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Applied and Environmental Microbiology, August 2008, p. 5263-5264, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00446-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

LETTER TO THE EDITOR

Reorganization and Monophyly of the Genus Rickettsiella: All in Good Time

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The genus Rickettsiella of intracellular bacterial pathogens of arthropods currently comprises the three recognized species Rickettsiella popilliae, R. grylli, and R. chironomi, together with numerous further pathotypes or "subjective synonyms." On the basis of ultrastructural data, these bacteria were originally assigned to the alphaproteobacterial order Rickettsiales. However, in 1997, the determination of a 16S rRNA-encoding sequence from R. grylli revealed the highest homology with the corresponding genes from Coxiella burnetii and related Gammaproteobacteria (12). Ten years later, a paper by Cordaux et al. (1) links up with this finding when asking "if only R. grylli has been misclassified" or "if the genus Rickettsiella is monophyletic," in which case its taxonomic position "needs to be reassessed." The study is based on three new 16S rRNA gene sequences from the crustacean-infecting pathotype "R. armadillidii." From a phylogenetic analysis including these together with the aforementioned R. grylli sequence, the authors firstly conclude that "Rickettsiella bacteria overall appear to form a monophyletic group" and secondly suggest that "therefore, the genus Rickettsiella as a whole (not just Rickettsiella grylli) should be classified among the Gammaproteobacteria instead of the Alphaproteobacteria." These claims surely deserve a closer look.

On the one hand, the above taxonomic suggestion simply comes late. During the last decade, 16S rRNA genes from several Rickettsiella pathotypes have been determined (e.g., 3, 7, 13). Despite the lack of sequences from both the species R. popilliae and R. chironomi, these data have motivated the previous taxonomic reorganization of the entire genus Rickettsiella in the class Gammaproteobacteria, order Legionellales (5), i.e., as suggested by Cordaux and colleagues. The authors use these additional sequences to calculate the evolutionary origin of Rickettsiella in the second part of their study but fail to mention the R. grylli genome sequence available since 2006 (provisional GenBank accession number NZ_AAQJ00000000) that convincingly supports the reorganization of R. grylli by phylogenetic inference from beyond the 16S rRNA gene level (7a).

On the other hand, the claim of monophyly of the genus Rickettsiella appears premature. The authors' contribution of 16S rRNA gene sequences from a further pathotype could, at best, trigger a negative result concerning "the monophyletic status of the genus Rickettsiella." Positively, it can only add to the cumulative evidence provided by the aforementioned gammaproteobacterial rRNA gene sequences from Rickettsiella pathotypes. However, there is contradicting evidence as well, as several ultrastructurally well-characterized presumed Rickettsiella pathotypes have been found to carry chlamydial instead of alpha- or gammaproteobacterial 16S rRNA genes (e.g., see references 2, 4, 6, and 11). These findings appear relevant within the framework of a monophyly argument, and that they pass unmentioned is the more astonishing as Cordaux and colleagues have included other chlamydial sequences in their phylogenetic analysis. In contrast to the all-or-nothing scenario conjured by the authors, the emerging picture is therefore that the genus Rickettsiella has, with good reason, been removed from the Alphaproteobacteria and provisionally been assigned to the Gammaproteobacteria but that it might, in its present state, comprise numerous misclassified pathotypes.

If so, then is there any sense in talking about monophyly of uncultured organisms as Rickettsiella bacteria with many of the ultrastructurally characterized specimens, among them the nomenclatural type strain, being no more available for molecular phylogenetic analysis? It might be seen as a reasonable minimal condition of the monophyly of this genus that—all loosely associated pathotypes aside—at least the recognized Rickettsiella species form a monophyletic group. As there has been no previous molecular evidence from either R. chironomi or R. popilliae and as the pathotype "R. armadillidii" is a subjective synonym of the latter species (5), the data assessed by Cordaux et al. in this sense make a considerable contribution to an elucidation of the problem under study. Curiously enough, the authors themselves seem to miss this point when stating incorrectly that ‘ "R. armadillidii" and R. grylli are considered synonymous species in the currently accepted nomenclature.’ Meanwhile, determined 16S rRNA-encoding sequences from further R. popilliae-synonymous pathotypes lend additional support to the taxonomic assignment of the genus Rickettsiella to the class Gammaproteobacteria (8-10). However, as long as there are no molecular data available from the third recognized species, R. chironomi, monophyly of the genus Rickettsiella remains a premature claim.

