Genome-Wide DNA Microarray Analysis of Francisella tularensisStrains Demonstrates Extensive Genetic Conservation within the Species but Identifies Regions That Are Unique to the Highly Virulent F. tularensissubsp. tularensis

ABSTRACTFrancisella tularensisis a potent pathogen and a possible bioterrorism agent. Little is known, however, to explain the molecular basis for its virulence and the distinct differences in virulence found between the four recognized subspecies, F. tularensissubsp. tularensis, F. tularensissubsp. mediasiatica, F. tularensissubsp. holarctica, and F. tularensissubsp. novicida. We developed a DNA microarray based on 1,832 clones from a shotgun library used for sequencing of the highly virulent strain F. tularensissubsp. tularensisSchu S4. This allowed a genome-wide analysis of 27 strains representing all four subspecies. Overall, the microarray analysis confirmed a limited genetic variation within the species F. tularensis, and when the strains were compared, at most 3.7% of the probes showed differential hybridization. Cluster analysis of the hybridization data revealed that the causative agents of type A and type B tularemia, i.e., F. tularensissubsp. tularensisand F. tularensissubsp. holarctica, respectively, formed distinct clusters. Despite marked differences in their virulence and geographical origin, a high degree of genomic similarity between strains of F. tularensissubsp. tularensisand F. tularensissubsp. mediasiaticawas apparent. Strains from Japan clustered separately, as did strains of F. tularensissubsp. novicida. Eight regions of difference (RD) 0.6 to 11.5 kb in size, altogether comprising 21 open reading frames, were identified that distinguished strains of the moderately virulent subspecies F. tularensissubsp. holarcticaand the highly virulent subspecies F. tularensissubsp. tularensis. One of these regions, RD1, allowed for the first time the development of an F. tularensis-specific PCR assay that discriminates each of the four subspecies.