SUMMARY: We describe methods for transposon mutagenesis and allelic replacement in the facultative intracellular pathogen Recombinant clones were constructed by insertion of partially cut or DNA into pUC19 and then mutagenized with a mini-Tn-Km transposon. could be transformed with these plasmids either by a chemical transformation method or by electroporation, whereas could be transformed only by electroporation. Transformation of by electroporation was enhanced in the absence of the capsule. Southern blot analysis showed that the Km marker was rescued either by integration of the plasmid into the 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 could also be transformed with chromosomal DNA carrying the Km marker and the transformation frequency obtained using chromosomal DNA was generally greater than that obtained using plasmid DNA. was also transformed by an IncQ plasmid containing an DNA insert, which replicated autonomously in this host.


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