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1
1. Cordaux, R., M. Paces-Fessy, M. Raimond, A. Michel-Salzat, M. Zimmer, and D. Bouchon. 2007. Molecular characterization and evolution of arthropod pathogenic Rickettsiella bacteria. Appl. Environ. Microbiol. 73:5045-5047.[Abstract/Free Full Text]
2
2. Corsaro, D., V. Thomas, G. Goy, D. Venditti, R. Radek, and G. Greub. 2007. ‘Candidatus Rhabdochlamydia crassificans’, an intracellular bacterial pathogen of the cockroach Blatta orientalis (Insecta: Blattodea). Syst. Appl. Microbiol. 30:221-228.[CrossRef][Medline]
3
3. Czarnetzki, A. B., and C. C. Tebbe. 2004. Diversity of bacteria associated with Collembola—a cultivation-independent survey based on PCR-amplified 16S rRNA genes. FEMS Microbiol. Ecol. 49:217-227.[Medline]
4
4. Drobne, D., J. trus, N. nidari, and P. Zidar. 1999. Morphological description of bacterial infection of digestive glands in the terrestrial isopod Porcellio scaber (Isopoda, Crustacea). J. Invertebr. Pathol. 73:113-119.[CrossRef][Medline]
5
5. Fournier, P.-E., and D. Raoult. 2005. Genus II. Rickettsiella Philip 1956, 267^AL, p. 241-247. In G. M. Garrity, D. J. Brenner, N. R. Krieg, and J. T. Staley (ed.), Bergey's manual of systematic bacteriology, 2nd ed., vol. 2, part B. Springer, New York, NY.
6
6. Kostanjsek, R., J. trus, D. Drobne, and G. Avgustin. 2004. ‘Candidatus Rhabdochlamydia porcellionis’, an intracellular bacterium from the hepatopancreas of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda). Int. J. Syst. Evol. Microbiol. 54:543-549.[Abstract/Free Full Text]
7
7. Kurtti, T. J., A. T. Palmer, and J. H. Oliver. 2002. Rickettsiella-like bacteria in Ixodes woodi (Acari: Ixodidae). J. Med. Entomol. 39:534-540.[Medline]
7
8. Leclerque, A. 2008. Whole genome-based assessment of the taxonomic position of the arthropod pathogenic bacterium Rickettsiella grylli. FEMS Microbiol. Lett. 283:117-127.[CrossRef][Medline]
8
9. Leclerque, A., and R. G. Kleespies. 2008. 16S ribosomal RNA-, GroEL-, and MucZ-based assessment of the taxonomic position of Rickettsiella melolonthae and its implications for the organization of the genus Rickettsiella. Int. J. Syst. Evol. Microbiol. 58:749-755.[Abstract/Free Full Text]
9
10. Leclerque, A., and R. G. Kleespies. 2008. Type IV secretion system components as phylogenetic markers of entomopathogenic bacteria of the genus Rickettsiella. FEMS Microbiol. Lett. 279:167-173.[CrossRef][Medline]
10
11. Leclerque, A., and R. G. Kleespies. 20 February 2008, posting date. Genetic and electron-microscopic characterization of Rickettsiella tipulae, an intracellular bacterial pathogen of the crane fly, Tipula paludosa. J. Invertebr. Pathol. doi:10.1016/j.jip.2008.02.005.
11
12. Radek, R. 2000. Light and electron microscopic study of a Rickettsiella species from the cockroach Blatta orientalis. J. Invertebr. Pathol. 76:249-256.[CrossRef][Medline]
12
13. Roux, V., M. Bergoin, N. Lamaze, and D. Raoult. 1997. Reassessment of the taxonomic position of Rickettsiella grylli. Int. J. Syst. Bacteriol. 47:1255-1257.[Abstract/Free Full Text]
13
14. Spaulding, A. W., and C. D. von Dohlen. 2001. Psyllid endosymbionts exhibit patterns of co-speciation with hosts and destabilizing substitutions in ribosomal RNA. Insect Mol. Biol. 10:57-67.[CrossRef][Medline]

