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Journal of Bacteriology, December 2008, p. 7830-7837, Vol. 190, No. 23
0021-9193/08/$08.00+0     doi:10.1128/JB.01188-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Genetic Dissection of the Francisella novicida Restriction Barrier ^,

Department of Genome Sciences, University of Washington, Campus Box 355065, 1705 NE Pacific St., Seattle, Washington 98195

Received 25 August 2008/ Accepted 15 September 2008

Francisella tularensis is the causative agent of tularemia and is a category A select agent. Francisella novicida, considered by some to be one of four subspecies of F. tularensis, is used as a model in pathogenesis studies because it causes a disease similar to tularemia in rodents but is not harmful to humans. F. novicida exhibits a strong restriction barrier which reduces the transformation frequency of foreign DNA up to 10⁶-fold. To identify the genetic basis of this barrier, we carried out a mutational analysis of restriction genes identified in the F. novicida genome. Strains carrying combinations of insertion mutations in eight candidate loci were created and assayed for reduced restriction of unmodified plasmid DNA introduced by transformation. Restriction was reduced by mutations in four genes, corresponding to two type I, one type II, and one type III restriction system. Restriction was almost fully eliminated in a strain in which all four genes were inactive. The strongest contributor to the restriction barrier, the type II gene, encodes an enzyme which specifically cleaves Dam-methylated DNA. Genome comparisons show that most restriction genes in the F. tularensis subspecies are pseudogenes, explaining the unusually strong restriction barrier in F. novicida and suggesting that restriction was lost during evolution of the human pathogenic subspecies. As part of this study, procedures were developed to introduce unmodified plasmid DNA into F. novicida efficiently, to generate defined multiple mutants, and to produce chromosomal deletions of multiple adjacent genes.

Published ahead of print on 3 October 2008.

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