cterised (Wayne et al., 1987; Wardson of the percentage of cul-ost obvious examplealenvironment.So, to obtaina better understand-e o f m i c r o b i a l d i v e r s i t y i n t h e m a i n t e n a n c e plorethemicrobialdiversityandtoanalysethestruc -eofmicrobialcommunities(e.g.,MuyzerandRamationsoftraditionalcultivationtech-.

y , t h e y h a v e n o t g i v e n a n y information on theergo by diel and seasonal uctuations or a f t e r ironmental perturbations. As microbial ecologyactions among microorganismsive, and hence impractical for multiple sampledisation of extracted rRNA (e.g., Stahl et al.,8 ; R a s k i n e t a l . , 1 9 9 5 ) o r i n w h o l e c e l l h y b r i d i s a t i o n cularmicroorganismsfor which probeshave been es y.One ofthengerprintingtechniquesthat hasbeene d i n m i c r o b i a l e c o l o g y f o r m o r e t h a n a d e c a d e i s t h e high resolution polyacry-, another genetic ngerprintingtechnique, ophoresis r o d u c e d i n t o m i c r o b i a l e c o l o g y ( M u y z e r e t a l . , GE and the related techniquecalled e gradient gel electrophoresis itationsoftheseapproachesforstudiesinmicrobial E(Fischer&Lerman,1979,1983;Myersetal.,7;Riesneretal., 1991)DNAfragmentsofthesam emamide)oralineartemperaturegradient.Themeltng temperature. Once a domain with the r i a n t s c a n b e d e t e c t e d i n D N A f r a g m e n t s u p t o ased to nearly 100% by the attachment of a GC-A fragment(Myerset al., 1985; Shefeld et al.,8 9 ) . A s e q u e n c e o f g u a n i n e s ( G ) a n d c y t o s i n e s ( C ) i s d o f o n e o f t h e P C R p r i m e r s , c o a m p l i - h sequence acts as a high melting domaint i o n i n t o s i n g l e s t r a n d s . T h e l e n g t h o f t h e G C clamprybetween30and50nucleotides(see Table2 ine P C R p r i m e r s i s l a b e l l e d a t i t s 5 d w i t h a p h o t o a c - pound,such as psoralene, which interca-covalentlylinkthemtogetherafterUV irradiation. mide (Muyzer et al., 1997). The advantag e chmakesitpossibletoobservelessdominantDN A o s t a i n s s i n g l e s t r a n d e d D N A , a n d s i l v e r s t a i n e d g e l s ean increasinggradientof denaturantsor tem-e.DNA moleculesat the left sideof thegel, whereentrationof denat forehalt.Atintermediateconcentrationsofdenat -s,themoleculeshavedifferentdegreesofmelting,r e s p o n d i n g t o themeltingtemperatureofthelowestltingdomainofthefragme n t . P e r p e n d i c u l a r g e l s a r e ents. In addition, from these gels the optimalean increasinggradientof denaturantsor tem-a t u r e f r o m t o p - t o - b o t t o m , p a r a l l e l t o t h e d i r e c t i o n o f minedto obtainmaximumresolutionbetweenthe

