adapt to

ABOUT THE AUTHORS.James O. McInerney andRichard Powell areuniversity lecturerswhose research groupsare interested inbioinformatics andmolecular microbialecology respectively.For several years, thetwo groups havecombined their interestsin a research study ofuncultured marinebacteria. Under theirsupervision, MariceMalarkey and MartinaE. Wernecke haverecently successfullycompleted theirpostgraduate studiesusing moleculartechniques to analyzeuncultured Archaea inIrish coastal waters.

T R O P I C A L C O N S E R V A N C Y4

SPUORGEFIL SEICEPSDEBIRCSED SEICEPSDETAMITSEFOREBMUN YCARUCCA

HIGH L OW

sesuriV 000,4 000,000,1 000,05 rooPyreV

setoyrakorP 000,4 000,000,3 000,05 rooPyreV

ignuF 000,27 000,007,2 000,002 etaredoM

aozotorP 000,04 000,002 000,06 rooPyreV

eaglA 000,04 000,000,1 000,051 rooPyreV

stnalP 000,072 000,005 000,003 dooG

sedotameN 000,52 000,000,1 000,001 rooP

snaecatsurC 000,04 000,002 000,57 etaredoM

sdinhcarA 000,57 000,000,1 000,003 etaredoM

stcesnI 000,059 000,000,001 000,000,2 etaredoM

scsulloM 000,07 000,002 000,001 etaredoM

setarbetreV 000,54 000,55 000,05 dooG

srehto 000,511 000,008 000,002 etaredoM

SLATOT 000,057,1 000,556,111 000,536,3 rooPyreV

Table 1.Approximate numberof species, describedand estimated, for the

major groups oforganisms

(adapted fromWatson et al 1995).The relevant figuresfor the prokaryotes

are highlighted.

growth under laboratory conditions may not be repre-sentative, or even major components of, the prokary-otic community of which they are natural members. Theresult is that prokaryotic diversity remains almost un-explored. A comparison of the numbers of identifiedspecies from other life groups (e.g. fungi, algae, plants,and animals) quickly highlights the fact that the cur-rent description of 5,163 validly named species of bac-teria (Garrity & Holt 2001) constitutes an almost in-significant number in terms of the inventory of all spe-cies currently residing on Earth (Table 1). Indeed, arecent estimate of the number of living prokaryoticspecies was between 105 - 107 (Hammond 1995).

This large numerical discrepancy is primarily becausemicrobiologists have relied on the traditional ecologi-cal tools of microscopy and bacterial culture. Prob-lematically, when the results from both approachesare compared, the number of bacteria observed fromthe microscopic analysis usually exceeds the numberof bacteria cultivated in the laboratory by at least twoorders of magnitude (Jannasch and Jones 1959; Kogureet al 1979). Current classification (i.e. phenetic clas-sification) of bacterial species also compounds thedifficulty as, crucially, it requires pure cultures ofbacterial strains for examination and is therefore lim-ited by the bias inherent in laboratory cultivation.Also, it is not designed to provide information on theevolutionary relatedness of different bacterial species.This is unfortunate as prokaryotes provide neither a

useful fossil record of past species nor rich anatomi-cal detail in living species for comparative studies.However, the few ancient bacteria-like fossils that doexist show the presence of bacterial cells or bacte-rial community activity in some of the Earth’s oldestrocks dated to over 3.5 billion years ago (Schopf1993). By comparison, the oldest microfossils ofmulti-cellular red algae date to 1.25 billion years ago(Butterfield et al 1990) while the oldest metazoanfossils date to the Ediacaran era of approximately 600million years ago (Schopf 1999).

The clear deduction from the limited fossil record isthat cell-based life arose comparatively quickly af-ter the planet’s formation 4.5 billion years ago, andfor two-thirds of the time since then, it was limited toprokaryotic-like life. Indeed, it was the impact andevolution of prokaryotic life that provided a suitableenvironment for the subsequent evolution of animaland plant species. For example, in geochemical terms,the formation of an oxygenated atmosphere suitablefor the evolution of many eukaryotic species was pri-marily due to bacterial photosynthesis. Or, in biologi-cal terms, think of the endosymbiotic events wherebybacteria provided the chloroplast and mitochondrialorganelles found in many current eukaryotic cells(Taylor 1974; Margulis 1993).

