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Microbiology (1997), 143,2983-2989 Printed in G reat Britain

Ribosome analysis reveals prominent activityof an uncultured member of the classActinobacteria in grassland soilsAndreas Felske,l Holger Rheims, Arthur Wokerink, Erko Stackebrandtand Antoon D. L. AkkermansAuthor for correspondence: Andreas Felske. Tel : +31 317 484250. Fax: +31 317 483829.e-mail : [email protected]

1 Wageningen AgriculturalUniversity, DepartmentofMicrobiology, Hesselinkvan Suchtelenweg 4,6703 CTWageningen,The Netherlands2 DSMZ- DeutscheSammlung vonMikroorganismen undZellkulturen GmbH,Mascheroder Weg 1b,38124 Braunschweig,Germany

A 16s rRNA-based molecular ecological study was performed to search fordominant bacterial sequences in Drentse A grassland soils (The Netherlands).In the fir st step, a library of 165 clones was generated from PCR-amplified165rDNA. By sequence comparison, clone DA079 andtwo ther identical clonescould be affilia ted to a group of recently described uncultured Actinobacteria.This group contained 165 rDNA clone sequences obta ined from differentenvironments across th e world. T o determine whether such unculturedorganisms were par t of the physiologically active populat ion in the soil,ribosomes were isolated from the environment and 16s rRNA was pa rtia llyampl if ied via RT-PCR using conserved primers for members of the domainBacteria. Subsequent sequence-specif c separation by temperature-gradientgel electrophoresis (TGGE) generated f ingerprints of th e amplicons. Suchcommunity fingerprints were compared with the TGGE pattern of PCR-amplif ied rDNA of clone DA079 which was generated with th e same set ofprimers. One of the dominant fingerprint bands matched with the bandobtained from the actinobacterial clone. Southern blot hybridizationwith aprobe made from clone DA079 confirmed sequence ident ity of clone andfingerp rint band. This is the fir st report that a member of the novelactinobacterial group may play a physiologically active role in a nativemicrobial community.

Keywords: 16s rDN A library, Actinobacteria, temperature-gradient gel electrophoresis(TGGE), ribosome extraction from soil, V6 probe

INTRODUCTIONOver the last few years, molecular ecological studies onmainly terrestrial environments (Liesack & Stacke-brandt, 1992a, b; Stackebrandt et al., 1993; Rheimset al., 1996a, b) have indicated the existence of tw omonophyletic groups of uncultured bacteria from theclass Actinobacteria (Stackebrandt et al., 1997). Asjudged from the analysis of PCR-amplified 16 s rDN Athese sequences showed only rath er rem ote similaritiesto sequences of cultured actinobacteria. The relevantclones were obtained from an Australian forested soiltaken from the Mount Coot-tha region in Brisbane,

Abbreviation: TGGE, temperature-gradient gel electrophoresis.The EMBL accession number for the sequence of DA079 reported in thispaper is Y11555.

Queensland (Liesack & Stacke brandt, 1992a, b ; tacke-brandt et al., 1993), a pea t bog sample from Germany(Rheims et al., 1996a, b), geothermally heated soil fromNew Zealand (Rainey et al., 1993) and a soil samplefrom Finland (Saano et al., 1995). Also, short 16 s rDNAclone sequences from a paddy field (M aid ak et al., 1994),a soybean field (Ueda et al., 1995) and a marineenvironment (Fuhrman et al., 1993) were demonstratedto cluster with these actinobacterial sequences. Thus ithas been concluded that this group of unculturedorganisms might contribute to ecologically importantprocesses (Rheims et al., 1996b).The presence of rDNA sequences in a clone libraryproves neither activity nor abundance of the micro-organism from which the DNA is represented in thelibrary. Previous investigations already indicated tha t alarge fraction of environmental microbial communities

