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-subunit-based phylogeny of Ehrlichia spp., Anaplasma spp., Neorickettsia spp. and Wolbachia pipientis
Unité des Rickettsies, CNRS UPRES-A 6020, Université de la Méditerranée, 27 Boulevard Jean Moulin, 13385 Marseille cedex 5, France
Correspondence
Didier Raoult
Didier.Raoult{at}medecine.univ-mrs.fr
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ABSTRACT |
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 TOP ABSTRACT MAIN TEXTREFERENCES |
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-subunit of RNA polymerase, was used in a phylogenetic investigation of nine species from the genera Ehrlichia, Neorickettsia, Wolbachia and Anaplasma. The complete nucleotide sequences obtained for Anaplasma phagocytophilum (HGE agent), Ehrlichia chaffeensis, Neorickettsia sennetsu, Neorickettsia risticii, Anaplasma marginale and Wolbachia pipientis were amongst the longest rpoB sequences in GenBank and ranged from 4074 bp for N. sennetsu to 4311 bp for W. pipientis. Additional partial rpoB sequences were obtained for Ehrlichia canis, Ehrlichia ruminantium and Ehrlichia muris. Identical phylogenetic trees were inferred from multiple sequence alignments of the nucleotide sequences and the derived amino acid sequences using either distance, maximum-likelihood or parsimony methods. This study confirms the phylogeny previously inferred from sequence analyses of the 16S rRNA gene, groESL and gltA and allows the confirmation of four monophyletic clades. The rpoB nucleotide sequences were more variable than the 16S rRNA gene and groESL sequences at the species level.
. Full descriptions of the rpoB genes determined in this study, the specific primers used for genome walking and their positions on the rpoB gene are available as supplementary material in IJSEM Online (http://ijs.sgmjournals.org).
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MAIN TEXT |
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TOPABSTRACTMAIN TEXTREFERENCES |
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). Taxonomic and phylogenetic studies of these bacteria were initially based on their morphological, antigenic and metabolic characters (Weiss & Moulder, 1984). Using such a previous classification scheme, the family Rickettsiaceae was found to be composed of, amongst others, the tribe Ehrlichieae, containing the genera Ehrlichia, Cowdria and Neorickettsia, and the tribe Wolbachieae, containing the genus Wolbachia. The genus Anaplasma was classified in a different family, the Anaplasmataceae. With the introduction of molecular techniques, new approaches became available to study the phylogenetic and taxonomic relationships amongst bacteria (Roux, 1999). Phylogenetic studies based on comparisons of 16S rRNA gene sequences of ehrlichieae showed that the bacteria belonged to the 
-subclass of the Proteobacteria and were closely related to the members of the genus Rickettsia (Weisburg et al., 1989). Ehrlichia, Wolbachia pipientis, Neorickettsia, Cowdria and Anaplasma grouped together in the same Ehrlichia cluster (Drancourt & Raoult, 1994), whereas Wolbachia persica grouped with the facultatively intracellular bacterium Francisella tularensis (Forsman et al., 1994). Additional phylogenetic trees made by comparing groESL (Sumner et al., 1997, 2000) and gltA (Inokuma et al., 2001) sequences confirmed the phylogenetic relationships derived from comparisons of 16S rRNA gene sequences. Recently, based on these data, Dumler et al. (2001) proposed to reorganize this clade into four genera, Anaplasma, Ehrlichia, Neorickettsia and Wolbachia, in a single family, the Anaplasmataceae.
It has been emphasized that reliable bacterial phylogeny is best obtained by comparing results obtained from multiple molecular tools, and it has been suggested that any alternative genes used should encode central cellular functions (Olsen & Woese, 1993). We chose to study rpoB as a suitable gene for additional phylogenetic studies of the ehrlichieae. The gene encodes the -subunit of RNA polymerase and belongs to an operon comprising rplL, rpoB and rpoC (Yura & Ishihama, 1979). These genes encode an enzyme complex that is highly conserved amongst the Bacteria (Ovchinnikov et al., 1981) and the Archaea (Klenk & Zillig, 1994) and their nucleotide sequences have been widely and successfully used to infer phylogenetic relationships amongst other bacteria (Klenk & Zillig, 1994; Mollet et al., 1997; Pühler et al., 1989; Rowland et al., 1993; Renesto et al., 2000a, b).
The objectives of our study were (i) to determine the complete sequences of the rpoB genes of six species currently belonging to the genera Ehrlichia, Neorickettsia, Wolbachia and Anaplasma and partial sequences from related species, (ii) to determine the rpoB-based phylogenetic relationships between these species using multiple methods of analysis and (iii) to compare the results with previously established phylogenies for these bacteria.
The strains used in our study are described in Table 1. Anaplasma phagocytophilum (HGE agent) was cultured in HL-60 cells while Ehrlichia chaffeensis, Neorickettsia sennetsu, Neorickettsia risticii, Ehrlichia canis and Ehrlichia ruminantium were co-cultured with DH82 cells. W. pipientis was co-cultured with Aa23 cells (mosquito cell line) (O'Neill et al., 1997). Organisms were purified on renografin gradients and DNA was extracted using the QIAamp Tissue kit (Qiagen) according to the manufacturer's recommendations. DNA extracted from Anaplasma marginale and Ehrlichia muris was kindly provided by Dr G. Palmer (Washington State University, Pullman, USA) and Dr M. Kawahara (Nagoya City Public Health Research Institute, Japan), respectively.