Therefore, although microbiologists were well awareof its potential significance, the technical limitations

B I O D I V E R S I T Y 3 ( 2 ) 5

Figure 1.Universal phyloge-netic tree based oncomparative SSUrRNA gene sequenceanalysis (Woese et al1990). The positionof the root wasdetermined bycomparing paralogousgene sequences thatdiverged before thethree primarylineages emergedfrom their commonancestral condition.The numbers of thebranch tips corre-spond to thefollowing groups oforganisms.Bacteria:1, the Thermotogales;2, the flavobacteriaand relatives; 3, thecyanobacteria; 4, thepurple bacteria; 5, theGram-positivebacteria; and 6, thegreen non-sulphurbacteria. Archaea:the kingdomCrenarchaeotaencompassing 7, thegenus Pyrodictium; 8,the genusThermoproteus; andthe kingdomEuryarchaeotaencompassing 9, theThermococcales; 10,the Methano-coccales; 11, theMethanobacteriales;12, the Methano-microbiales; and 13,the extremehalophiles.Eucarya:14, the animals; 15,the ciliates; 16, thegreen plants; 17, thefungi; 18, theflagellates; and 19,the microsporidia.

meant that an exploration of prokaryotic diversity wasimpossible to perform in any systematic manner.Thankfully, the advent of DNA-based methods andthe insightful ideas of a few researchers have recentlyprovided a radical solution to this problem.

A THREE DOMAIN RATHERTHAN FIVE KINGDOM CLASSIFICATIONIn 1987, Carl Woese summarized ten years of work andproposed a phylogenetic classification system forprokaryotic species based on the nucleotide sequences ofsmall subunit ribosomal RNA (SSU rRNA) molecules. Hereported that SSU rRNA gene sequences could be usedfor comparative analysis between different species toprovide a tree of relatedness based on common ancestryor genealogy. Significantly, as both prokaryotic andeukaryotic cells contain SSU rRNA genes, phylogeneticanalysis could also be used to compare both prokaryoticand eukaryotic species.

This sequence-based phylogenetic system representeda new model for evaluating the relatedness of anyspecies, in terms of shared ancestry and evolution.Figure 1 shows a copy of the first presentation of theuniversal phylogenetic tree (Woese et al 1990). Forthe first time, the placement of the prokaryotes wasfirmly positioned on a universal tree of life. The re-sulting picture radically changed previous perceptionsand convention by contradicting the five-kingdomclassification of cellular life, i.e. Prokaryotes, Pro-tists, Fungi, Plants, and Animals (Whittaker 1959).The phylogenetic tree of life supported the proposalthat the five-kingdom system be replaced with a three-domain system wherein the differences between eachdomain are of a more profound nature than the differ-ences that separate each kingdom. With a revolution-ary impact on microbiology, the prokaryotes were splitinto two domains, the Bacteria (previously termedeubacter ia ) and the Archaea (previous ly te rmedarchaebacteria). The third domain, the Eucarya, con-

tained the other four eukaryotic kingdoms of protists,fungi, animals, and plants. The innovative nature ofthe phylogenetic tree was clear: Now both prokary-otic and eukaryotic species could be analyzed togetherto give (a) a picture of the comparative genetic di-versity of all cellular life, and (b) a true view of therange of diversity accounted for by the prokaryotes.

The inferences that can be drawn from the universalphylogenetic tree are new and exciting for microbiol-ogy. As the geological evidence suggests the pres-ence of both thriving cyanobacteria-like and sulphate-reducing Bacteria 3.5 billion year ago (Schopf 1993;Shen et al 2001), the origin of the last common an-cestor and the division of prokaryotes into the Bacte-ria and Archaea-Eucarya lineages must have occurredbefore this time. This places the origin of life sur-prisingly early in the planet’s development at a timewhen it was inhospitable by today’s standards. Inter-estingly, these conditions correlate with inferencesthat can be deduced from the phylogenetic tree. Thecurrent members of the deepest branches of the do-mains Bacteria and Archaea live at high temperaturesand in oxygen-free environments.

The work of Woese and his colleagues had providedmicrobiologists with a new framework to examine the roleand impact of prokaryotes in the context of the evolutionand diversity of life on Earth. However, the challengeremained to solve, or at least manage, the problem posedby the fact that the vast majority of living bacterial specieswill not culture under laboratory conditions.