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is in a stage of low a ctivity or resting (Bakken & Olsen,1987; Roszak et af., 1984). DNA obtained fro m en-vironmental samples could thus originate from suchdormant cells, from dead cells (Josephson et al., 1993),or even from free DN A. Adso rption of DNA at mineralsurfaces, especially in soils, could h arb ou r m ore o r lessintact nucleic acids a long time after lysis of the sourceorganism (Lorenz & Wackernagel, 1987). Our strategyto obtain information about the presence of metabo-lically active bacteria in th e environm ent focuses on theanalysis of 16s rRNA from isolated ribosomes. As theribosome per cell rati o is roughly pro portiona l to growthrate of bacteria (Wagn er, 1994), rR NA is regarded a s anindicator of total bacterial activity.Here we repo rt on th e finding of a novel sequence of on eof the actinobacterial lineages from the Dre ntse A rDN Aclone library (A. Felske, A. Wo lterink, R. van Lis &A. D. L. Akkermans, unpublished results). The clonedsequence DA079 was investigated for its significance inthe environmental 16s rDNA population a nd 1 6s rRNAfractions of ribosomes th at were isolated from th e samesite (Drentse A agricultural test area, The N etherlan ds).After direct ribosome isolation from soil samples (Felskeet af., 1996), rRN A was purified and used for RT-PCRwith bacteria-specific primers. This partial 16s rRNAamplicon, representing the complex sequence popu-lation of the so il, was seq uence-specifically sep arat ed bytemperature-gradient gel electrophoresis (TGGE;Ros-enbaum & Riesner, 1987 ). Th e resulting fingerprint wasscreened for the cloned sequence DA079 by amplifyingthe plasmid DNA with the same primers as those usedfor soil rRNA. R unning this product next to the rRNAfingerprint indicated possible matches. Subsequent elec-trophoretic Southern blotting and hybridization with aclone-specific probe was applied to confirm the match.METHODSCollection of soil samples. Peaty, acid grasslands of theDrentse A agricultural research fields next to the AnlooerDiepje River, the Netherlands (06" 41' E, 53" 03' N), werethe sites of sample collection. A total of 120 surface samples( < 10 cm depth) were taken on three different testfields duringMarch 1996. Another 240 surface samples were taken on thesame and additional three testfields in October 1996. Sixdifferent testfields (A, B, C, F, K and 0) along the AnlooerDiepje River were investigated. Distances between the relevanttestfields were several hundred metres, with a maximum of1.5 km between testfields F and K. On each testfield, 40 soilcores of about 50 g were taken with a drill (0-10 cm depth)and transferred into sterile sample bags. The 40 samples ofeach testfield were pooled to four samples by sieving andmixing 10 single samples (5 g input each). Details of the soilproperties were published by Stienstra et al. (1994).Isolation, amplification, cloning and sequencing of 165 rDNAsequences. Total DNA was isolated from Drentse A soilsamples by a parallel pathway during ribosome isolation(Felske et al. , 1996). Amplification of 16s rDNA sequenceswas performed with a GeneAmp PCR System 2400 thermo-cycler (Perkin Elmer Cetus), using 35 cycles of 94 "C for 10s,54 "C for 20 s and 68 "C for 2 min. The PCR reactions (100 pl)contained 10 mM Tris/HCl (pH 8*3),50 mM KC1, 3 mM