						Andreas Leclerque Federal Research Centre for Cultivated Plants Julius Kühn Institute (JKI) Institute for Biological Control Darmstadt, Germany Phone: 496151407254 Fax: 496151407290 E-mail: leclerque{at}hotmail.com

Authors' Reply

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A. Leclerque appears to have no technical objection to our study (1), and he even ends up concluding that our "Rickettsiella armadillidii" 16S rRNA sequence data "make a considerable contribution to an elucidation of the problem under study." Yet, Leclerque also argues that our results are merely confirmatory because previously determined 16S rRNA sequences from other Rickettsiella pathotypes (3, 6, 12) already led to the conclusion we drew. In fact, the sequence data cited by Leclerque are irrelevant to the question of the taxonomic assignment of Rickettsiella bacteria. This is because the 16S rRNA sequences reported in these studies (3, 6, 12) constituted the actual basis for retrospectively inferring the presence of Rickettsiella bacteria in the arthropod hosts analyzed, based on high sequence similarity with an R. grylli sequence (11). A circular reasoning subsequently consisting of using these sequences to investigate Rickettsiella taxonomic position would necessarily lead to support for the assignment of Rickettsiella bacteria to Gammaproteobacteria but would be undermined by ascertainment bias. By contrast, we performed an unbiased molecular characterization of Rickettsiella bacteria by only including samples for which Rickettsiella infection has been ascertained independently of molecular information (see Fig. 1 in reference 1).

Leclerque regrets that we "fail(ed) to mention the R. grylli genome sequence available since 2006 (provisional GenBank accession number NZ_AAQJ00000000) that convincingly supports the reorganization of R. grylli by phylogenetic inference from beyond the 16S rRNA gene level." We humbly note that Leclerque himself fails to mention any tangible result or publication that would actually support his claim. In any event, the reassignment of R. grylli to Gammaproteobacteria is already well accepted (11) and not questioned in our study (1).

Leclerque also argues that our suggestion of Rickettsiella monophyly is premature, as evidenced by two formerly presumed Rickettsiella pathotypes (4, 10) that have subsequently been shown to be related to Chlamydiales instead of the Rickettsiella genus sensu stricto and renamed accordingly (2, 5). Again, this argument is irrelevant because if these pathotypes are unrelated to Rickettsiella bacteria sensu stricto and had initially been misclassified as Rickettsiella members, then they are not relevant anymore to the question of the monophyly of Rickettsiella bacteria sensu stricto. For example, it would seem absurd to include Rickettsiella bacteria in analyses addressing questions related to the monophyly of Rickettsiales, just because Rickettsiella bacteria used to be classified as Rickettsiales until they were shown be Gammaproteobacteria.

Since the publication of our study (1), Leclerque's own work based on additional well-characterized Rickettsiella strains and markers has provided further molecular support for the monophyly of Rickettsiella bacteria sensu stricto (7-9). We agree with Leclerque that additional molecular data will help further clarify the evolutionary history of Rickettsiella bacteria. Meanwhile, assuming Rickettsiella monophyly constitutes the framework that is most consistent with the currently available molecular evidence.

