phylogenetic analysis of anaerobic thermophilic bacteria:

8
JOURNAL OF BACTERIOLOGY, Aug. 1993, p. 4772-4779 Vol. 175, No. 15 0021-9193/93/154772-08$02.00/0 Copyright © 1993, American Society for Microbiology Phylogenetic Analysis of Anaerobic Thermophilic Bacteria: Aid for Their Reclassification FREDERICK A. RAINEY,lt* NAOMI L. WARD,1t HUGH W. MORGAN,2 ROSEMARY TOALSTER,' AND ERKO STACKEBRANDT1t Department of Microbiology, Centre for Bacterial Diversity and Identification, The University of Queensland, St. Lucia, Queensland 4072, Australia, 1 and Department of Biological Science, The University of Waikato, Hamilton, New Zealand2 Received 14 December 1992/Accepted 24 May 1993 Small subunit rDNA sequences were determined for 20 species of the genera Acetogenium, Clostridium, Thermoanaerobacter, Thermoanaerobacteinum, Theymoanaerobium, and Thermobacteroides, 3 non-validly described species, and 5 isolates of anaerobic thermophilic bacteria, providing a basis for a phylogenetic analysis of these organisms. Several species contain a version of the molecule significantly longer than that of Escherichia coil because of the presence of inserts. On the basis of normal evolutionary distances, the phylogenetic tree indicates that all bacteria investigated in this study with a maximum growth temperature above 650C form a supercluster within the subphylum of gram-positive bacteria that also contains Closridium thermosaccharolyticum and Clostridium thermoaceticum, which have been previously sequenced. This super- cluster appears to be equivalent in its phylogenetic depth to the supercluster of mesophilic clostridia and their nonspore-forming relatives. Several phylogenetically and phenotypically coherent clusters that are defined by sets of signature nucleotides emerge within the supercluster of thermophiles. Clostrdium thermobutyricum and Clostridium thernopainarium are members of Clostridium group I. A phylogenetic tree derived from transversion distances demonstrated the artificial clustering of some organisms with high rDNA G+C moles percent, i.e., Clotridium fervidus and the thermophilic, cellulolytic members of the genus Clostriium. The results of this study can be used as an aid for future taxonomic restructuring of anaerobic sporogenous and asporogenous thermophilic, gram-positive bacteria. The potential biotechnological use of thermophilic bacte- ria and their thermostable enzymes has led to extensive isolation studies in a wide variety of thermophilic environ- ments (18). Many of these isolates have been characterized and described as new genera or new species of existing genera. Examples for pheno- and genotypically well-defined taxa are the aerobic members of the genera Thermus (9) and Thermomicrobium (26), the microaerophilic member of the genus Aquifex (5), and the anaerobic members of the genera assigned to the order Thermotogales (11). However, the description of the majority of anaerobic, thermophilic isolates, with a temperature optimum for growth of 55 to 70°C, was based almost solely on phenotypic data, the determination of DNA base composition being the only genomic taxonomic measure. Gram-positive staining reaction and spore formation have been used as key criteria to allocate the isolates as new species of the genus Clostrid- ium, i.e., Clostridium fervidus, Clostridium cellulosi, and Clostridium thermosaccharolyticum. Those isolates for which spore formation had not been demonstrated at the time of characterization were described as members of new genera, i.e., Thermoanaerobacter, Thermobacteroides, and Thermoanaerobium. Apparent taxonomically important physiological traits such as homoacetogenesis, mixed acid fermentation, and polysaccharide degradation were not ex- clusive for certain taxa but were widespread features. Sev- eral species investigated in this study have recently been * Corresponding author. t Present address: German Collection of Microorganisms and Cell Cultures, Mascheroder Weg 1B, Braunschweig D-3300, Germany. reclassified (20), and the new nomenclature is used in the following text. Application of 16S rRNA sequence analysis to the anaer- obic thermophilic species provided surprising results. Firstly, thermophilic nonsporeformers such asAcetogenium kivui and Thennoanaerobacter brockii (since shown to form spores by Cook et al. [7]) were more closely related to some thermophilic members of the genus Clostridium than meso- philic and thermophilic clostridial species were related to each other (6, 39). Secondly, members of different genera, such as Thermobacteroides acetoethylicus and Thermo- anaerobacterbrockii were phylogenetically almost identical, as judged by 16S rRNA similarity values of more than 99.3% (2). A recent compilation of non-sulfate-reducing, thermo- philic, obligately anaerobic bacteria lists more than 25 spe- cies of eight genera (44). In this communication, we present phylogenetic data on available strains and additional isolates in order to determine the basis for future taxonomic rear- rangements by using a polyphasic approach. MATERIALS AND METHODS Bacterial strains. Bacterial strains were obtained from the respective culture collections as indicated in Table 1. Strains 0k9. B1 and RI2. B1 were isolated from thermal spring samples (Table 1) with CBM medium (31) containing amor- phous cellulose and incubated at 70°C. From cellulolytic enrichments, pure cultures were obtained by using the roll tube method of Hungate (14). To obtain cell mass for DNA isolation, all strains were cultivated in 2/1 cellobiose medium at the temperature optima determined in the original descrip- tion (Table 1). The growth medium 2/1 cellobiose contained 4772