MgCl,, 0.05o detergent W-1 (LifeTechnologies), 150 pMeach of dATP, dCTP, dGTP and dTTP, 100 pmol primers 8fand 1512r (as below), 2.5 U T a q DNA polymerase (Life-Technologies), and 1p1 template DNA. Amplification pro-ducts were confirmed by 1.4o agarose gel electrophoresis andthen separated from primers and dNTPs on a low-melting-point agarose gel. Subsequently they were cloned in pGEM-Tlinear plasmid vector and Escherichia coli JMl 09 competentcells according to the manufacturer's instructions (Promega).Isolated and purified plasmids (Wizard373 DNA purificationsystem; Promega) were sequenced using a Sequenase (T7)-terminator dsDNA sequencing kit (Pharmacia) on a Li-CorSequencer 4000L.Isolation of ribosomes and amplification of 165 rRNA.Ribosomes were isolated from Drentse A soil samples (1ginput) as previously described (Felskeet al. , 1996). RT-PCRwas performed with the rTth DNA Polymerase kit fromPerkin Elmer Cetus. Reverse transcription reactions (10 pl)contained 10 mM Tris/HCl (pH 8*3), 90 mM KCl, 1 mMMnCl,, 200pM each of dATP, dCTP, dGTP and dTTP,750 nM primer L1401 (as below),2 5 U rTth DNA polymeraseand 1p1 template RNA. After incubation for 15 min at 68 "C(reverse transcription), 40 pl PCR additive containing 10 mMTris/HCl (pH 8*3),100 mM KCl, 0.75 mM EGTA, 00 5 o(v/v) Tween 20,3-75 mM MgCl,, 50 pM each of dATP, dCTP,dGTP, and dTTP, 190 nM primer U968/GC (as below) wereadded. Amplification was performed in a GeneAmp PCRSystem 2400 thermocycler, using 35 cycles of 94 "C for 10 s,56 "C for 20 s and 68 "C for 40s. The correct size ofamplification products was checked by electrophoresis on a1.4YO agarose gel.Partial 165 rDNA amplification of clone DA079 for TGGE. Asingle DA079 clone colony (identified by sequence analysis)was taken up with a sterile toothpick and transferred to a1.5 ml microcentrifuge tube containing 50 p1 TE buffer. Thetube was heated for 15 min at 95 "C and then chilled on ice. ATGGE-suitable 16s rDNA amplicon was generated with aGeneAmp PCR System 2400 thermocycler, using 25 cycles of94 "C for 10s, 56 "C for 20 s and 68 "C for 40 s. The PCRreactions (20pl) contained 10 mM Tris/HCl (pH 8*3), 0 mMKC1,3 mM MgCl,, 50 pM each of dATP, dCTP, dGTP, anddTTP, 100pmol primers U968/GC and L1401 (as below),05 U T aq DNA polymerase and 1pl DA079 cell lysate.Dilution series of the PCR product (2, 1, 0.5 and 0.25 pl perlane) were used for subsequent TGGE and electrophoreticSouthern blotting.Preparation of the clone-specific probe V6-DA079. A probefor the clone DA079 was generated by amplification of thehighly variable V6 region of the 16s rDNA (Heuer& Smalla,1997) with a GeneAmp PCR System 2400 thermocycler, using30 cycles of 94 "C for 10 s, 46 "C for 20 s and 68 "C for 10 s.The PCR reaction (100 pl) contained 10 mM Tris/HCl(pH 8-3),50 mM KC1, 3 mM MgCl,, 25 pM each of dATP,dCTP, dGTP, and dTTP, 200 pmol primers V971 and R1057(as below), 2.5 U T a q DNA polymerase and 1p1 DA079 celllysate. Resulting PCR products were purified and concentratedby ethanol precipitation. The precipitated DNA was resolvedin 50 p1 nanopure water and then 5'-labelled using phage T4polynucleotide kinase (Promega) and 3.7 Bq of [y3'P]ATP(370 MBq m1-l; Amersham Buchler) in a 30 min reaction at37 "C.TGGE, electrophoretic Southern blot and hybridization. TheDiagen TGGE system was used for sequence-specific sep-aration of PCR products. The temperature gradient wasoptimized to 9 "C difference for improved resolution. Electro-