E e q u i p m e n t c a n b e o b t a i n e d f r o m d i f f e r - ercial companies, such as Bio-Rad (Her-,USA),INGENY(Leiden,TheNetherlands),andS. Scientic Co., Inc. (Del Mar, USA). TGGEi p m e n t o r i g i n a l l y s o l d by Diagen GmbH (Gercommunity complexityof a micro-l biolms (Muyzer et al., 1993).r t h i s p u r p o s e b a c t e r i a l g e n o m i c D N A w a s e x t r a c t e d resentinthe sample. TheindividualPCR productse s u b s e q u e n t l y s e p a r a t e d b y D G G E . T h e r e s u l t w a s the number of bandscor-ide probe specic for sulfate-reducing bacteriaCR-d hydrothermal vents. Denaturing gradient gelrophoresisofDNAfragmentsobtainedafterenzy-primers, showedonly 1 band for re1 w d i a g r a m o f t h e d i f f e r e n t s t e p s i n t h e s t u d y o f t h e s t r u c - ular markers isthe heart of a strategy tonce (DNA)andactivity (rRNAormRNA)ofbacterial e s i n t h i s h a b i t a t , w h i c h w a s a l s o f o u n d b y estigators (Moyer et al., 1994, 1995) for aobialcommunityfromanotherventsite. Sequences of ribosomal DNA fragments obtainedager Fjord water column and sediment samples. PCR prod-ned from DNA reect the presence of bacteria, while ducing bacteria in a stratied water column o f m e n v i r o n m e n t a l D N A w o u l d d e m o n s t r a t e t h e p r e s - , and that PCR products obtained after ampli-s e o b t a i n e d b y R T P C R w i t h n u c l e i c a c i d s f r o m d i f - ) for soil microbial communities. TGGE pro-res in a hot spring cyanobacterial comfragments from the rhizophere and phyllophere of several transgenic (T) and non-transgenic (N) potato y(Ferriset al., 1996).DGGE prolesof sampleslar bacterial populations. However, differentq u e n c i n g o f i n d i v i d u a l b a n d s f r o m t h e differentpro esrevealedknownbutalsonewbacterialphylotypes.ently,Murr ayetal. (1996)usedDGGEofPCR - icdiversityofbacterioplanktonassemblagesfromes. They found that the two assemblageslt of the availability of different kinds of organicE o f e n z y m a t i c a l l y a m p l i e d r D N A frag ntshasalsobeenusedtoidentifybacteriainabiode -dedwallpainting(Rekeetal.,1996).Sequencing hmenttechniquesfavoure dobacteria.CombinedwithDGGEanalysisoftheseldiversityin nonaxeniccultures, lichensandcom-ial assemblages, such as those present inferent membersbelonging to the rlands. Comparative DGGE analysis of DNA s , d e m o n s t r a t e d t h e p r e s e n c e o f t h e s e b a c t e r i a i n t h e s has been applied to compare bacterial popula-t r a n s g e n i c p o t a t o p l a n t s ( F i g u r e 3 ; H e u e r & S m a l - A fragments frombtainedfromthe phyllospheresampleswere lesse r e t a l . (1997) used DGGE and TGGE tor e n t s o i l s , a n d t o m o n i t o r

s h i f t s i n t h e i r a b u n - ces in the potato rhizosphere. In this study, the er and a reverse bacterial primer, followed byalled vironmentalDNA with the bac-al primers directly, made it possible to estimateR - p r o d u c t s o b t a i n e d f r o m t o t a l c o m m u n i t y e g e l a s t h e m a j o r b a n d i n t h e T G G E p r o l e d e r i v e d unityDNA as well as a band obtaineduleswere used to studyammoniaoxidizingbac-. Comparative DGGE analysis of PCR prod-nvironmentalDNA and fromclonedinsertsu s esrelativelyclosetothe focused on the analysisa i n e d f r o m f u n c t i o n a l g e n e s c a n a l s o b e u s e d . W a w - d Muyzer(1995)designedPCR primersto amplins couldeasily be separatedby DGGE. Inactedfroma microbialmat and fromdifferentbac-l biolmsdemonstrateda greatergenetic diversity awer and Muyzer, 1995; Wawer, 1996). obialecologicalstudiesoftenrequirethesamplingn, cloning techniques are no t ngDGGEorTGGEmanysamplestakenatdifferen t lysed.Thismakesthetechniquesapowerfultoolfo r oworkers(1996)followedthe succes-s a n d P C R D G G E t o m o n i t o r s u c c e s s i o n a l c h a n g e s , . Concomitantlywith the develop-t o f t h e b i o l m a n i n c r e a s i n g n u m b e r o f b a n d s w a s ysiswas also used to followthe spatial-oraldistributionofsulfatereducingbacterialpop-cleotide probe spem in the top layers of the microbial mat at 5 a.m. ard,1997).SimilarDGGEpatternswerefoundform p l e s c o l l e c t e datthesame siteandforsiteswithth e ferentproleswereseenforsamplesfromsiteswith as originally used to studyr simpler mixtures of microorganisms.asicapproach,includingphenotypicalanded strains of the cyanobacterium noplastes cia-Pichel et al., 1996).n t i c a l D G G E p a t t e r n s o f 1 6 S r D N A f r a g m e n t s f r o m ently,Teskeetal. (1996b)usedDGGEofrDN Aetodesignmoreselectiveconditionsandsucceeded zer (1997) to determine the success of isolaethesespeciesfromtheenvironmentsbyenrichment disationanalysisof DGGE patternsof16S rDNA c oligonucleotideprobefor which the target sitenedmolecularandmicrobiologicalapproach sis of PCR-amplied 16S rDNAled analysis, i.e., sequencing of the tota