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Page 1: Phylogenetic Analysis of Anaerobic Thermophilic Bacteria:

JOURNAL OF BACTERIOLOGY, Aug. 1993, p. 4772-4779 Vol. 175, No. 150021-9193/93/154772-08$02.00/0Copyright © 1993, American Society for Microbiology

Phylogenetic Analysis of Anaerobic Thermophilic Bacteria:Aid for Their Reclassification

FREDERICK A. RAINEY,lt* NAOMI L. WARD,1t HUGH W. MORGAN,2 ROSEMARY TOALSTER,'AND ERKO STACKEBRANDT1t

Department ofMicrobiology, Centre for Bacterial Diversity and Identification, The University ofQueensland, St. Lucia, Queensland 4072, Australia, 1 and Department ofBiological Science,

The University of Waikato, Hamilton, New Zealand2

Received 14 December 1992/Accepted 24 May 1993

Small subunit rDNA sequences were determined for 20 species of the genera Acetogenium, Clostridium,Thermoanaerobacter, Thermoanaerobacteinum, Theymoanaerobium, and Thermobacteroides, 3 non-validlydescribed species, and 5 isolates of anaerobic thermophilic bacteria, providing a basis for a phylogeneticanalysis of these organisms. Several species contain a version of the molecule significantly longer than that ofEscherichia coil because of the presence of inserts. On the basis of normal evolutionary distances, thephylogenetic tree indicates that all bacteria investigated in this study with a maximum growth temperatureabove 650C form a supercluster within the subphylum of gram-positive bacteria that also contains Closridiumthermosaccharolyticum and Clostridium thermoaceticum, which have been previously sequenced. This super-cluster appears to be equivalent in its phylogenetic depth to the supercluster of mesophilic clostridia and theirnonspore-forming relatives. Several phylogenetically and phenotypically coherent clusters that are defined bysets of signature nucleotides emerge within the supercluster of thermophiles. Clostrdium thermobutyricum andClostridium thernopainarium are members of Clostridium group I. A phylogenetic tree derived fromtransversion distances demonstrated the artificial clustering of some organisms with high rDNA G+C molespercent, i.e., Clotridium fervidus and the thermophilic, cellulolytic members of the genus Clostriium. Theresults of this study can be used as an aid for future taxonomic restructuring of anaerobic sporogenous andasporogenous thermophilic, gram-positive bacteria.

The potential biotechnological use of thermophilic bacte-ria and their thermostable enzymes has led to extensiveisolation studies in a wide variety of thermophilic environ-ments (18). Many of these isolates have been characterizedand described as new genera or new species of existinggenera. Examples for pheno- and genotypically well-definedtaxa are the aerobic members of the genera Thermus (9) andThermomicrobium (26), the microaerophilic member of thegenus Aquifex (5), and the anaerobic members of the generaassigned to the order Thermotogales (11).However, the description of the majority of anaerobic,

thermophilic isolates, with a temperature optimum forgrowth of 55 to 70°C, was based almost solely on phenotypicdata, the determination of DNA base composition being theonly genomic taxonomic measure. Gram-positive stainingreaction and spore formation have been used as key criteriato allocate the isolates as new species of the genus Clostrid-ium, i.e., Clostridium fervidus, Clostridium cellulosi, andClostridium thermosaccharolyticum. Those isolates forwhich spore formation had not been demonstrated at thetime of characterization were described as members of newgenera, i.e., Thermoanaerobacter, Thermobacteroides, andThermoanaerobium. Apparent taxonomically importantphysiological traits such as homoacetogenesis, mixed acidfermentation, and polysaccharide degradation were not ex-clusive for certain taxa but were widespread features. Sev-eral species investigated in this study have recently been

* Corresponding author.t Present address: German Collection of Microorganisms and Cell

Cultures, Mascheroder Weg 1B, Braunschweig D-3300, Germany.

reclassified (20), and the new nomenclature is used in thefollowing text.

Application of 16S rRNA sequence analysis to the anaer-obic thermophilic species provided surprising results.Firstly, thermophilic nonsporeformers such asAcetogeniumkivui and Thennoanaerobacter brockii (since shown to formspores by Cook et al. [7]) were more closely related to somethermophilic members of the genus Clostridium than meso-philic and thermophilic clostridial species were related toeach other (6, 39). Secondly, members of different genera,such as Thermobacteroides acetoethylicus and Thermo-anaerobacterbrockii were phylogenetically almost identical,as judged by 16S rRNA similarity values of more than 99.3%(2).A recent compilation of non-sulfate-reducing, thermo-

philic, obligately anaerobic bacteria lists more than 25 spe-cies of eight genera (44). In this communication, we presentphylogenetic data on available strains and additional isolatesin order to determine the basis for future taxonomic rear-rangements by using a polyphasic approach.