DNA-BASED STUDIES OF PROKARYOTIC ECOLOGYConcurrently with the development of the phylogeneticclassification, Norman Pace and his colleagues weredeveloping a new approach for the study of bacterialecology. Their aim was to study natural bacterialcommuni t i e s by d i rec t ly r e t r i ev ing in fo rmat ivemolecu l e s , i . e . DNA sequences , a s opposed t o

T R O P I C A L C O N S E R V A N C Y6

bacter ia l ce l l s for cu l t iva t ion (Pace e t a l 1985;1986). As their informative molecule of choice wasthe SSU rRNA gene , any such gene s equencesretrieved directly from a natural community couldbe used to produce a phylogenetic description of then a t u r a l b a c t e r i a l c o m m u n i t y i n c l u d i n g b o t hculturable and non-culturable members.

Initially, this molecular approach to bacterial ecol-ogy was relatively slow to gain a place among thetools used by microbial ecologists. Technically, it waslaborious and required the construction of gene librar-ies from the total genomic DNA isolated from naturalcommunities. Subsequently, large numbers of clonesfrom these libraries had to be screened in order toidentify individual clones containing SSU rRNA genesfrom the various community members. However, thedevelopment of PCR amplification technology (Saikiet al 1988) removed this technical obstruction. Theinitial reports using this PCR-based approach showedthat the majority of SSU rRNA genes sequences re-covered from natural communities were derived fromnovel bacterial species (Ward et al 1990; Giovannoniet al 1990). Since then, the large number of similarreports analyzing diverse bacterial communities haverepeatedly demonstrated and confirmed the existenceof novel bacteria and the limitations of traditionalgrowth-dependent methods. With as many environmen-tally derived SSU rDNA sequences described as thosederived from cultured bacterial species, the phyloge-netic tree for the Bacteria has flowered in detail sinceits initial description. The current tree contains atleast 36 phyla, 13 of which do not as yet have a cul-tured representative (Hugenholtz et al 1998).

THE SURPRISING ARCHAEAT h e d e s c r i p t i o n o f t h eArchaea in the first univer-s a l p y l o g e n e t i c t r e e w a snovel and surprising. Knownbeforehand as archaebacteria(Balch et al 1997; Woese andFox 1977), they representeda s m a l l g r o u p o f h i g h l ya typ ica l bac te r ia l spec iesthat inhabited unusual or ex-treme environmental niches(e.g. thermophilic springs,hydrothermal vents, high-sa-l i n e w a t e r s , a n o x y g e n i cmuds). Even today, there areon ly 217 accred i ted , cu l -tured archaebacter ia l spe-cies (Garrity and Holt 2001).In biochemical terms, thesecultured species constitutethree groups: methanogens,

extreme halophiles, and extreme thermophilic sulphurmetabolizers. Phylogenetic analysis of approximately50 archaeal SSU rRNA genes derived from these cul-tured species showed that rather than simply beingc o m p o s e d o f o b s c u r e b a c t e r i a l s p e c i e s , t h earchaebacteria constituted a taxonomic rank of thehighest order, i.e. the domain Archaea . Within thisdomain, the Archaea split into two major lineages(termed kingdoms): the Euryarchaeota containing themethanogens, extreme halophiles, and sulphur reduc-ers, and the Crenarchaeota containing the extreme ther-mophiles (Woese 1987; Woese et al 1990).The Archaea held other surprises for microbiologists. Theunusual habitats of cultured archaeal species led to the pre-sumption that these living Archaea represented ancient orunchanged bacterial forms limited today to niches that re-flect early Earth conditions and are devoid of, or limited in,competition from other Bacteria and Eucarya species. Thesemisconceptions were overturned by the unexpected identi-fication of the presence of Archaea SSU rRNA genes incold oxygenated seawaters (DeLong 1992; Fuhrman et al1992). These uncultured marine Archaea probably play amajor role in the bacterioplankton community as other DNA-based analysis suggests that they are responsible for between2% and 30% of the total bacterial activity in these oceanwaters. Furthermore, a recent study calculated that thesemarine Archaea may constitute approximately 1.3 x 1028

