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1 R. M. Alden Research Laboratory, Santa Monica, CA 90404, USA
2 Lehr- und Forschungsgebiet Orale Mikrobiologie und Immunologie der Klinik für Zahnerhaltung, Parodontologie und Präventive Zahnerhaltung und Institut für Medizinische Mikrobiologie, Universitätsklinikum (RWTH), D-52057 Aachen, Germany
Correspondence
Georg Conrads
gconrads{at}ukaachen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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-fucosidase-positive (like the type strain) while the other was -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 -fucosidase-negative P. asaccharolytica strains as a new species should also be considered.
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
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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.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). It has also been shown that Oribaculum catoniae, although saccharolytic, is phylogenetically a member of the genus Porphyromonas; thus, it has been reclassified as Porphyromonas 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 classification, both of which are from humans: PLLOs (‘Porphyromonas 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 et al., 1997). It should also be mentioned that there is a 16S rRNA gene sequence for ‘Porphyromonas canis’ strain JCM 10100 in GenBank/EMBL/DDBJ (accession no. AB034799), but this name has never been formally proposed or validated. Within the proposed order ‘Bacteroidales’, Tannerella forsythensis (a species related to Bacteroides distasonis and Bacteroides merdae) is grouped within the proposed family ‘Porphyromonadaceae’, so that all three might be close relatives of Porphyromonas species (Sakamoto et al., 2002).
Sequence polymorphisms and length variations found in the 16S–23S rRNA gene internal transcribed spacer (ITS) are increasingly being used as tools for the differentiation of bacterial species and subspecies (Guasp et al., 2000; Motoyama & Ogata, 2000; Conrads et al., 2002). This is because the higher number of variable sites typical for the ITS sequence (Soller et al., 2000) can overcome the apparent limitations of the resolution of 16S rRNA-based phylogenies in some genera where amplification and sequencing of this region is easy, almost free of ambiguities and reproducible, as has been described for Fusobacterium (Conrads et al., 2002).
The current study was performed to generate ITS data for most of the type strains of the genus Porphyromonas and for T. forsythensis, B. distasonis and Prevotella melaninogenica (outgroup), and to compare a phylogenetic tree deduced from these data with one deduced from corresponding 16S rRNA gene data. The ITS sequences were further used to clarify the phylogenetic relationship between Porphyromonas gingivalis and Porphyromonas gulae, as well as between atypical -fucosidase-negative isolates of Porphyromonas asaccharolytica (which are phenotypically indistinguishable from the PELO group; Vaisanen et al., 1997) and typical -fucosidase-positive Porphyromonas asaccharolytica.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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PCR amplification and DNA sequence analysis.
The 16S primer SPFPorph (5'-GTA CAC ACC GCC CGT CAA GCC-3', corresponding to Escherichia coli positions 1390–1411) and the 23S primer SPRPorph (5'-TCG CAG CTT ATC ACG TCC TTC-3', corresponding to E. coli positions 62–42) were designed based on the complete genome of P. gingivalis W83 (GenBank/EMBL/DDBJ accession no. NC_002950); however, the respective regions among bacterial small-and large-subunit sequences (RDP) are relatively conserved. PCR was carried out using an Uno I (Biometra) thermocycler in a volume of 100 µl containing 1x PCR buffer, 1·5 mM MgCl2, 2 units Taq polymerase, 0·2 mM each of dATP, dCTP, dGTP and dTTP (Roche Biochemicals), 10 pmol SPFPorph forward primer, 10 pmol SPRPorph reverse primer and 100 ng template DNA. Primer oligonucleotides were synthesized using a DNA synthesizer (OLIGO 1000; Beckman). The amplification was performed using the following temperature profile and 30 cycles: denaturation, 1 min at 94 °C; annealing, 1 min at 52 °C; elongation, 2·5 min at 72 °C. Amplification products (aliquots of 10 µl) were separated electrophoretically on a 2 % macro agarose gel in 1x TPE (80 mM Tris/phosphate, 2 mM EDTA, pH 7·5) for a minimum of 18 h at 30 V.
