1887

Abstract

This paper is the first detailed description of the development and use of new genetic tools specifically for the safe manipulation of highly pathogenic subsp. . Most of these tools are also demonstrated to work with other subspecies. Kanamycin and hygromycin resistance determinants that function as genetic markers in subsp. strain Schu and sets of episomal shuttle vectors that are either unstable or stably maintained in the absence of selection were developed. In addition, the gene, expressed from the promoter, was successfully used as a marker for transposon mutagenesis. This work also includes the development of -based suicide plasmids expressing kanamycin resistance that can be used for electroporation-mediated allelic exchange of unmarked mutations in Schu and the live vaccine strain (LVS). Using these plasmids, the two predicted -lactamase genes, and , in Schu and LVS were deleted. Only the Δ mutants had increased susceptibility to ampicillin, and this phenotype was complemented by a plasmid expressing . The results suggest that the -lactam antibiotic resistance phenotype of Schu and LVS is likely due to only one of the two -lactamase genes present and that ampicillin resistance can be used as an additional selectable marker in -lactamase deletion mutants. The collection of tools presented in this report will be helpful for the genetic analyses of subsp. pathogenesis.

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2006-11-01
2019-12-10
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