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phoresis was performed with a 0.8 mm polyacrylamide gel(6 % , w/v, acrylamide; 0.1 "/o, w/v, bis-acrylamide; 8 M urea;20 '/o, v/v, formamide ; /o, v/v, glycerol) with 1x TA buffer(40 mM Tris-acetate, pH 8-0) at a fixed current of 9 mA(about 120 V) for 16 h. A temperature gradient from 37 to46 "C wa s established in the direction of electrophoresis.Samples for RT-PCR of soil rRNA a nd PCR of plasmid DN Awere applied twice in symmetrical order.After electrophoresis one-half of the gel was used for silverstaining (Engelen et al., 1995), the other half for Southernblotting. This half was shaken for 15 min in 0 5 x TBE buffer(Sambrook et al., 1989). Tw o pieces of gel-sized Whatm anfilter paper and one sheet of nylon mem brane (Hy bon d-N + ;Amersham ) were treated in the same way. O ne filter paper, t hemembrane, the gel and finally the other filter paper wereplaced above each other into a T ransBlot SD ElectrophoreticTransfer Cell (Bio-Ra d). After closing the transfer cell acurrent of 400 mA w as applied for 1h. After this electro-phoretic blot the mem brane was briefly washed in 0 5 x TB Eand placed on top of another Whatman filter paper (pre-soaked with 0 4 M N aO H) for 10m in. After shaking thememb rane for 10 min in 2 x SSC (Sambrook et al., 1989), theDNA was immobilized by baking at 12 0C for 30 min.Prehybridization (1h at 56 "C) and hybridization (16 h at56 "C) were performed in 5 x SSC with 2 % (w/v ) blockingreagent (Boehringer), 0 1 /o N-lauroylsarkosine, 0.02 'o SDSand 20% (v/v) formamide. For hybridization, 10 p1 labelledprobe V6-DA079 were added. Subsequent stringent washingsteps were twice fo r 5 min in 2 x SSC with 0.1 "o SDS and twicefor 15 min in 0.1 x SSC with 0.1 "o SDS on a shaker a t roomtemperature. A detection screen (Molecular Dynamics) wasincubated with the hybridized membrane and th e probe signalswere detected with a Phosphor Imager SF (Molecular Dy-namics). Quantification was performed with image analysissoftware ImageMaster 1D Elite version 2.0 (Pharma cia).Oligonucleotides.All oligonucleotides used in this study w erespecific for bacterial 16s rRNA. Th e numbers in the primernames indicate the position of the 5' nucleotide in the 16srRNA of E. coli (Brosiuset al., 1978). Th e sequence for primerU968/GC is 5'-(GC clamp)-AAC GCG AAG AAC C T T AC-3', and for primer L1401 it is 5'-CGG T G T GT A CAA GACCC-3'. These 17mers are specific for highly conserved 16srRNA regions from bacteria (Niibe let al., 1996).Th e sequenceof the GC clamp, linked to the 5' terminus of the PCR-amplified product is 5'-CGC CCG GGG CGC GCC CCGGGC GGG G CG GG G GCA CGG GGG G-3'. This 40mer isuseful for accurate separation of PCR products in th e gradientgel electrophoresis (Muyz er et al., 1993).Th e primers used for generating th e clone library are were 8f,CT C AG-3', and 1512r, 5 '-GTG AAG CT T ACG G (C /T )TAGC T T G T TA CGA CTT-3'. Both are specific for highlyconserved 16s rRNA regions of bacteria (tak en an d modifiedfrom Weisburg et al., 1991).Probe V6-DA079 was amplified with primers V971, 5'-GCGAAG AAC CTT ACC-3', and R1057, 5'-CAT GCA GCACC T G T-3'; both are specific for highly conserved 1 6s rRN Aregions from bacteria (Hartung, 1996).Phylogenetic analysis for clone DA079. The 16s rDNAsequence of clone DA079 was transferred to the alignmenteditor A E ~Maidak et al., 1994) and compared t o the DSM Z16s rDNA database of Actinoba cteria. Th e sequence of DA079was then compared to th e most closely related m embers ofuncultured peat organisms of groups I1 and I11 and theirclosest cultivated relatives. Sequences of clones from other

