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We examined the nature of the interactions between the facultative intracellular pathogens Francisella tularensis and F. novicida and rodent macrophages. Growth of F. tularensis LVS was observed in macrophage monolayers from mice, guinea pigs, or rats. In contrast, F. novicida grew in macrophages from mice and guinea pigs but not in macrophages from rats. Transmission electron microscopy studies indicated that both Francisella species survive within macrophage phagosomes that are unfused with lysosomes.

We describe methods for transposon mutagenesis and allelic replacement in the facultative intracellular pathogen Francisella Recombinant clones were constructed by insertion of partially cut F. tularensis or F. novicida DNA into pUC19 and then mutagenized with a mini-TnlO-Km transposon. F. novicida could be transformed with these plasmids either by a chemical transformation method or by electroporation, whereas F. tularensis could be transformed only by electroporation. Transformation of F. tularensis by electroporation was enhanced in the absence of the capsule. Southern blot analysis showed that the KmR marker was rescued either by integration of the plasmid into the Francisella chromosome or by allelic replacement. Allelic replacement was found to be the mechanism underlying a site-specific mutation affecting FopA, an outer-membrane protein of Francisella. F. novicida could also be transformed with chromosomal DNA carrying the KmR marker and the transformation frequency obtained using chromosomal DNA was generally greater than that obtained using plasmid DNA. F. novicida was also transformed by an IncQ plasmid containing an F. novicida DNA insert, which replicated autonomously in this host.

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