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Genome-wide DNA microarray analysis of Francisella tularensis strains demonstrates extensive genetic conservation within the species but identifies regions that are unique to the highly virulent F. tularensis subsp. tularensis - PubMed

Genome-wide DNA microarray analysis of Francisella tularensis strains demonstrates extensive genetic conservation within the species but identifies regions that are unique to the highly virulent F. tularensis subsp. tularensis

Martien Broekhuijsen et al. J Clin Microbiol. 2003 Jul.

Abstract

Francisella tularensis is 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. tularensis subsp. tularensis, F. tularensis subsp. mediasiatica, F. tularensis subsp. holarctica, and F. tularensis subsp. novicida. We developed a DNA microarray based on 1,832 clones from a shotgun library used for sequencing of the highly virulent strain F. tularensis subsp. tularensis Schu 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. tularensis subsp. tularensis and F. tularensis subsp. holarctica, respectively, formed distinct clusters. Despite marked differences in their virulence and geographical origin, a high degree of genomic similarity between strains of F. tularensis subsp. tularensis and F. tularensis subsp. mediasiatica was apparent. Strains from Japan clustered separately, as did strains of F. tularensis subsp. 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. tularensis subsp. holarctica and the highly virulent subspecies F. tularensis subsp. 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.

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Figures

FIG. 1.
FIG. 1.

Hierarchical cluster analysis of F. tularensis DNA microarray hybridizations. Strain identity is indicated by FSC number (Table 1) and subspecies.

FIG. 2.
FIG. 2.

Schematic depiction of the variable chromosomal region, RD1, identified among the different F. tularensis subspecies with insertions and deletions. Two direct repeats were found to define the boundaries of each deleted genomic segment. Within RD1, two complete open reading frames, designated A and B, were present. Both open reading frames A and B were found in strains Schu S4, ATCC 6223 (F. tularensis subsp. tularensis), and FSC147 (F. tularensis subsp. mediasiatica), although in strain FSC147 open reading frame A was truncated by 10 amino acids from the C terminus and open reading frame B was truncated by 12 amino acids from the N terminus. Only open reading frame B was found to be present in the LVS (F. tularensis subsp. holarctica), and only open reading frame A was found to be present in strain FSC075 (F. tularensis subsp. holarctica, Japan). Four open reading frames, designated C, D, E, and F, were found in strain FSC040 (F. tularensis subsp. novicida). Searches of the sequences in the GenBank database with the BLAST program could not identify any known gene function of the open reading frames of RD1.

FIG. 3.
FIG. 3.

PCR with primers flanking RD1 resulted in amplicons of different sizes for the different subspecies of F. tularensis. Lanes 1 to 3, strains ATCC 6223, Schu S4, and FSC199 (F. tularensis subsp. tularensis), respectively; lanes 4 to 6, strains FSC147, FSC148, and FSC149 (F. tularensis subsp. mediasiatica), respectively; lanes 7 to 9, strains FSC035, FSC155, and FSC200 (F. tularensis subsp. holarctica, non-Japanese), respectively; lanes 9 to 12, strains FSC021, FSC022, and FSC075 (F. tularensis subsp. holarctica, from Japan), respectively; lane 13, strain FSC040 (F. tularensis subsp. novicida). Molecular sizes (in kilobases) are indicated on the left.

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References

    1. Björkholm, B. M., J. L. Guruge, J. D. Oh, A. J. Syder, N. Salama, K. Guillemin, S. Falkow, C. Nilsson, P. G. Falk, L. Engstrand, and J. I. Gordon. 2002. Colonization of germ-free transgenic mice with genotyped Helicobacter pylori strains from a case-control study of gastric cancer reveals a correlation between host responses and HsdS components of type I restriction-modification systems. J. Biol. Chem. 277:34191-34197. - PubMed
    1. Boom, R., C. J. Sol, M. M. Salimans, C. L. Jansen, P. M. Wertheim-van Dillen, and J. van der Noordaa. 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28:495-503. - PMC - PubMed
    1. Clarridge, J. E., III, T. J. Raich, A. Sjöstedt, G. Sandström, R. O. Darouiche, R. M. Shawar, P. R. Georghiou, C. Osting, and L. Vo. 1996. Characterization of two unusual clinically significant Francisella strains. J. Clin. Microbiol. 34:1995-2000. - PMC - PubMed
    1. De Groote, M. A., T. Testerman, Y. Xu, G. Stauffer, and F. C. Fang. 1996. Homocysteine antagonism of nitric oxide-related cytostasis in Salmonella typhimurium. Science 272:414-417. - PubMed
    1. de la Puente-Redondo, V. A., N. G. del Blanco, C. B. Gutierrez-Martin, F. J. Garcia-Pena, and E. F. Rodriguez Ferri. 2000. Comparison of different PCR approaches for typing of Francisella tularensis strains. J. Clin. Microbiol. 38:1016-1022. - PMC - PubMed

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