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(5'-TIATGGGIICIAAIATGCA-3'; positions 2108–2127 relative to the A. phagocytophilum rpoB sequence) and R7u (5'-GCCCAICATTCCATITCICC-3'; positions 3944–3924 relative to the A. phagocytophilum rpoB sequence) by reference to an alignment of rpoB sequences of R. prowazekii, Bartonella henselae, Bartonella quintana and A. phagocytophilum (determined in this study). The Genome Walker method was used to determine the 5'and 3' ends of the genes of Ehrlichia chaffeensis, N. risticii, N. sennetsu, A. marginale and W. pipientis. The specific primers used for genome walking and their positions on the rpoB gene are available as supplementary material in IJSEM Online (http://ijs.sgmjournals.org). With primers D4 and R7u, partial sequences were obtained for three other species, Ehrlichia canis, Ehrlichia ruminantium and Ehrlichia muris. Amplification and sequencing were performed under the conditions described above.
Phylogenetic trees were constructed based on the rpoB gene sequences and deduced amino acid sequences after alignment using the CLUSTAL W program (Thompson et al., 1994) supported within the Bisance workstation (Dessen et al., 1990). Phylogenetic trees were inferred using the PHYLIP software package (Felsenstein, 1989). Evolutionary distance matrices, generated by DNADIST and PROTDIST, were constructed by the method of Kimura (1980). The matrices were used to infer dendrograms using the neighbour-joining method (Saitou & Nei, 1987). The data were also examined using parsimony (DNAPARS and PROTPARS in PHYLIP) and maximum-likelihood analysis (DNAML in PHYLIP). Bootstrap values were obtained for a consensus based on 100 randomly generated trees using SEQBOOT and CONSENSE. Tree figures were generated using TreeView version 1.61 (Page, 1996) for parsimony and maximum-likelihood analysis and using MEGA (http://evolgen.biol.metro-u.ac.jp/mega/) for the neighbour-joining method (Sudhir et al., 1993). Sequence similarity comparisons were made with the GAP program of the GCG package (Genetics Computer Group), again supported within the Bisance workstation.
In this study, we developed rpoB consensus primers by comparing the sequences of the rpoB genes of various bacteria in GenBank. We used these primers in PCRs to amplify the rpoB gene from ehrlichieae. The primer pairs D420GL/R1820AGB and D1760AGB/R4060AGB enabled the amplification of 3890 bp of the rpoB of A. phagocytophilum in two fragments, F1 (1230 bp) and F2 (2650 bp). These extended from rplL (upstream of rpoB) beyond a part of rpoB. The primer pair D4 and R7u enabled the amplification of fragments of rpoB of 2000 bp from A. marginale, Ehrlichia chaffeensis, N. sennetsu, N. risticii and W. pipientis. The 5' and 3'ends were determined using the Universal Genome Walker kit, resulting in rpoB gene sequences of 4074 bp in N. sennetsu, 4092 bp in N. risticii, 4137 bp in Ehrlichia chaffeensis, 4146 bp in A. marginale, 4185 bp in A. phagocytophilum and 4311 bp in W. pipientis. The G+C content of the rpoB gene varied from 33·1 mol% for Ehrlichia chaffeensis to 48·4 mol% for A. marginale and deduced amino acid sequences varied from 1357 to 1436 aa. The percentage similarity varied from 57·1 % (A. marginale vs N. sennetsu) to 98·1 % (N. sennetsu vs N. risticii) for the nucleotide sequences and from 60·2 % (Ehrlichia chaffeensis vs N. sennetsu) to 95·2 % (N. sennetsu vs N. risticii) for the deduced amino acid sequences. The percentages of similarity of the rpoB sequences between the two species N. sennetsu and N. risticii and other ehrlichial species were lower (58·5–65·4 %) than those between other Ehrlichia species and B. henselae (44·8–66·3 %). Using the primer pair D4 and R7u, we also established partial rpoB nucleotide and deduced amino acid sequences for Ehrlichia canis, Ehrlichia ruminantium and Ehrlichia muris. Full descriptions of the rpoB genes determined in this study are available as supplementary material in IJSEM Online (http://ijs.sgmjournals.org).
The topologies observed in the rpoB trees obtained using the three methods of analysis were identical, and showed the ehrlichieae to be closely related to the genus Rickettsia in the -subclass of the Proteobacteria. The bootstrap values showed greatest support using the neighbour-joining method and ranged from 99 to 100 %, except at the node where Ehrlichia chaffeensis diverged, which had a bootstrap value of 70 %. The phylogenetic tree for the ehrlichieae had four branches. The first branch comprised the genus Anaplasma, although there was relatively low similarity (74·5 %) between A. marginale and A. phagocytophilum. The second branch contained Ehrlichia chaffeensis, the third W. pipientis and the fourth branch contained N. sennetsu and N. risticii, which had very high similarity (98·1 %). When more ehrlichieae, namely Ehrlichia canis, Ehrlichia muris and Ehrlichia ruminantium, were included in a partial rpoB-based tree, the same topologies were found and had high bootstrap values (Fig. 1).
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and are in agreement with the findings of groESL- and gltA-based phylogenetic studies (Sumner et al., 1997, 2000; Inokuma et al., 2001) and those postulated previously (Drancourt & Raoult, 1994). The number of clades depends on the level of rpoB sequence divergence used to define them (Fig. 1): three clades would be delineated using a 5 % divergence level, four clades would be delineated at a divergence level between 4·5 and 5 % and five clades would be delineated at a divergence level of >2·3–4·5 %. Biological and phenotypic properties may help to resolve the question of the appropriate level of divergence. Phylogenetic relationships often cannot be accurately established using sequence information from only a single gene. Sequence analysis of the rpoB gene, which has more sequence variation than the 16S rRNA gene, is an additional tool on which to base the phylogenetic relationships of the ehrlichieae.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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