l upAandB,respectively.Theauthorsindicatedtha t DGGEapproachisan easy, andtimesavingmean s tocolsusedforthephysicalseparationofbacteriaomsoilmatrice s a n d t h e r h i z o s p h e r e ( H e u e r & S m a l - oduciblepatternsofPCRamplied16Stly, JaspersandOvermann(1997)usedPCRealedmicroheterogeneityinthedifferentrRNA g.,F,1996;Heuer&Smalla,1997;Liesacketal. , productsfrombacterialgenomicDNA extractiction fragment length polymorphisme been applied to screen clone libraries. TGGEed to determiney in clone libraries, and to estimate thement. For this purpose, the nearly,the inserts are reamts and those obtained from environmental DNA,but not necessarE h a s b e e n u s e d t o d e t e r m i n e t h e e r r o r r a t e ny Smalla (pers. comm.) for example use d NAgenesfrombacterialDNAextractedfromdif - ntenvironmentalsamples.Comparativeanalysisof ctswith nestedDGGE/TGGEprimersshowedin d l i n g . R o c h e l l e e t a l . ( 1 9 9 4 ) f o u n d t h a t d i f f e r - A sequence analysis. For instance, a sediment tanaerobicallyandfrozenwithin2hoursaftersamular characterizations. Problems arenteredwith the reliable and reproduciblelysis of l b a c t e r i a l c e l l s a s w e l l a s w i t h t h e e x t r a c t i o n o f i n t a c t ate instead of extracted DNA can beiologicalstate of the cells (Silva &N A g e n e s b y P C R h a s b e e n d e s c r i b e d b y R e y s e n - ctionwasused to facilitate templatedenat-cation (Rey-chet al., 1992).Cosolvents, such as glycerolandstrated the effect of genome size and the copyd u c t s . A n o t h e r p r o b l e m i n t h e u s e o f P C R t o a m p l i , 1 9 9 4 ) . C o m p u t e r a l g o r i t h m s , s u c h a s t h e s. Raineyet al. (1994)describeddifferentcloningicationprocessmightcontribute9 ; F e r r i s e t a l . , 1 9 9 7 b ) . A h e t e r o d u p l e x D N A ng temperature is lower than for homoduplexionof heteroduplexformation will result in fouroduplex and homoduplex molecules.E a n d T G G E a n a l y s i s t o i n c r e a s e t h e r e s o l u t i o n i n , Murray et al. (1996) concluded that the f o r m a - n t h e r e l a t i v e f o r m a t i o n o f h e t e r o d u p l e x D N A i n t h e ed by using a higher ionic strength,e , P C R p r o d u c t s c a n b e t r e a t e d w i t h a s i n g l e - s t r a n d i t a t i o n s i s t h e s e p a r a t i o n o f o n l y r e l a t i v e - its the amountof sequenceinformatione n e t i c i n f e r e n c e s a s w e l l a s f o r p r o b e d e s i g n . E a l t h o u g h t h e y h a d s u b s t a n t i a l s e q u e n c e v a r i - . A similar result was described by Buchholz-s i b l e t o s e p a r a t e r D N A f r a g m e n t s d i f f e r i n g i n t w o t o d separate DNA fragments

997)nicelydemonstratedthatdoublebandsintheEpatternswer earesultofthepresenceaso-called ers were used in separate PCR reactionserent resolutions of separation. As the melt-ts for which sequences are e the hybridisation potentialw e e n p r i m e r s a n d p r o b e s a n d S S U r R N A s e q u e n c e s A reannealing experiments Torsvik ferentgenomespresentinsoilsamples.Itwillbeioustothereader thatDGGEorTGGEcannotsepa teallofthe16SrDNAfragmentsobtainedfromsuc h oretictechniqueswillonlydisplaytherDNAfrag-ving clean sequencesfrom indi-u a l b a n d s . A n o t h e r p r o b l e m i n t h e s t u d y o f c o m m u equenceheterogeneity(Nl e t a l . , 1996)whichhtleadtoanoverestimationofthenumberofbacte -alcommunities.Thesameistrueforthe ertheless,substantialinformationaboutthespeciesp o s i t i o n c a n b e o b t a i n e d f r o m v e r y c o m p l e x m i c r o - ties by DGGE or TGGE analysis. Bac-l cells can be dislodged from soil (e.g. Priemss (e.g. Jaspers & Overmann, 1997). Further-b e n & H a r r i s , 1 9 9 5 ) . v r e a s e t a l . ( 1 9 9 5 ) u s e d achto fractionate complexmixturesof DNAactedfromsoil samplespriorto PCR amplicationningbisbenzimideto which long chains of poly-eneglycol(PEG)were covalentlycoupledto sep-R products.This electrophoreticapproachhasr useful approach to obtain information of lex DGGE or TGGE proles is hybridizationo t i d e probes (Muyzer et al., 1993; Teske et a l . , 6 a ; B r i n k h o f f & M u y z e r , 1 9 9 7 ) . H e u e r e t a l . ( 1 9 9 5 ) geninlabelledpolynucleotideprobesunder E o r T G G E p a t t e r n s . T h e s e p r o b e s w e r e p r o - tic amplicationof the hypervariable nceinformationisrequiredtoproducetheprobes.p l e x b a n d i n g patterns can also be reducede a n a l y s i s o f P C R p r o d u c t s o b t a i n e d w i t h etes (Heuer. , 1 9 9 7 ) . A s e c o n d s t r a t e g y i s t h e a m p l i c a - dby a reamplicationof these PCR productswithmine the ecological importance of partic-r t h e r m o r e , s i m p l e r D G G E p a t t e r n s c a n b e a i n e d using PCR primers for functional genes,ch are only present in particular bacterial popawer and Muyzer, 1995). , but their future perspectives aresing.CombinedwithPCRamplicationofmark esortheirtranscripts(rRNAandmRNA)DGGEd T G G E c a n g i v e a d i r e c t d i s p l a y o f t h e p r e d o m i n a n t ted out; a lot of e x p e r i e n c e , e s p e c i a l l y eon the thermodynamicsof melt-viourof double-strandedDNA in solutionandcommunitiesor certain indicatormicroorganism-nds or the release of geneticall y .i t i n g n e w d i r e c t i o n w i t h i n t h e e l d o f m o l e c - r m i c r o b i a l e c o l o g y i s theuseoffunctionalgenesas s (Wawer et al., 1997). By comparative analy-