MATERIALS AND METHODS

Bacterial strains. Bacterial strains were obtained from therespective culture collections as indicated in Table 1. Strains0k9. B1 and RI2. B1 were isolated from thermal springsamples (Table 1) with CBM medium (31) containing amor-phous cellulose and incubated at 70°C. From cellulolyticenrichments, pure cultures were obtained by using the rolltube method of Hungate (14). To obtain cell mass for DNAisolation, all strains were cultivated in 2/1 cellobiose mediumat the temperature optima determined in the original descrip-tion (Table 1). The growth medium 2/1 cellobiose contained

4772

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PHYLOGENY OF ANAEROBIC THERMOPHILIC BACTERIA 4773

TABLE 1. Source, characteristics, and accession numbers of the strains described in this study

Temp ('C) Spres Cellulose/ G+C (mol%) GenBanklBacterial strain Source' optimum/ detected xylan Reference EMBL

maximum utilization DNA rDNA no.

Acetogenium kivuiT DSM 2030 67/75 - -/- 38 57.3 21 L09160Thermoanaerobacter thermohydrosulfuricusT DSM 567b 69/78 + -/+ 35-37 56.7 20 L09161E100-69

Thermoanaerobacter ethanolicusT strain ATCC 31550 69/78 - -/+ 32-38 56.8 47 L09162JW-200

Thermobacteroides acetoethylicusT ATCC 33265b 65/75 - -/- 31 57.3 3 L09163Thermoanaerobacter ethanolicus 39E ATCC 33223b NDC + ND ND 59.2 20 L09164Thermoanaerobacter brockiiT DSM 1457 68/77 + -/+ 30-31.4 59.1 20, 52 L09165ThermoanaerobacterfinniiT DSM 3389 65/75 + -/ND 32 58.3 34 L09166Clostridium thermocopriae JT-3T IAM 13577 60/<75 + +/+ 37.2 59.6 15 L09167Clostridium thermoautotrophicumT DSM 1974 58/68 + -/- 53-55 57.5 45 L09168Clostridium thermoaceticumT 58/65 + -/- 54-56 56.0 44 RDPdClostridium thennosaccharolyticumT 55/67 + -/+ 29-32 53.2 44 RDPThermoanaerobacterium saccharolyticumT DSM 7060 60/70 - -/+ 36 54.2 20 L09169Thermoanaerobacterium thermosulfurigenes ATCC 3374b 60/75 + -/+ 36 54.0 20 L09171Thermoanaerobactenum xylanolyticumT DSM 7097 60/70 + -/+ 36 53.6 20 L09172Thermoanaerobium lactoethylicum ZE-1 Kondratieva 65/75 - -/- 34.6 53.7 19 L09170"Anaerocellum thermophilum" Z-1320 Svetlichny 72-75/83 - +/+ 36.7 58.7 38 L09180"Caldocellum saccharolyticum" Tp8T.6331 ATCC 43494 70/<80 - +/+ 36 58.7 35 L09178"Thernoanaerobacter cellulolyticus" NA10 IFO 14436 75/<80 - +/+ 37.7 59.1 10, 40 L09183Strain COMP.B1 Morganb ND - +/+ ND 58.1 12 L09179Strain 0k9.Ble Raineyb ND - +/+ ND 58.8 This study L09186Strain RI2.B1e Raineyb ND - +/+ 36 58.5 This study L09181Strain Rt8.B7 Morganb ND - +/+ 35.5 58.6 12 L09184Clostridium thermolacticum TC 21 DSM 2911b 65/<75 + +/+ 42.3 56.9 22 L09176Clostridium stercorariumT NCIMB 11754 65/>70 + +/+ 39 57.1 23 L09174Strain RtS1.B1 Morganb ND + +/+ ND 57.1 12 L09175Clostridium thernocellumT DSM 1237b 60/68 + +/+ 38-39 55.3 41 L09173Clostridium cellulosiT Jian-hua 55-60/65 + +/ND 35 55.4 50 L09177Clostridium fervidusT ATCC 43204" 68/<78 + -/+ 39 58.8 28 L09187Clostridium thennobutyricumT Wiegel 57/61.5 + -/- 37 53.3 46 X72868Clostridium thermopalmanumT DSM 5974 55/60 + -/ND 35.7 53.3 36 X72869

a Bacterial strains were obtained from the following sources: DSM, German Collection of Microorganisms and Cell Cultures; ATCC, American Type CultureCollection; NCIMB, National Collection of Industrial and Marine Bacteria, Aberdeen, United Kingdom; IAM, Institute of Fermentation, Tokyo, Japan; IFO,Industrial Fermentation Organization, Osaka, Japan. Strains were also obtained from the collections of V. Svetlichny (Institute of Microbiology, Moscow,Russia), M. Jain-hua (Tianjin Institute of Light Industry, Tianjin, China), J. Wiegel (University of Georgia, Athens), E. N. Kondratieva (Moscow StateUniversity, Moscow, Russia), H. W. Morgan (University of Waikato, Hamilton, New Zealand)."The strain sequenced is held at the collection of H. W. Morgan, University of Waikato, Hamilton, New Zealand.C ND, not determined.d RDP, sequence data obtained from the Ribosomal Database Project (24).' Abbreviation used for new isolates from New Zealand thermal sites: Ok, Orakei Korako, RI, Raoul Island.

