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

The 16S ribosomal DNAs (rDNAs) of two strains of Francisella tularensi s and one strain of Francisella philomiragia were sequenced. On the ba sis of phylogenetic analysis data, the genus Francisella was placed in the gamma subclass of the Proteobacteria. The most closely related or ganism was the intracellular bacterium Wolbachia persica. The sequence d 16S rDNA molecules of the Francisella species exhibited very high le vels of similarity (98.5 to 99.9%). Two variable regions, comprising 3 90 to 450 nudeotides of the 16S rDNA molecules of 17 additional Franci sella strains, including members of the species F. tularensis and F. p hilomiragia, were also sequenced. At most, six nucleotide differences were observed among the sequences of the F. tularensis strains. The se quence of Francisella novicida was virtually identical to the sequence s 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 strain s by using the PCR at the genus, species, and subspecies levels. This permitted sensitive identification of strains belonging to the genus F rancisella and discrimination of the species F. tularensis and F. phil omiragia.