tions,but onlythe preferentialexpressionofthe s p a r t i c u l a r p o p u l a t i o n m i g h t b e b e t t e r a d a p t e d t o t h e th on hydrogenthan the other on. This might point to a niche differentiation of o n s i n t h e c o m m u n i t y u n d e r d i f f e r e n t e n v i r o n te reductase gene (Karkhoff et. , 1 9 9 5 ) , w e w i l l s o o n b e a b l e t o u s e D G G E a n d o t h e r rprintingtech niquestorelatestructuretofunction ,r o b i a l c o m m u n i t i e s a n d t h e i r p e r f o r m a n c e i n t h e r o b i a l p o p u l a t i o n s . L e e a n d c o w o r k e r s ( 1 9 9 6 ) hism(SSCP; Oritaet al., 1989)ofPCR-ampliedal communities. Furthermore, SSCP of mal DNA restric -illiams, 1 9 9 0 ) n g e r p r i n t i n g t o f o l l o w t h e r e s p o n s e o f d i f - ent soil microbial communitiesto the applicationof 9 5 ) u s e d a s i m p l e a n d r a p i d e l e c t r o p h o r e s i s m e t h o d entsobtainedafter enzymaticamplicationmation,while sequencingof bands obtainedates gel-to-gel comparison, and imagel y s i s o f t h e g e l s . F u r t h e r m o r e , t h e u s e o f u o r e s c e n t roductscombinedwithcapillaryelectrophoresis rated PCR products, which can than b e wever,regardingthebiasesandlimitationsofallhodsonlyaninte gratedapproachcombiningmole -vealtheroleofmicrobialdiversityinecosystemfunce are gratefulto all participantsof the EU-sponsoredy. We thank Hendrik Schr , U l r i c h N ger Heuer forcritically readingthe nnRI,StromleyJ,Devereux R,KeyR&StahlDA(1992)Moleells withou tr n s S M , F u n d y g a R E , J e f f r i e s M W & P a c e N R ( 1 9 9 4 ) R e m a r k a b l e t EW (1996) Molecular microbialersity of an agricultural soil in Wisconsin. Appl. Environ. elloNF,KeohavongP,SandersonBJS&ThillyWG(1988)DNAis MJ, Muyzer G & Ward DM (1996) Denaturing gradient gelbial mat community. Appl. Environ.ityfollowing disturbance. Appl.Environ.Microbiol.is MJ & Ward DM (1997) Seasonal distributions of dominantke A,Wolterink A,van LisR&AkkermansADL(1998)Screen-her SG &Lerman LS(1983) DNAfragments differing by singlersuchungen zu derBiodiversitanJA,McCallum K&DavisAA(1993)Phylogenetic diversiannoni SJ,Britschgi TB,MoyerCL,&FieldKG(1990)Geneticcommunities associated withpotato plantsbyBIOLOGuring gradients. Appl. Environ. Microbiol. 63: 3233 139 Hoe MG (1988) Identifcation of bacteria by low molecular weightRNA proles: a new chemotaxonomic approach. J. Microbiol.Meth. 8: 235248HoeMG (1990) Transfer RNAsas genotypic ngerprints of eubacteria. Arch. Microbiol. 153: 299304Holben WE, Calabrese VGM, Harris D, Ka JO & Tiedje JM (1993)Analysis of structure and selection in microbial

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