INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, OCt. 1997, p. 1134-1139 0020-7713/97/$04.00 + 0 Copyright 0 1997, International Union of Microbiological Societies Vol. 47, No. 4 Phylogenetic Analysis of the Genus Desulfotomaculum: Evidence for the Misclassification of Desulfotomaculum guttoideum and Description of Desulfotomaculum orientis as Desulfosporosinus orientis gen. nov., comb. nov. ERKO STACKEBRANDT," CATHRIN SPROER, FREDERICK A. RAINEY, JUTTA BURGHARDT, ORSOLA PAUKER, AND HANS HIPPE DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, 38124 Braunschweig, Germany Almost complete 16s ribosomal DNA (rDNA) sequences were determined for the type strains of nine species belonging to the genus Desulfotomaculum and for seven strains described as strains of this genus. The sequences were compared with previously published 16s rDNA and rRNA sequences of the type strains of the other species of the geous. The majority of the species form a phylogenetically coherent cluster within the Clostridium-Bacillus subphylum of gram-positive bacteria. The cluster consists of phylogenetically well-sepa- rated lineages containing (i) Desulfotomaculumnigrificans,Desulfotomaculumaeronauticum, and Desulfotomacu- lum ruminis, (ii) Desulfotomaculum geothermicum, Desulfotomaculum thermosapovorans, and Desulfotomaculum sapomandens, (iii) Desulfotomaculum kuznetsovii, Desulfotomaculum australicum, and Desulfotomaculum thermo- cisternurn, (iv) Desulfotomaculum thermobenzoicum and Desulfotomaculum thermoacetoxiduns, and (v) Desulfo- tomaculum acetoxidans. Some as-yet-undescribed Desulfotomaculum strains are phylogenetically well-separated from strains of the described species. Desulfotomaculumguttoideum shares extremely high 16s rDNA similarity with certain Clostridium species (e.g., Clostridium sphenoides and Clostridiurn celerecrescens) and is most likely a misidentified species. Desugotomaculum orientis represents a new genus which branches most closely to the genus Desugtobacterium. The name Desulfosporosinus orientis gen. nov., comb. nov., is proposed for this taxon. The genus Desulfotomaculum comprises 15 validly described species, more than half of which have been described during the past 7 years. The majority of the more recently described species originate from thermal environmental sites which are presently among the prime habitats in the search for novel prokaryotic biodiversity (38). Although several Desulfotomaculum species show gram-neg- ative staining behavior, their cell walls possess the typical ul- trastructure of gram-positive bacteria (29, 36). Species are defined by sulfate reduction and the presence of spores, but the shape of spores (spherical to oval) and the location of spores (central, subterminal, or terminal) vary. Desulfotomacu- lum species differ from each other in physiology; e.g., some species are autotrophic, while others grow by fermentation of glucose and other organic substrates. A few species perform homoacetogenesis by converting substrates, such as H, plus CO, and a few others, to acetate (24, 44). Several studies involving comparative sequence analysis of 16s rRNA and 16s ribosomal DNA (rDNA) have shown that Desulfotomaculum species are related to members of the Clos- tridium-Bacillus subphylum (4, 9, 10, 16, 38). However, fine details concerning the phylogenetic coherence of this genus and the relatedness of its species have not yet been fully ex- plored. Most previous studies included only a few species, and even the most recently published phylogenetic trees did not include more than eight or nine species (14, 18, 31). These studies showed that the majority of species cluster together to form a major subline of descent among the several clusters encompassing clostridia and their non-spore-forming and/or coccoid relatives (4). Consistently, the main Desulfotomaculum * Corresponding author. Mailing address: DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg lB, 38124 Braunschweig, Germany. Phone: 49 531 2616 352. Fax: 49 531 2616 418. E-mail: erko@gbf-braunschweig.de. species cluster adjacent to members of the genera Moorella, Thermoanaerobacterium, and Thermoanaerobacter. In contrast, Desulfotomaculum orientis branched outside the main Desulfo- tomaculum cluster, showing moderate relatedness to members of the genus DesulJitobacterium (31,42). However, the branch- ing order of higher taxa within the Clostridium-Bacillus sub- phylum has not yet been unambiguously determined, because certain factors affect the branching pattern of lineages; these factors include the selection and number of reference organ- isms, the length of the sequences included in the analysis and the regions of the molecule compared, differences in the DNA base compositions of rDNA from mesophilic and thermophilic species, and the quality of the sequences in the database, which is related to the various sequencing approaches used (32). The classification of the genus Clostridium and its relatives (e.g., the genera Peptococcus, Eubacterium, Ruminococcus, and Bacillus) is presently undergoing a dramatic revision which is based on the results of comparative 16s rDNA sequence analyses. In this paper we describe the phylogenetic relatedness of the type strains of all available species of the genus Desulfotomaculum and some undescribed strains of this genus; our data led to the conclusion that, with two exceptions, the results of phyloge- netic analysis match the phenotype-based classification of De- sulfotomaculum species. MATERIALS AND METHODS Bacterial strains. The strains analyzed in this study, their growth media, and their growth temperatures (11, 12) are listed in Table 1. All strains were culti- vated anaerobically as described previously (11, 12). 16s rDNA sequence determination and analysis. Genomic DNAs were ex- tracted from the strains investigated in this study and were used for PCR- mediated amplification of 16s rDNA (34). The purified PCR products were cloned, the 16s rDNA inserts were reamplified and sequenced as described previously (33), and the sequence reaction mixtures were electrophoresed by using a model 373A automatic DNA sequencer (Applied Biosystems, Foster City, Calif.). In order to analyze the closest relatives of Desulfotomaculum strains, their 1134