cells throughout the global oceans, a number that is close tohalf of the estimated 3.1 x 1028 bacterial cells present in thesame waters (Karner et al 2001). In fact, as ocean watersconstitute one of the largest planetary niches, the marineArchaea may be one of the most dominant prokaryotic groupson Earth (Mestel 1994).The surprising identification of non-cultured Archaeainhabiting a relatively non-extreme environment has primedfurther DNA-based searches for Archaea. To date, thepresence of uncultured Archaea have been reported in avariety of general terrestrial and aquatic environmentsincluding soybean and rice field soils, forest soils, coastalsalt marshes, lake waters and sediments, and the deepplanet subsurface. This data shows that our previouspicture of archaeal ecology was l imited from itsdependence on the analysis of cultured species. Clearly,the Archaea are ubiquitous, occur in great abundance, andinhabit both unusual niches as well as a full range of largeand non-extreme environments containing robustcompetition from other Bacteria and Eucarya species.

A CURRENT VIEW OFTHE PHYLOGENY OF THE ARCHAEAAs initially described, and based on the SSU rRNAgene sequences of approximately 50 cultured species,the Archaea constituted two kingdoms, Crenarchaeotaand Euryarchaeota (Woese 1987). As with the domainBacteria, the addition of the uncultured archaeal SSUrRNA gene sequences is now changing this view of

Prokaryotesare ubiquitous!

Numerous species arefound in extreme

environments, such asthis hot spring in

YellowstoneNational Park, U.S.A.

(a thermophilicenvironment).

However, excitingly, inthe last decade, many

other prokaroytes havebeen identified in

“ordinary” environ-ments such as soybean

and rice field soils,forest soils, coastal

salt marshes, lakewaters and sediments,

and the deep planetsubsurface.

(Photo takenin 1996 by

German Jurgens,Department of

Applied Chemistryand Microbiology,

University of Helsinki,Finland).

B I O D I V E R S I T Y 3 ( 2 ) 7

Figure 2.SSU rRNAsequence-basedunrooted universalphylogenetic treeshowing theplacement of theproposed newArchaeal kingdom,Korarchaeota(Barns et al 1996).The numbers indicatepercentage bootstrapre-sampling scores.Paralogous genesequence analysisplaces the root ofthe tree on the branchat the base of theBacteria.

a rchaeal evolut ion. Phylogenet ic analysis of themarine archaeal SSU rDNA sequences indicates thatthe p lank ton ic Archaea cons t i tu te two separa teevolutionary groups. Group I represents a novel deep-branching lineage that is either loosely associated withthe Crenarchaeota (DeLong 1992) or, perhaps, repre-senting a new archaeal kingdom (McInerney et al1997). Group II marine Archaea represent a series ofnovel l ineages within the Euryarchaeota (DeLong1992). Interestingly, the other environmental archaealSSU rRNA genes sequences predominantly tend toassociate with the Group I marine Archaea and occa-sionally with the Group II marine Archaea . A furtherg roup o f uncu l t u r ed Archaea SSU rRNA gene

sequences recovered from a hot spr ing sediment(Barns et al 1994) showed even greater genetic diver-g e n c e f r o m t h e c u l t u r e d E u r y a r c h a e o t a a n dCrenarchaeota than the other environmentally derivedarchaeal SSU rRNA gene sequences (Figure 2). Thislineage has been proposed as a new kingdom-leveltaxon within the Archaea and termed the Korarchaeota(Barns et al 1996).

Therefore, our current view of archaeal evolution,although limited when compared to the Bacteria, alsoshows that the vast majority of the Archaea remainuncul tured, i .e . there are as yet no cul t ivatedrepresentatives of two of the four Archaea kingdoms.

ARCHAEA

EURYARCHAEOTA

CRENARCHAEOTA

KORARCHAEOTA

Marine Archaea

!!!!!