5 '-CAC GGA TCC AGA CT T TGA T (C/T) (A /C) TG G

environments could no t be shown within the sa me tree (exceptclone MC58), as they do not have enough sequence in-forma tion or overlap w ith the othe r clone sequences. For theconstruction of a phylogenetic tree, sequences from othermore remotely related organisms were also included in thecomparison.The similarity values for these sequences were transformedinto phylogenetic distance values that compensate for m ultiplesubstitutions at any given site in the sequence (Jukes &Can tor, 1969). Th e phylogenetic dendro gram w as constructedwith the neighbour-joining method included in the PHYLIPpackage (Felsenstein, 1993).RESULTS A N D DISCUSSIONDetection of clone DA079 rDNA and relatedenvironmental sequences in soilsClone DA079 originates from a D r e n t s e A 16s r D N Aclone l ib ra ry , compr i s in g 165pos i t ive clones (A. Felske,A . Wol te r ink , R. va n Lis & A. D. L. A k k e r m a n s , u n -publ ished resul ts ) . Two other c lones were ident ical ins equence , so f inal ly thre e of 165clones represented thisDA079 sequence. Some c l o s e l y r e l a t e d G e r m a n p e a tclones (des ignated TM c lones ) we re p rev ious ly fou nd tohave relatives in geograph ica l ly w ide ly s epara ted soilenv i ro nme n ts , i .e . Australia, Finland, Japan, New Z e a -l a n d , a n d in t h e A t l a n t i c and Pac if ic oceans ( s um ma r izedb y Rheims et al., 1996b). With t h e d e t e c t i o n of t h eu n c u l t u r e d organism DA079 in D r e n t s e A g r a s s l a n dsoils another e x a m p l e for the wide d is t r ibu t ion of thisn o v e l g r o u p of uncu l tu red ac t inobac te r ia in d i f f e ren tsoil types is given.In detail, c l o n e DA079 is a m e m b e r of peat c l o n e g r o u pI1 (as defined b y R h e i m s et al., 1996b), s h o w i n g as imilar i ty of 95-3YO to clone TM208 (Fig. 1).No closerela t ionship exists b e t w e e n t h e peat c l o n e g r o u p I1( inc lud ing c lone DA079) a n d t h e n e a r e s t c u l t u r e drelative, Acidimicrobium ferrooxidans (Clark& N o r r i s ,1996).According to th e c u r r e n t t a x o n o m i c s t r u c t u re oft h e class A c t i n o b a c t e r i a ( S t a c k e b r a n d t et al., 1997), vena r e l a t i o n s h i p at f ami ly l eve l s e ems un l ike ly .Quantification of sequences in clone librariesTo d r a w a c o n c l u s i o n o n t h e i m p o r t a n c e of theseo rgan i s ms , as detected by ana lys i s of amplif icat ionproducts, th e f o l l o w i n g factors s h o u l d b e c o n si d e re d .D e d u c t i o n of the n u m b e r of o r g a n i s m s c h a r a c t e r i z e d bya u n i q u e 16sr D N A s e q u e n ce i n t h e c o m m u n i t y f r o m t h en u m b e r of s equences in a 16s r D N A l ib r ar y is difficult.As most 16s rDNA s e q u en c e s a p p e a r o n l y o n c e d u r i n gs equence ana lys i s of c lones (Wel le r et al. , 1991;Borne-man et al., 1996), the q u e s t i o n is r a i s e d w h e t h e r thisreflects the real a b u n d a n c e or whether t h e s e u n i q u es e q ue n c e s w e r e r a n d o m l y r e c ov e r ed from a l o w - a b u n -d a n c e b a c k g r o u n d ?One m a y i m a g i n e a m o d e l i n w h i c h 50% of al l t h e 16srDNA within a n a t i v e b a ct e ri a l c o m m u n i t y o r i g i n a t e sf r o m a few dozen d o m i n a n t s p e c i e s . T h e other 50%cons is ts of t h o u s a n d s of r a r e s e q u en c e s o c c u r r i n g with

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Rubmbacter mdmtolerensEubacteriumfossor

Coriobactedum glomemns- -

Acidimicrobium hmxidans"CandidatusMicrothrixpawicella"

- T M 6 2 + 2 d o ~ ~ 1

-DA079t 2

- M81 + 4 clones- M208-,-TM214- - M232 + 1 clone

Fig, 7. Dend rogram showing phylogenetic positions of 165rDNA related groups II and Il l and the clone DA079 within theActinobacteria. The sequence of Bacillus subtilis served as anoutg roup sequence. The analysis is based on abo ut 1100 5' n t ofthe 165 rDNA sequence (limited by the length of the sequenceof clone MC58). The bar represents 10nt substitutions per100 nt.

a very low mean abundance each. This model is notunreasonable as the presence of thousands of differentbacterial species can be expected to occur in a single soilsample (Torsvik e t al., 1990). Extraction of DNA andsubsequent PCR amplification with universal bacterialprimers yields a 16s rDNA amplicon of a very complexsequence composition, optimally reflecting the nativecomposition. In the subsequent steps of ligation andtransformation, the selection of sequences is random.Hence rare sequences have a realistic chance of ap-pearing in a successfully cloned insert. In our model,one-half of all clones will represent the few dominantspecies, while the other half is composed of randomlyselected members of a low-abundance background. Thehigher the microbial diversity, the more likely is theappearance of such randomly selected rare sequences.Thus the normally limited size of environmentalclone libraries (a few hundred clones) has the dangerof overestimating unique clones. Multiple detection ofidentical clone sequences within one clone library, onthe other hand, might indeed indicate abundance in theoriginal population. In this study three identical 16srDNA sequences of the type DA079 were identifiedwithin the Drentse A library of 165 clones.In contrast to the uncertain meaning of sequencequantities in clone libraries, the semi-quantitative as-

Fig. 2. Amplicon fingerprints on silver-stained TGGE gels. RT-PCR products from ribosomes and a partia l 165 rDNA PCRproduct from genomic DNA from the same probing site,obtained with the corresponding set of primers, each showbands of eq ual position, matchin g with the DA0 79 signal asindicated. Fingerprints represent two of four pooled sampleseach from Drentse A sites A, F and K. 55' indicates the positionand size of the single-stranded cDN NrD NA fraction.