(per liter) 0.9 g of NH4Cl, 0.9 g of NaCi, 0.2 g of MgCl2, 0.75g of KH2PO4, 1.5 g of K2HPO4, 0.75 g of cysteine-HCl, 2 gof Trypticase peptone, 1 g of yeast extract, 1 g of cellobiose,1 ml of resazurin (0.2% [wt/vol]), 1 ml of FeCl3 (0.15%[wt/vol]), and 1 ml (each) of trace element solution SL10 andselenite-tungstate solution (43). The medium with a final pHof 7.0 was dispensed as 10-ml amounts in Hungate tubesunder an atmosphere of N2-H2 (95%:5% [vol/vol]).

Sequencing methods. The extraction of genomic DNA,amplification of the gene encoding small subunit rRNA(named 16S rDNA in the following) and purification ofpolymerase chain reaction (PCR) products were performedas described previously (30, 32). The double-stranded PCRproducts were sequenced by the manual sequencing method(30) or the automated method (32). The primers used forsequencing were those published by Stackebrandt and Char-freitag (37). The sequences are available from GenBank orEMBL under the accession numbers shown in Table 1.Data analysis. The 16S rDNA sequences were aligned

manually against homologous sequences of prokaryotes con-tained in the ribosomal RNA Database Project (24) orobtained as recent data bank releases. Following a prelimi-

nary analysis to allocate the organisms to a phylum, a secondanalysis, in which the number of reference strains wasrestricted to the closest relatives of the organisms of thisstudy, was performed. Because of gaps in certain previouslysequenced molecules and regions of alignment uncertainty,the following regions of the 16S rDNA were omitted from theanalysis: 1 to 19, 69 to 97, 206 to 218, 452 to 480, 838 to 848,923 to 953, 1021 to 1044, 1135 to 1144, and 1440 to 1542(position numbering defined by the Escherichia coli se-quence). Pairwise evolutionary distances were computed byusing the correction of Jukes and Cantor (17). The least-squares distance method of De Soete (8) was used in theconstruction of phylogenetic dendrograms from distancematrices. Transversion distances were computed by usingthe modified correction of Jukes and Cantor (17) as indicatedby Weisburg et al. (42).

RESULTS AND DISCUSSION

The 16S rDNA of 28 thermophilic species was sequencedalmost completely (>98% of the E. coli 16S rRNA se-quence). 16S rDNA sequences of Acetogenium kivui, Ther-

VOL. 175, 1993

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4774 RAINEY ET AL.

ClusterA

Cluster B

Cluster C

&Theroanaembacler ethanolicui sitrinS39tThermoanaerobactebrockiiThermoaaerobacterfinniiThenoanaerobacterethanolicus strain JW-200Thermobacteroldes acetoethylicusThermoaerobacter thermohydrmsulurcus

Acetogenhum ktmiClostridltu thermocopriaerCmX~~~etfoum

uLClosfidam temdoatrophicumThmnoanaerobacterwnu ylanoycum

7Thermoanaerobacterium thermosuofrigenes7Thermoanaerobacterium saccharolyticwn

Thennoamaeroblum lactoethylicumClostridlum thermosaccharolyticum

stmainOk9.B1strainRt8. B7

MThermoanaerobacter celluloycusS strainNAI0r stain CO . B1

_ ~~~ClusterD { L CaldocellumsaccharolydcunsIrain RI2. B

TMnaerocellum thermophilum"Clostrdldumfervidus

Clostridium thermolacticumCluster E t E Clostrdldum stercorartum

inRMS. B1Clostrdum thermocellwn

Clostrlum cellulosiSym bomii

Sp.pauclvoransHeliobacterium chlorumBacillus subtills

Clostridium barkeClostrdidum acdiduricI

Clostridwn amlnov

m srain Tp8T.6331

strain Z-1320

ri

valericwn_ Josroawum sttcraawnu

Group I Clostridium tyrobutyrlcumClostridum butyrlcum

Clostridium thermobutyncumClostridium thermopalmarium

FIG. 1. Phylogenetic tree derived from normal evolutionary distances (see Methods and Materials). Scalesubstitutions per 100 nucleotides.