BACTERIA

EUCARYA

T R O P I C A L C O N S E R V A N C Y8

Figs. 1-11. 1a-b, Thermotoga maritima represents a genus of extremely thermophilic bacteria growing up to 90°C [R. Huber et al.1986. Archives of Microbiology 144: 324-333]; 2, Aquifex pyrophilus represents a group of marine hyperthermophilic hydrogen-oxi-dizing bacteria [R.Huber et al. 1992. Systematic and Applied Microbiology 15:340-351]; 3a-b, Thermocrinis ruber , a pink-filament-forming hyperthermophilic bacterium isolated from Yellowstone National Park, U.S. [R.Huber et al. 1998. Applied and EnvironmentalMicrobiology 64:3576-3583]; 4, Pyrobaculum aerophilum is a nitrate-reducing hyperthermophilic archaeum [P.Völkl et al. 1993. Ap-plied and Environmental Microbiology 59: 2918-2926]; 5a-b, Pyrolobus fumarii represents a group of Archaea, extending the uppertemperature limit for life to 113°C [E.Blöchl et al. 1997. Extremophiles 1:14-21]; 6a-b, Sulfur-inhibited Thermosphaera aggregans ,a genus of hyperthermophilic Archaea isolated after its prediction from environmentally derived 16S rRNA sequences [R.Huber et al.1998. Internat ional Journal of Systematic Bacteriology 48:31-38]; 7a-b , Thermococcus chitonophagus , a chi t in-degrading,hyperthermophilic archaeum from a deep-sea hydrothermal vent environment [From R.Huber et al. 1995. Archives of Microbiology164:255-264]; 8a-b, Ignicoccus , a genus of hyperthermophilic, chemolithotrophic Archaea, represented by two species, Ignicoccusislandicus and Ignicoccus pacificus [H.Huber et al. 2000. International Journal of Systematic and Evolutionary Microbiology. 50:2093-2100]; 9a-b, Methanopyrus kandleri, a genus of abyssal, hyperthermophilic, methanogenic archaea growing at 110°C [R.Huber et al. 1989].Nature 342:833-83; M.Kur et al. 1991. Archives of Microbiology 156: 239-247]; 10a-b, Archaeoglobus veneficus , a facul ta t ivechemolithoautotrophic hyperthermophilic sulfite reducer, isolated from abyssal black smokers [H.Huber et al. 1997. Systematic andApplied Microbiology 20: 374-380]; 11, Methanothermus fervidus , an extremely thermophilic methanogen isolated from an Icelandichot spring [K.O.Stetter et al. 1981. Zentralbl. Bakterial. Parasitenkde. Infektionskr. Hyg. Abt. 1 Orig. Reiche C 2:166-178].

A DIVERSITYOF HIDDEN LIFE!With thanks to

Prof. Dr. Karl Stetterand Dr. Reinhard

Rachel, UniversitaetRegensburg,

LehrstuhlMikrobiologie,

for allowing us toreprint the followingselected images fromtheir website. http://

www.biologie.uni-regensburg.de/

Mikrobio/Stetter/bilder.html

1a 1b

3b

43a

2

5b5a

9b

8b7b

9a

8a7a

6a 6b

11

10a

10b

B I O D I V E R S I T Y 3 ( 2 ) 9

TAPPING THE HIDDEN RESOURCEOF UNCULTURED PROKARYOTESThe latest challenge for microbiologists is to developtechniques that will allow much better access to non-culturable prokarytic species rather than simply theretrieval of their SSU rRNA gene sequences. The com-bined use of both DNA-based methods and traditionalfermentation technology has already proved success-ful for the laboratory cultivating of previously un-known species (Huber et al 1995; Kane et al 1993).The basic experimental strategy is to use the SSUrRNA gene sequence in fo rma t ion de r ived f romu n c u l t u r e d s p e c i e s t o ( a ) d e s i g n a n d m o n i t o rlaboratory fermentation protocols that selectivelytarget the unknown species, or (b) modify the actuali n o c u l u m s o t h a t t h e d e s i r e d s p e c i e s h a s l e s scompetition from the more readily adaptable species.

As an alternative strategy, and in this era of genomics,it has become possible to clone and determine the nu-c leot ide sequences of many genes , i f not wholegenomes, of non-culturable bacteria. This strategy isbased on the direct cloning of the genomic DNA fromentire natural communities into routine laboratorys t r a i n s s u c h a s Escher i ch ia co l i . T h e t e r m“metagenome” has been coined for th is s t ra tegy(Rondon et al 1999). These E. coli clones can then bescreened directly for novel biological activity ofinterest, e.g. new enzyme activity of biotechnologicalimportance. The result is the direct acquisition ofnovel genes and proteins without ever attempting tocultivate the hidden species.

