Summary auto-generated
Roux and colleagues developed citrate synthase gene (gltA) sequencing as a phylogenetic tool for analyzing Rickettsia species. They amplified and sequenced a 1,234-bp gltA fragment from 28 rickettsiae using PCR and automated DNA sequencing. The spotted fever group rickettsiae divided into two major subgroups: one containing tick-only isolates (R. massiliae, R. rhipicephali, and others) and a larger subgroup containing most human pathogens and additional tick isolates. Phylogenetic analysis revealed that R. bellii, R. canada, and the AB bacterium represent divergent lineages separate from classical typhus and spotted fever groups. The gltA approach proved superior to 16S rRNA sequencing for rickettsial phylogeny, providing stronger bootstrap support for most nodes. When applied broadly to bacterial sequences from GenBank, gltA sequencing showed reliable phylogenetic signal across domains, with sequence similarity varying appropriately by taxonomic distance. The authors conclude that gltA sequence comparison offers a valuable complementary approach to ribosomal RNA analysis, particularly when 16S sequences yield ambiguous relationships due to high similarity among closely related bacteria.
Key findings
- gltA gene sequencing provides superior phylogenetic resolution compared to 16S rRNA for Rickettsia species, with higher bootstrap support for most nodes
- Spotted fever group rickettsiae divide into two subgroups: one with tick-only isolates and another with most human pathogens
- R. bellii, R. canada, and the AB bacterium represent distinct evolutionary lineages separate from classical typhus and spotted fever groups
- Citrate synthase gene shows appropriate sequence divergence across bacterial taxa, with similarity levels varying by evolutionary distance
- gltA sequencing is recommended as a complementary molecular marker to 16S rRNA for resolving bacterial phylogeny
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Abstract
Using PCR and an automated laser fluorescent DNA sequencer, we amplified and sequenced a 1,234-bp fragment of the citrate synthase-encoding gene (gltA) of 28 bacteria belonging to the genus Rickettsia. Comparative sequence analysis showed that most of the spotted fever group (SFG) rickettsiae belonged to one of two subgroups. The first subgroup included Rickettsia massiliae, strain Bar 29, Rickettsia rhipicephali, "Rickettsia aeschlimanni," and Rickettsia montana, which have been isolated only from ticks. The second subgroup was larger and included the majority of the human pathogens and also rickettsiae isolated only from ticks; the members of this subgroup were strain S, Rickettsia africae, "Rickettsia mongolotimonae," Rickettsia sibirica, Rickettsia parkeri, Rickettsia conorii, Rickettsia rickettsii, the Thai tick typhus rickettsia, the Israeli tick typhus rickettsia, the Astrakhan fever rickettsia, "Rickettsia slovaca," and Rickettsia japonica. The sequence analysis also showed that the tick-borne organisms Rickettsia helvetica and Rickettsia australis and the mite-borne organism Rickettsia akari were associated with the SFG cluster; that Rickettsia prowazekii and Rickettsia typhi, two representatives of the typhus group, clustered together; and that Rickettsia canada, Rickettsia bellii, and the AB bacterium probably represent three new groups. We compared the phylogenetic trees inferred from citrate synthase gene sequences and from 16S ribosomal DNA (rDNA) sequences. For rickettsial phylogeny, the citrate synthase approach was more suitable, as demonstrated by significant bootstrap values for all of the nodes except those in the larger subgroup defined above. We also compared phylogenetic analysis results obtained in a comparison of the sequences of both genes for all of the representatives of the domain Bacteria for which the gltA sequence was determined. We believe that comparison of gltA sequences could be a complementary approach to 16S rDNA sequencing for inferring bacterial evolution, especially when unstable phylogenetic models are obtained from ribosomal sequences because of high levels of sequence similarity between the bacteria studied.