After purification using the Wizard DNA Clean-up system (Promega), the spacer DNA was directly sequenced in duplicate using a Big Dye-Deoxy terminator cycle sequencing kit (Applied Biosystems) and an automated capillary DNA sequencer (API Prism 310; Applied Biosystems). Sequences were assembled using the program Vector NTI Suite 9.0 (InforMax) and aligned using the program GeneDoc (Nicholas & Nicholas, 1997) A phylogenetic tree was constructed by using the neighbour-joining method and the programs CLUSTAL W (Chenna et al., 2003) and TreeView (Page, 1996), using Prevotella melaninogenica as an outgroup. The robustness of tree topologies and the statistical significance levels of interior nodes were determined by performing bootstrap analyses (1000 iterations) using the ARB software package (Ludwig et al., 2004).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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-fucosidase-negative strain) to 960 bp (Porphyromonas asaccharolytica ATCC 25260T, -fucosidase-positive strain) (Fig. Ab, available as supplementary material in IJSEM Online). In general, it was not possible to differentiate Porphyromonas species by comparing ITS gel-electrophoretic profiles alone (for length of amplicons see supplementary material). Further discrimination without need of sequencing might be possible by ITS restriction digest with endonucleases, since we found considerable variation in restriction sites (e.g. AvaI, ApaLI, ClaI, EcoRI, HindIII and SmaI).
Sequencing the purified ITS amplicons of the Porphyromonas strains using SPFPorph and SPRPorph as primers led to nearly ambiguity-free sequence determination by comparing both runs and directions. Transfer RNA genes (type Ile and Ala) were found in all ‘Porphyromonadaceae’ ITS sequences but not in Prevotella melaninogenica, using the program tRNAscan-SE, version 1.21 (Lowe & Eddy, 1997).
Phylogenetic tree reconstruction based on the ITS sequences (short version only in the case of Prevotella melaninogenica) is demonstrated in Fig. 1. As mentioned above, the different strains of Porphyromonas gingivalis matched on a 97–99 % level and the two Porphyromonas macacae ATCC strains (ATCC 49407T is the type strain of Porphyromonas salivosa, which has been reclassified) matched on a 94 % level; however, Porphyromonas asaccharolytica was more heterogeneous (80–99 % range in similarity level). Even more interesting, this latter species, which phenotypically differed in -fucosidase activity, showed two main clusters. Intercluster similarity was only 80–87 %, whereas intracluster similarity was 92–99 % and thus more comparable with the phylogenetic relatedness of Porphyromonas gingivalis strains.
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). The fourth group was formed by Porphyromonas macacae, Porphyromonas asaccharolytica and Porphyromonas cansulci, which showed a match between 66 and 68 %.
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-fucosidase-positive and 16 -fucosidase-negative strains of Porphyromonas asaccharolytica and clearly showed that both groups diverged into individual phylogenetic branches (Fig. 3a). While the -fucosidase-negative strains phenotypically resemble the previously studied extra-oral isolates of PELOs, it is obvious that they are not genetically related to Porphyromonas endodontalis (Fig. 3b).
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), especially when comparing species that are closely related. The phylogenetic differences are not only expressed in the sequence information itself but also by the different lengths of amplicons and, in some cases, the formation of distinct band patterns by gel electrophoresis resulting from variations among the rrn operons in the same strain (Christensen et al., 2000; Iteman et al., 2000). However, in some members of the Proteobacteria and in low-G+C Gram-positive genera such as Bacillus and Clostridium, six to ten rRNA operons exist, interfering with the reproducibility of ITS sequencing (Boyer et al., 2001). Furthermore, it was demonstrated by the same group that one strain might possess several ITS regions which are identical in length but differ by sequence. As our ITS sequences – deduced from 38 Porphyromonas strains – were almost ambiguity-free, we conclude that the existence of multiple rRNA operons with significant difference in sequence in this genus is not very likely, which is in accordance with the findings of Nadkarni et al. (2002), who studied Porphyromonas endodontalis and other species using rRNA-operon-directed real-time quantitative PCR.