moanaerobacter thernohydmosulfiuicus, Thennoanaerobacterethanolicus, Thermoanaerobacterium thermosulfurigenes,Thermoanaerobacterium saccharolyticum, Thermoanaero-bacterium xylanolyticum, Thermoanaerobium lactoethyli-cum, and Thermobacteroides acetoethylicus contain inser-tions of various lengths in one or more regions aroundposition 80, 1040, 1140, or 1440. The only bacterium forwhich an insertion of comparable size in the region aroundposition 80 has been reported is Desulfotomaculum australi-cum, a gram-positive, spore-forming sulfate reducer (27).More than 1,100 unambiguously alignable nucleotides

were analyzed by two different methods for computingevolutionary distances. One method generates normal evo-lutionary distances, while the other makes corrections forthe high G+C content found in the rRNA genes of thermo-philic organisms. A 40-by-40 similarity matrix was computedand is available upon request from the corresponding author.The phylogenetic tree, based on normal evolutionary

distances (Fig. 1), shows that all species sequenced fell intothe radiation of clostridia and their non-spore-forming rela-

bar at bottom indicates 5

tives (6), Bacillus spp. and relatives (1), the phototrophicHeliobactenum chlorum (49), the mycoplasmas (42), and thegram-negative-staining members of the Sporomusa/Seleno-monas cluster (4, 33). While 2 species, Clostridium ther-mobutyricum and Clostridium thernopalmawum, are actu-ally members of group I clostridia (nomenclature of Johnsonand Francis [16]), the other 26 thermophilic organismsinvestigated are members of an apparently phylogeneticallycoherent supercluster that constitutes a major branch withinthis subphylum. As judged from the similarity values foundfor the most unrelated strains of the supercluster, its phylo-genetic depth (>84% sequence similarity) is as large as thatfound for representatives of several major groups of meso-philic clostridia (>84%). Twenty-six of the thermophilicorganisms fall into five major groups, as well as the singlelineages of Clostridium fervidus and Clostridium cellulosi(Fig. 1). The arabic letters A, B, C, D, and E were used toavoid nomenclatural conflicts with the Johnson and Francisgroups I to VI (16) and the groups I to III as defined by Leeet al. (20) for certain thermophilic clostridia and their non-

J. BACTERIOL.

Page 4: Phylogenetic Analysis of Anaerobic Thermophilic Bacteria:

PHYLOGENY OF ANAEROBIC THERMOPHILIC BACTERIA 4775

Them7noanaerobacter ethanolicus strain 39EThermoanaerobacter brockiiThermoanaerobacterfinniiThermoanaerobacter ethanolicus strain JW-200Thermobacteroldes acetoethylicus

ClusterA Thermoanaerobacter thermohydrosulfuricusAcetogenium kivuiClostridium thermocopriae

Cluster B L Clostridium thermoautotrophicumClostridium thermoaceticum

Thermoanaerobacterium saccharolyticumThermoanaerobacterium xylanolyticum

ClsterC ALLThermoanaerobacterium thermosulfurigenesThermoanaeroblum lactoethylicumClostridium thermosaccharolyticum

strain Ok9. B1strain Rt8. B7"Thermoanaerobacter cellulolyticus" strain NAl0

_ - "Caldocellum saccharolcum"strain Tp8T.6331Cluster D _- strain COMP. BI

strain RI2. B1"Anaerocellum thermophilum" strain Z-1320

Syn. bryantiiHellobacterium chlorum

Bacillus subtilisClostridium barkeri

Clostridium acidiuriciClostridium aminovalericum

Clostridium sticklandiiClostrdium cellulose

Clostridium thennolacticumCluster E | 1 Clostridium stercorarium

| ~~~stieRt5 1.B1Clostridium thermocellum

Clostridiumfervidus|Group I I Clostridium tyrobutyricumGroupI Clostridium butyricum

lostridium thernobutyricumClostridium thermopalmarium

FIG. 2. Phylogenetic tree derived from transversion distances (see Methods and Materials). Scale bar at bottom represents 5 substitutionsper 100 nucleotides.

spore-forming relatives. Although the phylogenetic coher-ency of the thermophiles is not affected, the branching pointof the supercluster within the radiation of the major lines ofthe subphylum depends to some extent upon the selection ofreference organisms. In any case, the length of the branchseparating the thermophiles from the mesophiles (Fig. 1) isextremely short, and the branching pattern has no statisticalsignificance, according to bootstrap analysis (data notshown).By using transversion distances for the generation of a

phylogenetic tree (Fig. 2), it is clear that the high G+C molespercent of the rDNA has introduced bias resulting in theclustering together of most of the thermophilic clostridia andrelatives (transversion distances not shown). While thepositions of clusters A to D are not greatly affected, clusterE, Clostridium fervidus, and Clostridium cellulosi show a

significant rearrangement. These organisms appear to beperipherally related to group I clostridia (16) that containboth mesophilic and thermophilic members. Other changesintroduced by the transversion analysis affect the relation-ship of Clostridium sticklandii and Clostridium aminovalen-cum, the branching of Heliobacterium chlorum, and in somecases the internal composition of clusters A, B, and D. Forthe following discussion, the results of the transversionanalysis are considered.