A series of other DNA-based methods have also beend e v e l o p e d t h a t p r o v i d e u s e f u l i n f o r m a t i o n o np r o k a r y o t i c c o m m u n i t y s t r u c t u r e i n c l u d i n g i t sd ive r s i t y w i thou t ac tua l ly hav ing to de t e rmineindividual SSU rRNA gene sequences. Methods suchas denaturing gradient gel electrophoresis (Muyzeret al 1993), amplified ribosomal DNA restrictionanalysis (Acinas et al 1997), or terminal-restrictionf ragment length polymorphism (Liu e t a l 1997)amplify the SSU rRNA gene sequences of all speciespresent in a natural prokaryotic community. Thisresults in a DNA fingerprint that is characteristic ofthe diversity of the community at the time of sam-pling. These DNA fingerprints can then be used incomparative analysis to monitor temporal or spatialchanges in community composition.

Finally, with the advent of DNA microarray tech-nology, it is now possible to build DNA microarrays,which are glass slides containing several thousanddifferent synthetic DNA molecules that are specificfor the various bacterial phyla or groups describedon the phylogenetic tree and allow you to examinethem simultaneously. DNA or rRNA isolated directly

from natural communities can then be used to screenthese bacter ial “genosensors” producing a rapid,culture-independent view of community composition.Ultimately, these genosensors may have the abilityto provide both a quantitative assessment in termsof the number of different groups within a naturalcommunity, and also qualitative data with respectto the comparative abundance and activity of thedifferent groups. Guschin et al (1997) demonstratedthe utility of this approach in an analysis of nitrifyingbacteria that are known for their difficulty to culturedue to their long generation times and poor platinge f f i c i e n c i e s . I n t e r e s t i n g l y, D N A m i c r o a r r a ytechnology could plausibly allow the design of theultimate bacterial genosensor containing a sufficientnumber of different SSU rRNA-based synthetic DNAmolecules to cover every theoretical SSU rRNA genesequence combination. Such an approach might wellunearth other currently hidden prokaryotic phyla.

CONCLUSIONSIn the last decade, the use of DNA-based technol-ogy has provided a major st imulus for studies ofboth prokaryotic classification and ecology. It hasproduced a much clearer and perhaps a more equi-table view of the contribution of prokaryotic or-ganisms to life’s evolution and current biodiversity.In spectacular fashion, i t has uncovered the hiddensignificance of the Archaea and placed them as anequivalent taxonomic rank to the Bacteria and theEucarya . I t has provided a natural system for clas-sifying the various bacterial groups, many of whichwe note are more distantly related to each other inevolutionary terms than are plants and animals. I thas also produced a rat ional framework for eco-logical studies that are independent of the bias as-sociated with laboratory cultivation. The result isthe now constant f low of publications describingnovel SSU rRNA gene sequences isolated from anever- increasing range of natural populat ions andenvironments.

“If I could do it all over again, and re-live my vision in the twenty-first century,I would be a microbial ecologist. Ten bil-lion bacteria live in a gram of ordinarysoil, a mere pinch held between thumb andforefinger. They represent thousands ofspecies, almost none of which are knownto science. Into that world I would go withthe aid of modern microscopy and mo-lecular analysis.” E. O. Wilson. 1994. Natu-ralist. Island Press, Washington D.C., U.S.A.

T R O P I C A L C O N S E R V A N C Y10

Microbiologists have never had a better t ime to dis-cover new species. The current prokaryote worldrepresents a major biological resource that is asyet hardly described, although biotechnology com-panies have already focused i t in their sights forexploitation. Alternatively, as concern is expressedabout the condi t ion of b iosphere Ear th , and theimpact of anthropogenic activity, the challenge ofecosystem s tudy remains d i ff icul t i f one wishesreasonably to include the hidden prokaryotes.

ACKNOWLEDGEMENTSThe authors acknowledge the support provided bythe Enterprise Ireland Basic Research Programme(Contract SC/95/102) and the NUI Galway Millen-nium Research Fund for their research on archaealdiversity.

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Barns, S.M., R.E. Fundyga, M.W. Jeffries, and N.R. Pace.1994. Remarkable archaeal diversity detected in a YellowstoneNational Park hot spring environment. Proceedings of the Na-tional Academy of Sciences U.S.A. 91: 1609-1613.

Barns, S.M., C.F. Delwiche, J.D. Palmer, and N.R. Pace. 1996.Perspectives on archaeal diversity, thermophily and monophylyfrom environmental rRNA sequences. Proceedings of the Na-tional Academy of Sciences U.S.A. 93: 9188-9193.

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