Analysis of 16S Ribosomal DNA Sequences of Francisella Strains and Utilization for Determination of the Phylogeny of the Genus and for Identification of Strains by PCR

Abstract

The 16s ribosomal DNAs (rDNAs) of two strains of Franchella dularensis and one strain of Francisella philomiragia were sequenced. On the basis of phylogenetic analysis data, the genus Franciselk was placed in the y subclass of the Proteobacteria. The most closely related organism was the intracellular bacterium Wolbachia persica. The sequenced 16s rDNA molecules of the Francisella species exhibited very high levels of similarity (98.5 to 99.95%). Two variable regions, comprising 390 to 450 nucleotides of the 16s rDNA molecules of 17 additional Francisella strains, including members of the species F. tularensis and F. philomiragiu, were also sequenced. At most, six nucleotide differences were observed among the sequences of the F. tularensis strains. The sequence of Francisella novicida was virtually identical to the sequences of the F. tularensis strains, thereby supporting the hypothesis that these organisms are members of the same species. On the basis of the observed differences, primer pairs were designed to distinguish strains by using the PCR at the genus, species, and subspecies levels. This permitted sensitive identification of strains belonging to the genus Francisella and discrimination of the species F. tularensis and F. philomiragia.

The complete genome sequence of Francisella tularensis, the causative agent of tularemia

“…The Francisellaceae have no close pathogenic relatives, as inferred from sequence similarity or 16S rRNA phylogenies 14 . Instead, 16S rRNA data suggests that F. tularensis is a sister clade with arthropod endosymbionts like Wolbachia persica 14 and only distantly related to the human pathogens Coxiella burnetii and Legionella.…”

“…The Francisellaceae have no close pathogenic relatives, as inferred from sequence similarity or 16S rRNA phylogenies 14 . Instead, 16S rRNA data suggests that F. tularensis is a sister clade with arthropod endosymbionts like Wolbachia persica 14 and only distantly related to the human pathogens Coxiella burnetii and Legionella. This suggestion is supported by a phylogenomic analysis of more than 200 genes with homologs in F. tularensis and 15 other g-proteobacterial genomes, as previously undertaken for a smaller set of g-proteobacterial genomes 15 .…”

Tularemia: emergence/re-emergence

“…16S rDNA sequencing played an important role in the first identification of F. tularensis in the Southern Hemisphere and also in the tularemia outbreak associated with crayfish in Spain [4,69]. For identification of recovered bacterial isolates, the universal 16S rDNA primers as well as the Francisella specific 16S rDNA primers, provide good sequence data [28]. For diagnostic identification of Francisella spp.…”

Phylogenetic analysis of Calymmatobacterium granulomatis based on 16S rRNA gene sequences

“…There are a number of possibilities for the observed sequence differences. There is evidence of very little diversity of the 16s rDNA sequences of Francisella [26] and Legionella pneumophila [27], whereas for Borrelia burgdorferi strains, up to 1.0% divergence has been demonstrated [28]. Eckloff et al [29] compared the 16s rDNA sequences from five isolates of Helicobacter pylori and found deviations from the consensus sequence at 13 separate positions which could be attributed to clonal origins.…”

“…Forsman et al [26] observed unusually high levels of sequence homology between the 16s rRNA sequences of Wolbachia persica and Francisella and suggested that instead of placing K persica within the genus Francisella, the species should be m h e r characterised phenotypically to determine its relationship to Francisella species. In a similar way, although the 16s rDNA sequences of C. granulomatis are closely related to those of Klebsiella, they form a distinct group within the taxon Enterobacteriaceae.…”