Top LETTER REFERENCES LETTER{nbsp} REFERENCES{nbsp}

 

1
1. Cordaux, R., M. Paces-Fessy, M. Raimond, A. Michel-Salzat, M. Zimmer, and D. Bouchon. 2007. Molecular characterization and evolution of arthropod-pathogenic Rickettsiella bacteria. Appl. Environ. Microbiol. 73:5045-5047.[Abstract/Free Full Text]
2
2. Corsaro, D., V. Thomas, G. Goy, D. Venditti, R. Radek, and G. Greub. 2007. ‘Candidatus Rhabdochlamydia crassificans’, an intracellular bacterial pathogen of the cockroach Blatta orientalis (Insecta: Blattodea). Syst. Appl. Microbiol. 30:221-228.[CrossRef][Medline]
3
3. Czarnetzki, A. B., and C. C. Tebbe. 2004. Diversity of bacteria associated with Collembola—a cultivation-independent survey based on PCR-amplified 16S rRNA genes. FEMS Microbiol. Ecol. 49:217-227.[Medline]
4
4. Drobne, D., J. trus, N. nidari, and P. Zidar. 1999. Morphological description of bacterial infection of digestive glands in the terrestrial isopod Porcellio scaber (Isopoda, Crustacea). J. Invertebr. Pathol. 73:113-119.[CrossRef][Medline]
5
5. Kostanjsek, R., J. trus, D. Drobne, and G. Avgustin. 2004. ‘Candidatus Rhabdochlamydia porcellionis’, an intracellular bacterium from the hepatopancreas of the terrestrial isopod Porcellio scaber (Crustacea: Isopoda). Int. J. Syst. Evol. Microbiol. 54:543-549.[Abstract/Free Full Text]
6
6. Kurtti, T. J., A. T. Palmer, and J. H. Oliver, Jr. 2002. Rickettsiella-like bacteria in Ixodes woodi (Acari: Ixodidae). J. Med. Entomol. 39:534-540.[Medline]
7
7. Leclerque, A., and R. G. Kleespies. 2008. 16S ribosomal RNA-, GroEL-, and MucZ-based assessment of the taxonomic position of Rickettsiella melolonthae and its implications for the organization of the genus Rickettsiella. Int. J. Syst. Evol. Microbiol. 58:749-755.[Abstract/Free Full Text]
8
8. Leclerque, A., and R. G. Kleespies. 20 February 2008, posting date. Genetic and electron-microscopic characterization of Rickettsiella tipulae, an intracellular bacterial pathogen of the crane fly, Tipula paludosa. J. Invertebr. Pathol. doi:10.1016/j.jip.2008.02.005.
9
9. Leclerque, A., and R. G. Kleespies. 2008. Type IV secretion system components as phylogenetic markers of entomopathogenic bacteria of the genus Rickettsiella. FEMS Microbiol. Lett. 279:167-173.[CrossRef][Medline]
10
10. Radek, R. 2000. Light and electron microscopic study of a Rickettsiella species from the cockroach Blatta orientalis. J. Invertebr. Pathol. 76:249-256.[CrossRef][Medline]
11
11. Roux, V., M. Bergoin, N. Lamaze, and D. Raoult. 1997. Reassessment of the taxonomic position of Rickettsiella grylli. Int. J. Syst. Bacteriol. 47:1255-1257.[Abstract/Free Full Text]
12
12. Spaulding, A. W., and C. D. von Dohlen. 2001. Psyllid endosymbionts exhibit patterns of co-speciation with hosts and destabilizing substitutions in ribosomal RNA. Insect Mol. Biol. 10:57-67.[CrossRef][Medline]

						Richard Cordaux Didier Bouchon* Université de Poitiers CNRS UMR 6556 Ecologie, Evolution, Symbiose 40 Avenue du Recteur Pineau 86022 Poitiers, France
						* Phone: 33 549 45 38 95 Fax: 33 549 45 40 15 E-mail: didier.bouchon{at}univ-poitiers.fr

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Applied and Environmental Microbiology, August 2008, p. 5263-5264, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00446-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Leclerque, A. Articles by Bouchon, D. Search for Related Content PubMed PubMed Citation Articles by Leclerque, A. Articles by Bouchon, D. Agricola Articles by Leclerque, A. Articles by Bouchon, D.