PCR amplification of the ITS region using newly designed primers, and subsequent gel electrophoresis of 11 different Porphyromonas reference strains plus three clinical isolates of Porphyromonas gingivalis and 24 of Porphyromonas asaccharolytica, showed larger heterogeneity in length of amplicons in comparison to Fusobacterium species (Conrads et al., 2002). Furthermore, only one distinct amplification band was produced with Porphyromonas species as well as with the relatives T. forsythensis and B. distasonis, unlike Fusobacterium species (one to four bands; Conrads et al., 2002), Prevotella melaninogenica, Prevotella intermedia (two bands, data not shown) or many other genera analysed so far (Graham et al., 1997; Gurtler et al., 1999; Motoyama & Ogata, 2000). However, judging by the electrophoretic pattern of Porphyromonas asaccharolytica strains, where a 50 bp shorter, ‘shadow-like’ second band can be seen (Fig. Ab, available as supplementary material in IJSEM Online), a second 16S–23S rRNA gene ITS sequence might exist. Within a species, the length of amplicons and the deduced sequence is relatively constant as we have shown for Porphyromonas gingivalis (four strains), Porphyromonas macacae (two strains) and for fusobacterial species and subspecies. Thus, the heterogeneity found between the 25 Porphyromonas asaccharolytica strains could harbour an unrecognized species.
The high resolution of ITS sequences led to a separation of Porphyromonas asaccharolytica strains into two clusters: one cluster was -fucosidase-positive, as is typical of the type strain, and the other was -fucosidase-negative and biochemically resembled the previously studied unusual extra-oral isolates of PELOs. PELOs have been isolated as part of the mixed flora from extra-oral infections in adults (for example, from appendicitis with or without peritonitis, infected sacral decubitus ulcer, infected mastoid bone and pilonidal abscess) and have also been found in faecal specimens from children (Finegold & Jousimies-Somer, 1997). However, the isolation of PELOs from extra-oral sites is in contrast to the oral origin of Porphyromonas endodontalis but correlates with the Porphyromonas asaccharolytica-related cluster. By current biochemical methods, -fucosidase-negative Porphyromonas asaccharolytica strains cannot be differentiated from Porphyromonas endodontalis (Jousimies-Somer et al., 2002). Both are indole-positive, lack the typical glycosidase and peptidase enzymes, demonstrate fluorescence at 365 nm and do not ferment carbohydrates. In peptone/yeast/glucose (PYG) medium, acetic, propionic, butyric and isovaleric acids are produced as major metabolic products as well as isobutyric and succinic acids as minor products. The main cellular fatty acid is iso-C15 : 0, followed by 3-OH iso-C17 : 0, C16 : 0, iso-C13 : 0, 3-OH iso-C15 : 0, C14 : 0 and minor amounts of anteiso-C15 : 0. This profile is not distinctively different from that of Porphyromonas asaccharolytica (Vaisanen et al., 1997) but it is different from that of Porphyromonas endodontalis, which produces neither C14 : 0 nor 3-OH iso-C15 : 0, and less iso-C13 : 0. Again, this supports our theory that PELOs are not related to P. endodontalis but are identical to -fucosidase-negative P. asaccharolytica strains.
The separation between Porphyromonas gingivalis and Porphyromonas gulae as distinct species was supported by our ITS data; thus, Porphyromonas gulae should not be referred to as the ‘animal strain of Porphyromonas gingivalis’ as it is genetically related but not identical to Porphyromonas gingivalis. Fournier et al. (2001), describing Porphyromonas gulae, pointed out the paradox that although this species could be distinguished from Porphyromonas gingivalis phenotypically and by DNA–DNA similarity, the differences between genes encoding 16S rRNA were small. They also concluded that the recent divergences of ancestral phyla (for example, after colonizing different mammalian hosts) could not be sufficiently discerned by 16S information. Again, at least in some genera, ITS data give additional information and enhance phylogenetic resolution if discrepancies between DNA–DNA hybridization and 16S sequencing results are observed.
In conclusion, the ITS region is being used increasingly as an important tool for classification and differentiation of bacterial species. Our study is the first to provide this sequence information for most of the Porphyromonas species and their relatives. The higher resolution of ITS sequences helped clarify some of the current problems in molecular taxonomy. In addition, we found that the name PELO should no longer be used for -fucosidase-negative strains of Porphyromonas asaccharolytica that resemble Porphyromonas endodontalis. Instead, the name PALO (‘Porphyromonas asaccharolytica-like organism’) would be more suitable. Moreover, because of the reduced ITS similarity (80–85 %) between the -fucosidase-positive Porphyromonas asaccharolytica ATCC 25260T and the -fucosidase-negative cluster, the proposal of a new species seems to be warranted (Finegold et al., 2004).
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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