sessment of PCR amplicons in TGGE fingerprints isonly based on staining intensity. A silver-stained TGGEgel would show a background of thousands of differentsequences as a faint background smear, somewherebetween invisibility and extremely faint bands. Henceestimation of sequence abundance in genetic materialisolated from the environment is more likely to berepresentative by application of the TGGE approach.Detection of sequence DA079 by TGGE in total rRNAand rDNA from soilDirect ribosome isolation yielded purified rRNA [1.5&0.6 pg (g soil)-'] which could be used for RT-PCR withbacteria-specific primers. Ribosome isolation and sub-sequent RT-PCR were reproducible as demonstrated byTGGE. Partial sequences of the 16s rRNA were revers-ibly transcribed into cDNA, amplified by PCR and theproducts separated by high-resolution TGGE. TheTGGE fingerprint (Fig. 2 ) reflects the diversity ofdominant rRNA sequences of pooled soils from differentsampling sites. Fingerprints from the same testfield werehighly reproducible. Pooled samples from the sametestfield, as presented in this study, appeared to beidentical (Fig.2 ) .This indicates a high spatial constancyof the dominant bacteria in these grassland soils.To determine whether one of the major bands could beaffiliated to 16s rDNA of clone DA079, its plasmid insertwas amplified with TGGE primers U968/GC and L1401.The migration distance of the fragment obtained from

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Fig. 3.Comparison between a silver-stainedTGGE gel (left-hand side) and the secondhalf of the same gel after an electrophoreticSouthern blot and hybridization with theV6-DA079 probe (right-hand side). The DNAsamples were loaded onto correspondingpositions. RT-PCR products from ribosomesof the native bacterial community representpooled samples from Drentse A sites A, B, C,F K and 0. Next to this, dilution series ofthe DA079 PCR product (2, 1, 0.5 and 0.25pl)were applied. On top of the gel lanes,single-stranded DNA appears (position andsize marked 'SS'). At this position the V6-DA079 probe gives hybridization signals aswell. The 'smile correction' bar indicates animage correction carried out to compensatefor distortions of the blot signals.

the DA079 clone was identical to one of the strongestbands obtained from amplified cDNA. Here the defini-tion of total activity allows two scenarios for abundanceand state of the bacterial cells: this species counts forhigh total activity within the community by only lowactivity per cell, but extraordinarily high cell number ; rthis species could show an extraordinarily high activityper cell, but only low cell number within the community.The TGGE fingerprints obtained from amplified geno-mic rDNA from soil (Fig. 2) point more to the firstscenario. They indicate that sequence DA079 is not onlyone of the most abundant 16s rRNA but also 16s rDNAsequences. The relative abundance of DA079 in therDNA fingerprints appears comparable to the 16s rRNAin the ribosomal RT-PCR fingerprints. Thus it seemslikely that the prominent ribosome number from thisspecies is caused more by the high number of active cellsrather than by extraordinarily high activity per cell. Thehigh reproducibility of the TGGE fingerprints furtherindicates a homogeneous distribution of this activity.Confirmation of sequence DA079 by V6 probingAs even the high resolution of TGGE does not absolutelyexclude the possibility that two different 16s cDNAsequences might migrate to exactly the same position inthe gel, the authenticity of the two corresponding bandsneeded to be verified. A TGGE gel was symmetricallyloaded, each half containing cDNA samples preparedfrom ribosomes of different sampling sites and dilutionsof the PCR products obtained from 16s rDNA of cloneDA079. One-half of the gel was stained with silver,while the material of the second half was blotted onto amembrane and hybridized with a DA079-specific, radio-actively labelled probe (Fig. 3 ) . Comparison of thesignals of both approaches allows the conclusion that97f % of the silver-stained fingerprint band wasindeed composed of the DA079 16s rDNA fragment(Table 1 ).

