16S–23S rRNA gene internal transcribed spacer sequences for analysis of the phylogenetic relationships among species of the genus Porphyromonas Georg Conrads, 1,2 Diane M. Citron, 1 Kerin L. Tyrrell, 1 Hans-Peter Horz 2 and Ellie J. C. Goldstein 1 Correspondence Georg Conrads gconrads@ukaachen.de 1 R. M. Alden Research Laboratory, Santa Monica, CA 90404, USA 2 Lehr- und Forschungsgebiet Orale Mikrobiologie und Immunologie der Klinik fu ¨r Zahnerhaltung, Parodontologie und Pra ¨ ventive Zahnerhaltung und Institut fu ¨ r Medizinische Mikrobiologie, Universita ¨ tsklinikum (RWTH), D-52057 Aachen, Germany The 16S–23S rRNA gene internal transcribed spacer (ITS) regions of 11 reference strains of Porphyromonas species, together with Bacteroides distasonis and Tannerella forsythensis, were analysed to examine interspecies relationships. Compared with the phylogenetic tree generated using 16S rRNA gene sequences, the resolution of the ITS sequence-based tree was higher, but species positioning and clustering were similar with both approaches. The recent separation of Porphyromonas gulae and Porphyromonas gingivalis into distinct species was confirmed by the ITS data. In addition, analysis of the ITS sequences of 24 clinical isolates of Porphyromonas asaccharolytica plus the type strain ATCC 25260 T divided the sequences into two clusters, of which one was a-fucosidase-positive (like the type strain) while the other was a-fucosidase-negative. The latter resembled the previously studied unusual extra-oral isolates of ‘Porphyromonas endodontalis-like organisms’ (PELOs) which could therefore be called ‘Porphyromonas asaccharolytica-like organisms’ (PALOs), based on the genetic identification. Moreover, the proposal of a-fucosidase-negative P. asaccharolytica strains as a new species should also be considered. INTRODUCTION The genus Porphyromonas currently includes 13 recog- nized species of asaccharolytic, obligately anaerobic, non- spore-forming, Gram-negative, non-motile, pleomorphic bacilli. Of human origin are three catalase-negative species: Porphyromonas asaccharolytica, Porphyromonas endo- dontalis and Porphyromonas gingivalis. Most of the known species are, however, of animal origin, including the catalase- positive Porphyromonas canoris, Porphyromonas cangingivalis, Porphyromonas cansulci, Porphyromonas circumdentaria, Porphyromonas gingivicanis and Porphyromonas macacae (which includes Porphyromonas salivosa strains), and the catalase-negative Porphyromonas levii and Porphyromonas crevioricanis (Jousimies-Somer & Summanen, 2002). It has also been shown that Oribaculum catoniae, although saccharolytic, is phylogenetically a member of the genus Porphyromonas; thus, it has been reclassified as Porphyro- monas catoniae (Willems & Collins, 1995). Finally, animal strains of P. gingivalis that were catalase-positive were reclassified as Porphyromonas gulae; however, the difference between Porphyromonas gingivalis and Porphyromonas gulae appeared questionable by analysis of 16S rRNA gene sequences (Fournier et al., 2001). There are two additional candidates for new species classi- fication, both of which are from humans: PLLOs (‘Porphyro- monas levii-like organisms’; Jousimies-Somer, 1995, 1997; Jousimies-Somer et al., 1995) and PELOs (‘Porphyromonas endodontalis-like organisms’, isolated from extra-oral sites, whereas Porphyromonas endodontalis is almost exclusively isolated from endodontic infections; Jousimies-Somer, 1997; Jousimies-Somer & Summanen, 2002; Vaisanen Abbreviations: ITS, internal transcribed spacer; PELO, ‘Porphyromonas endodontalis-like organism’. Published online ahead of print on 24 September 2004 as DOI 10.1099/ijs.0.63234-0. The GenBank/EMBL/DDBJ accession numbers for the ITS and 16S rRNA gene sequences discussed in this study can be found in Figs 1 and 2. A similarity matrix table (.xls), an alignment file (.aln) and a multi sequence file (.msf) of DNA–DNA hybridization data for all sequences discussed in this article are available as supplementary material in IJSEM Online. A figure showing representative gel-electrophoretic patterns of Porphyromonas species is also available. 63234 G 2005 IUMS Printed in Great Britain 607 International Journal of Systematic and Evolutionary Microbiology (2005), 55, 607–613 DOI 10.1099/ijs.0.63234-0