Irrespective of their classification into different genera, allclusters (embracing thermophilic clostridia and relatives) arephylogenetically well defined. In most examples, 16S rDNAsimilarities (in the regions compared) between the clustersare less than 90%. This value is similar to those foundbetween members of mainly mesophilic clostridia (Fig. 1 and2). Intracluster similarity values are generally high, ranging

I an Daucivrnrsn~

VOL. 175, 1993

Page 5: Phylogenetic Analysis of Anaerobic Thermophilic Bacteria:

4776 RAINEY ET AL.

TABLE 2. 16S rDNA sequence signatures defining the five main clusters of thermophilic anaerobes

Sequence signature'Positiona Cluster Clostridium Clostridium Other

A B C D E cellulosi fervidusG A G G GAU AU AU GC AUG G A G GUA UA GU CG AUMK CG UA AU UACG UG UG GG GCCG CG CG GC CGCG CG CG GC UGGC GC GC GC AUA A A G AGU GU GU GG GUGC AG GC GU AAUA UA UA AU UAUA AU UA GC GCA C A G CGC GC GC AU GCG G G A GCG CG CG UA CGGC GC AU GC GCCG CG CG UR CGGY GU GC AU GCGC AU AU GY RUUA UA UA CG UAA A A U AAU AU AU GC YRGU AU AU AU AUGC GC GC CG AUGU GC GU GC GCGC GC GC GC AUG G A G GGC GC AU AU AUGC AU GC GC AUYA GC UM GC GCK A U U UCG UA CG CG CGGU GC GU GC GCUA UA GC CG GCCG UA GC UA URGC GC AG GC GCKW GC UA YG GUA A A G ACG CG CG GC CGG G A G GCG CG CG CG GCGC GC GC GC AUAG AG AG AG GAUA GU UA AU KMGC GC AU GC GYC C C U CUG UG UG CG UG

G

AUGGAACGCUAUGGCAGUUAUACGCGCG

UGGCUAGUAUUAAGCCGAUGCGCGUU

GCGCU

GO

AUAU

GCUAACGACGGCAGGCGUCUG

GAUG

AUAUGCGCCGGCAGUUGUACGCGCGUAGCGCGUGCUAAAUAUAUGCGCG

GCGUGCU

CGGCGCUAGCGCACGG

CGGCAGGUGCCGC

G

AURNNNNNNNNYRGCAGUNNUANNM

GCG

NNgcNNGU

NNNN

AauauRUGYww

G

NNNNNNU

NGGO

NNUR

GYNN

ANN

GCGRYAG

RY

GY

YG

a Position numbering defined by Escherichia coli sequence.b R, purine; Y, pyrimidine; K, G or U; M, C or A; W, A or U; N, A, C, G, or U.c Dominant composition in the majority of members of the Clostridium/Bacillus subphylum.

between 92.4 and 99.7%. The phylogenetic coherency ofmembers of clusters A to E and the significant phylogeneticdistance between them and single-line strains are supportedby the presence of signature nucleotides (Table 2), foundonly in members of the respective cluster.The following paragraphs outline the composition of indi-

vidual clusters and discuss these findings in the light ofphylogenetic and phenotypic properties, some of which are

compiled in Table 1.The cluster A thermophiles. Cluster A comprises five

species of the genus Thermoanaerobacter, as well as Ther-mobacteroides acetoethylicus,Acetogenium kivui, and Clos-tridium thennocopnae. Members of this cluster are pheno-typically incoherent with respect to spore formation,polysaccharolytic activity, and DNA base composition (31to 38 mol% G+C). On the other hand, they show highsimilarity in their rDNA base composition of 56.7 to 59.6mol% G+C, and they are defined by unique 16S rDNAsignature nucleotides (Table 2). The intracluster similarityvalues range from 95.2 to 99.5%. This confirms the results of

3153-35869139-224140-223145-178155-166316-337408-434414417-426441-493442-492444 490449450-483484578-763579-762599-639601-637602-636603-635641660-745668-738669-737673-717682-708730823-877834-852838-848863990-12151047-12101120-11531121-11521127-11451164-117211691243-129413041352-13701355-13671357-13651422-14781423-147714311514-1521

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PHYLOGENY OF ANAEROBIC THERMOPHILIC BACTERIA 4777

Bateson et al. (2), who found that partial 16S rRNA se-

quences of some members of this cluster also demonstrateda high degree of sequence similarity (98.3 to 99.6%), and ofLee et al. (20), who found that four members of this cluster(Thennoanaerobacter brockii, Thermoanaerobacter etha-nolicus, Thermoanaerobacter thermohydrosulfuricus, andThermoanaerobacter ethanolicus 39E) had DNA-DNA sim-ilarities greater than 57%. The data also support the view ofWiegel and Ljungdahl (48) that Thermoanaerobacterbrockii,Thermoanaerobacter ethanolicus JW-200, and Thermo-anaerobacter ethanolicus 39E were close taxonomic rela-tives, since they had similar substrate ranges and tempera-ture ranges for growth and displayed a biphasic growthcurve. Interestingly, the two strains of Thennoanaerobacterethanolicus are not identical; strain 39E appears to be more

closely related to Thermoanaerobacter brockii (Fig. 2),which is in accord with the findings of Bateson et al. (2) andthe fact that strain 39E (51), Thermoanaerobacter brockii(7), and Thennoanaerobacter finnii (34) form spores,whereas Thermoanaerobacter ethanolicus JW-200 (47) andThernobacteroides acetoethylicus (3) do not.Acetogenium kivui is a homoacetogen, but it does not

group with the other thermophilic homoacetogenic organ-isms, i.e., Clostridium thermoaceticum and Clostridiumthermoautotrophicum, which define cluster B. The lack ofrelationship between Acetogenium kivui and these two ho-moacetogens was suggested previously (44) on the basis ofthe large differences in the G+C values of DNA (38 and 54mol%, respectively). Clostridium thermocopriae is the onlymember of cluster A capable of cellulose degradation (15), acharacteristic shared with members of clusters D and E.As reported previously (32), only one of the two available

