Construction of gene replacement vectors for Gram? bacteria using a genetically modified sacRB gene as a positve selection marker

Abstract

Based on the wide-host-range suicide vector pSUP102 (Simon et al. 1983), gene replacement vectors (pWS232/pWS233) have been developed that facilitate the identification by a positive selection procedure of double recombination events in Gram− bacteria. The vectors contain the tetracycline and gentamicin resistance gene as selectable markers, as well as a modified sacRB gene mediating sucrose sensitivity. In order to increase the versatility of the sacRB gene as a positive selection marker and, hence, of vectors that carry this gene, an EcoRI as well as a HindIII site located within the coding region of the sacRB gene were removed by in-vitro mutagenesis. To test the suitability of the vectors, a Tn5-carrying EcoRI fragment of Rhizobium leguminosarum biovar. viciae VF39 was homogenotized into the wild-type strain, resulting in double recombinants with a Lac− phenotype. Although the Tn5 insertion was flanked on one side by only approximately 100 bp of VF39 homologous DNA, this was sufficient for homologous recombination to occur, and double recombinants could readily be isolated.