1887

Abstract

The ability to generate mutants of will be important if we are to understand the biology of this major pathogen. However, allelic replacement methods have only recently achieved success. We have developed a reproducible method for generating defined mutants of using homologous recombination. The transforming DNA was used following pre-treatment either with UV light or alkali denaturation in order to stimulate homologous recombination and abolish illegitimate recombination. Suicide vectors carrying one of nine amino acid biosynthesis genes were electroporated into , and homologous recombinants were obtained in all nine genes; eight resulted from single-crossover events (SCOs) and one from a double-crossover event (DCO) (in the gene). The remaining colonies were spontaneous hygromycin-resistant mutants; no products of illegitimate recombination were observed. To more efficiently distinguish spontaneous mutants, the gene was cloned into five vectors (two containing genes not previously tested), and the transformations were repeated. SCO mutants were identified by screening for blue colonies on indicator plates. White transformants were tested for auxotrophy and , and auxotrophic mutants were obtained. Only blue SCOs were obtained for and . Thus, using this methodology we have obtained homologous recombination in 11 genes, and DCOs in 4 genes, showing that it is possible to generate targeted mutants in a reproducible way.

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/content/journal/micro/10.1099/00221287-145-12-3497
1999-12-01
2020-01-28
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