Thennobacteroides species, Thernobacteroides acetoethyl-icus, is a member of cluster A. The second species, Ther-mobacteroides proteolyticus, is a representative of a novel,deep-rooting main line of descent of the domain Bacteriathat branches adjacent to members of the Thennotogales.The cluster B thermophiles. The two homoacetogenic,

spore-forming members of cluster B, Clostridium thennoau-totrophicum and Clostridium thennoaceticum, are equidis-tantly related to members of clusters A and C (88.4 to 90.7%sequence similarity). The two species are highly related(99.2%), which is in accord with the results of Bateson et al.(2) on partial 16S rRNA sequences (99.8%) and of Wiegel(44) on DNA hybridization (50% similarity), and they sharesome signature 16S rRNA nucleotides (Table 2). Their DNAbase composition (53 to 55 mol%) is significantly higher thanthat of other thermophilic and mesophilic clostridia, reflect-ing their distinct phylogenetic position.The cluster C thermophiles. Cluster C is also taxonomi-

cally heterogenous, comprising organisms of the generaClostridium, Thernoanaerobium, and Thermoanaerobacte-ium. Organisms of this cluster have a DNA base composi-

tion of 29 to 36 mol% G+C and a slightly higher rDNA basecomposition than mesophilic clostridia (53.2 to 54.2 and 50 to54 mol% G+C, respectively). The signature nucleotides areshown in Table 2. Intracluster similarity values range be-tween 98.0 and 99.7%.Only one of the organisms of cluster C was included in the

study of Bateson et al. (2), namely, Thermoanaerobacternumthenmosulfurigenes, and this species was found to groupseparately from the organisms of clusters A and B (describedabove). This finding is confirmed by this study, which extendsthis cluster to contain five species. Thermoanaerobacternumthennosulfuiigenes, Thenmoanaerobacteriwn saccharolyti-cwn, and Thennoanaerobacterium xylanolyticum were pre-

viously assigned to the same group, designated II, on thebasis of DNA hybridization studies (43 to 92% DNA simi-larity) (20); these authors indicated that the reduction ofthiosulfate to elemental sulfur may be a key characteristic ofmembers of this group, but this property has not beeninvestigated for the additional members as determined bythis phylogenetic study.

Since the type species of the genus Thernoanaerobium,Thermoanaerobium brockii, has been transferred to thegenus Thermoanaerobacter (20), the genus Thermoanaero-bium no longer exists and so Thermoanaerobium lactoeth-ylicum has no taxonomic status. This study indicates that itis phylogenetically closely related to the members of thegenus Thermoanaerobacterium. The assignment of Ther-moanaerobium lactoethylicum to the genus Thermoanaero-bium on the basis of its asporogenous nature may have beenwarranted at the time of description (19), but in light of theresults of this study it requires reclassification.The cluster D thermophiles. This cluster is composed of

seven extremely thermophilic, non-spore-forming, highlypolysaccharolytic organisms (13), none of which has been asyet validly described. Intracluster 16S rRNA similarity valuesrange between 96.3 and 99.7%, with values between 96.6 and97.8% separating two subclusters (Fig. 1 and 2). For thoseorganisms for which the base composition of DNA has beenreported, the G+C content is around 36 to 37 mol%. The basecomposition ofrDNA is in the range 58.1 to 59.1 mol%, whichis similar to the high values found for members of cluster A.The phylogenetic coherency of cluster D is reflected by alarge number of signature nucleotides (Table 2).

In addition to the four recent isolates (which are a selec-tion of 47 thermophilic cellulolytic isolates with high pheno-typic similarities [29]), this cluster contains three isolates forwhich genus names have been proposed but not validated.The close phylogenetic and phenotypic relatedness (Table 1)of all members of this group is a strong argument forassignment to a single genus.