The 95 nt probe used in Southern hybridization wasderived from the 16s rDNA insert of clone DA079.Comparison of the probe sequence with the homologousregion of clone sequences from highly related actino-bacteria detected in a peat bog (Rheims et al., 1996b)gave highest similarity values above 98% (data notshown). The question is raised whether the hybridi-zation results indicate the presence of sequence DA079in the ribosome fraction and rDNA clone library to theexclusion of other highly related actinobacterial se-quences, as found in the peat bog clone library. Mostlikely, such sequences would migrate to different posi-tions in the TGGE gel. It must be assumed that sequenceDA079 is the only prominent member of this group inthe ribosome fraction. Other highly related actinobac-terial sequences might also be present in the minoritypopulation, but they are not detectable in the TGGEfingerprints.ConclusionsThis study suggests that the cloned sequence DA079originated from one of the most active bacterial speciesin Drentse A grassland soils. The strength of the DA079band in the TGGE fingerprints and the hybridizationsignal demonstrated a prominent abundance of thissequence within the ribosomal 16s rRNA and genomic16s rDNA fractions from soil. Hence, it can beconcluded that the as-yet-uncultured actinobacteria area potentially important part of the native bacterialcommunity in Drentse A grassland soil. This leads to thespeculation that their role is similar in the otherenvironments where their presence had been describedpreviously. The 1 6 s rDNA clone libraries generated inthe past provided first indications of the enormousdegree of prokaryotic diversity by revealing hithertounknown sequences of unidentified organisms. Afteranalysis of the cloned sequences we can now turn backto the environment to reveal the ecologically relevant

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Table 1. Pixel volume quantification ratios for silver staining vs hybridization signals,respectively, soil fingerprint vs DA079

Lane Image pixel volume for: Ratio A/B DA079 signal/soil( = C ) fingerprint ratioSilver staining (A ) Hybridization (B) (1*96/C)

A 114.12B 55.54C 116.92F 86-92K 93.150 11432Mean of all soil samples (SD)2 d 135.441 d 99.050.5 p1 44-240.25 pl 20.35Mean of all DA079 samples (SD)

57.7422.59596540.655 -2554.6777.4550.832404

9-00

1.982 461.962.141-702 0 92 0 6 ( 1 1 )1-751.971-842 2 61.96 (10)

0 9 90 8 01.000 9 21.150 9 40.97 ( 5 )-----

organisms. Application of ribosome isolation, subse-quent RT-PCR and separation of amplicons by TGGE,in combination with taxon-specific probing , leads to theidentification of the metabolically dom inant portio n ofthe community. This allows us to more specificallydiscuss the composition of environmental microbialcommunities.ACKNOWLEDGEMENTSThis work was supported by a grant f rom the EuropeanCommunity project 'High Resolut ion Automated MicrobialIdentificat ion' (EC-HR AM I project BI02-CT94-3098). H.R.was supported by a grant from the Germ an research council(DFG-Sta184/13-1). We want to thank the Dutch StateForestry Commission, who al lowed us access to the naturereserve.REFERENCESBakken, L. R. & Olsen, R. A. (1987). Th e relationship between cellsize and v iability of soil bacteria. Mic rob Ecol 13, 103-114.Borneman, J., Skroch, P. W., O'Sullivan, K. M., Palus, J. A.,Rumjanek, N. G., Jansen, J. L., Nienhuis, J. &Triplea E. W. (1996).Mo lecular microb ial diversity of an agricultura l soil in Wisconsin.Appl E nviron Mic robio l62, 1935-1943.Brosius, J., Palmer, M. L., Kennedy, P. J. & Noller, H. F. (1978).Com plete nucleotide sequence of a 16s ribosomal RN A gene fromEscherichia coli. Proc Natl Acad Sci U SA 75, 480 1-4 805 .Clark, D. A. & Norris, P. R. (1996). Acidimicrobium ferrooxidansgen. nov., sp. nov. : mixed-culture ferrous iron oxidation withSulfobacillus species. Microbiology 142,785-790.Engelen, B., Heuer, H., Felske, A., NUbel, U., Smalla, K. &Backhaus, H. (1995). Protocols for the TGG E. In Abstracts for theWorkshop on Application of DGGE and TGGE in MicrobialEcology 1995. BBA for Agriculture And Forestry, Braunschweig.Felsenstein, J. (1993). PHYLIP (Phylogeny Inference Package)version 3.5.1. Seattle : Department of Genetics, University ofWashington.Felske, A., Engelen, B., NUbel, U. & Backhaus, H. (1996). Direct