Cluster E thermophiles. The phylogenetic position of thiscluster was significantly affected by the use of the transver-sion distances. Support for the relationship to the group I(16) (mainly mesophilic) clostridia is found in the phenotypicproperties such as spore formation and moderately thermo-philic growth temperature optimum. This cluster containsfour cellulolytic clostridia, of which three have been validlydescribed. Intracluster 16S rDNA similarity values rangebetween 92.4 and 99.0%, the lower values reflecting thedifferences that separate Clostridium thermocellum from theother three strains (Fig. 1 and 2). The lack of relatednessbetween Clostridium thernocellum and members of clustersA and C has previously been suggested by Lee et al. (20) onthe basis ofDNA hybridization experiments. Compared withthe cellulolytic members of cluster D, members of cluster Ehave lower growth temperature optima and, possibly as aconsequence thereof, a lower G+C base composition ofrRNA (55.3 to 57.1 mol% G+C). The G+C content of theirDNA ranges between 38 and 42.3 mol%. A number of 16SrDNA signature nucleotides support the phylogenetic dis-tinctness of this cluster (Table 2).Clostriwm cellulosi is remotely related to members of

cluster E (86.7 to 88.6% similarity), with which it shares theproperties of cellulose hydrolysis and spore formation. Theisolated phylogenetic position which is defined by a set ofsignature nucleotides (Table 2) confirms the phenotypic dis-tinctness of the species originally described by Yanling et al.(50). Compared with members of cluster E, Clostfidium cellu-

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4778 RAINEY ET AL.

losi has a lower temperature optimum and maximum, a lowerDNA G+C content, and a slightly lower rDNA content.

Clostridium fervidus. While the normal evolutionary dis-tance tree suggested the presence of a main subline ofdescent for this species, the transversion distance tree placesit with the group I clostridia (16). This relationship supportsearlier observations of Wiegel (44), who highlighted thesimilarities between Clostridium fervidus and Clostridiumthennobutyricum with respect to morphology, spore forma-tion, and DNA G+C content. The phylogenetic distinctnessof Clostridium fervidus can be supported by its fermentationend products, higher temperature range, and the presence of16S rRNA signature nucleotides (Table 2).

Clostridium thennobutyricum and Clostridium thernopalmar-ium. These two thermophilic, butyrate-producing spore-formers, with temperature optima of only 55 to 570C, arehighly related (98.3% similarity). They do not group withmembers of the individual lineages of thermophilic membersof clusters A to E but are members of the group I clostridia(16). This is in accord with the lower rDNA G+C value of53.3 mol%, which falls within the range of values of 50 to 54mol% determined for mesophilic clostridia. Their branchingpoint within the radiation of mesophilic clostridia, adjacentto other butyrate-producing organisms defining group I-E(Clostridium fallax) and group I-F (Clostridium sporogenes)(16), is deep (data not shown) and thus a confirmation oftheir genuine species status.

General considerations. Although thermophilic clostridial-type organisms have been considered evolutionarily themost ancient form of life (25), the data of this study excludethat the direct ancestor of these organisms originated in earlyevolutionary times. As members of the Clostridium/Bacillussubphylum, these thermophiles evolved significantly laterthan did the deep-branching, asporogenous thermophilictaxa such as Aquifex, the thermotogales, or thermi. Thegram-positive clostridial-type organisms do not constitute aphylogenetically coherent group that clearly originated froma common thermophilic ancestor. Although this possibilitycannot be excluded, because of lack of knowledge of thetotal microbial diversity and our restriction to the informa-tion available on cultured isolates, at present the data pointtowards an independent adaptation of mesophilic organismsto thermophilic environments.The extremely thermophilic organisms of clusters A, B,

and C that phenotypically match the description of Clostrid-ium spp. are not in the same phylogenetic confines as themesophilic type species, and therefore the placement ofthese organisms in the same genus seems unwarranted.Phylogenetic studies over the last 15 years have demon-strated the inadequacy of spore formation as a criterion forthe determination of taxonomic status in the ClostndiumlBacillus subphylum of gram-positive bacteria. This problemhas been previously discussed (6, 44), and the present studyhighlights the need to reconsider the current idea thatsporulating and nonsporulating organisms have to be placedin separate taxa. Lack of spore formation may only be theresult of the inability of the taxonomist to initially provideconditions to induce sporulation, as was found in the case ofThermoanaerobacter brockii (7). Even if the inability to formspores has a genetic basis, the use of the characteristicssporogeny and asporogeny could be restricted to the specieslevel. This study also recognizes the problems associatedwith the allocation of gram-positive, anaerobic, non-sulfate-reducing bacteria to the appropriate genus without priorinvestigation of their phylogenetic positions. The first exam-ple of a new concept has been provided by Lee et al. (20),

who, on the basis of DNA similarities and phenotypic data,transferred Clostridium thermohydrosulfuricum strains to thegenus Thermoanaerobacter (cluster A). Similarly, Clostrid-ium thermosulfurogenes was transferred to Thermo-anaerobactenum (cluster C). Both genera are now composedof sporeforming and non-spore-forming organisms, which forthe designation of taxa is more convincing from a biologicallypolyphasic point of view than from the traditional sole em-phasis of spore formation. It is likely that, in the light of recentresults, the phylogenetically coherent clustersA to E, definedin this study, will be reclassified as new genera and thesubclusters will be defined as new species.

ACKNOWLEDGMENTS

We thank J. G. Zeikus for permission to use information prior topublication.

This work was supported in part by a grant from the AustralianResearch Council to E.S.

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