ribosome isolation from soil to extract bacterial rRNA forcommunity analysis. Appl Environ Microbiol62, 41624167.Fuhrman, J. A,, McCallum, K. & Davis, A. A. (1993). Phylogeneticdiversity of subsurface marine microbial communities from theAtlantic a nd Pacific oceans. Appl Environ Microbiol 59, 1294-1302.Hartung, K. (1996). Entwicklung von Sonden zur lnterpretationvon TGGE-Gelen fur die Analyse mikrobieller Gemeinschafien.Diploma thesis, Fachhochschule Lippe.Heuer, H. & Smalla, K. (1997). Application of denaturing gradientgel electrophoresis (D GG E) 2nd temperature gradient gel electro-phoresis (T GG E) for studying soil microbial communities. InModern Soil Microbiology, pp . 353-373. Edited by J. D. vanElsas, E. M. H. Wellington & J. T. Trevors. New York: M arcelDekker.Josephson, K. L., Gerba, C. P. & Pepper, T. L. (1993). Polymerasechain reaction of nonviable bacterial pathogens. Appl EnvironMicrobiol59, 3513-3515.Jukes, T. H.& Cantor, C. R. (1969). Evolution of protein molecules.In Mamm alian Protein Metabolism , pp . 21-132. Edited by H. N.Munro. New York: Academic Press.Liesack, W. & Stackebrandt, E. (1992a). Occurrence of novelgroups of the do main Bacteria as revealed by analysis of geneticmaterial isolated from an Australian terrestrial environment.J Bacteriol 174,5072-5078.Liesack, W. & Stackebrandt, E. (1992b). Unculturable microbesdetected by molecular sequences and probes. Biodivers Conserv 1 ,250-262.Lorenz, M. G. & Wackernagel, W. (1987). Adsorption of DNA tosand and variable degradation rates of adsorbed DNA. ApplEnviron Microbiol53,2948-2952.Maidak, B. L., Larsen, N., McCaughey, M. J., Overbeck, R., Olsen,G. J., Fogel, K., Blandy, J. & Woese, C. R. (1994). The ribosomaldatabase project. Nucleic Acids Res 22, 3483-3487.Muyzer, G., De Waal, E. C. & Uitterlinden, A. G. (1993). Profilingof complex microbial populations by denaturing gradient gelelectrophoresis analysis of polymerase chain reaction-amplifiedgenes coding for 16s rRNA. Appl Environ Microbiol59,695-700.NUbel, U., Engelen, B., Felske, A., Snaidr, J., Wieshuber, A.,Amann, R.I., Ludwig, W. & Backhaus, H. (1996). Sequence

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Activity of uncultured Actinobacteria in soilheterogeneities of genes encoding 16 s rRNAs in Paenibaciffuspofymyxa detected by temperature gradient gel electrophoresis.J Bacteriof 178,5636-5643.Rainey, F. A., Ward, N. L. & Stackebrandt, E. (1993). Molecularecology study of a New Zealand acidothermal soil. In Thermo-phifes '93, Hamilton, New Zealand, abstract A7. Hamilton :University of Waik ato.Rheims, H., Rainey, F. A. & Stackebrandt, E. (1996a). A molecularapproach to search for diversity among bacteria in the en-vironment. ] Znd Microbiof 17, 159-169.Rheims, H., Sprbr, C., Rainey, F. A. & Stackebrandt, E. (1996b).Molecular biological evidence for the occurrence of unculturedmembers of the actinomycete line of descent in different environ-ments and g eographical locations. Microbiology 142,2863-2870.Rosenbaum, V. & Riesner, D. (1987). Temperature-gradient gelelectrophoresis - hermodynamic analysis of nucleic acids andproteins in purified form an d in cellular extracts. Biophys ChemRoszak, D. B., Grimes, D. J. & Colwell, R. R. (1984). Viable butnonrecoverable stage of Salmonella enteritidis in aquatic systems.Ca n ]Microbiof 30,334-338.Saano, A., Lindstrdm, K. & Van Elsas, J. D. (1995). Eubacterialdiversity in Finnish forest soil. In 7th ZnternationafSymposium onMicrobial Ecology, Santos, Brazil, abstract P1-3.9. Santos :Brazilian Society for Microbiology.Sarnbrook, J., Fritsch, E. F. & Maniatis, T. (1989). MolecularCloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor,NY : Cold Spring Harb or Laboratory.

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Received 13 March 1997; revised 27 May 1997; accepted 3 June 1997.

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