FIG. 3.

Strategy for site-directed mutagenesis of the exoP gene from R. meliloti. At the top the structure of the operon comprising the genes exoH to exoP of the exo gene cluster from R. meliloti Rm2011 (5) is shown. Promoters directing the transcription of exoP (5) are indicated by black dots. Mutants RmΔexoP and RmΔPII15 were constructed by deletion of the complete exoP gene and a part of the exoN coding region of wild-type R. meliloti Rm2011 and the expA1 mutant RmAR1015 (8), respectively. The deleted fragment was replaced by a spectinomycin resistance cassette (spc). Due to the integration of pExoP-XnZ plasmids into the genomes of these mutants by homologous recombination, the native structure of the exoN-exoP region was restored. Incomplete genes are printed in parentheses. The pExoP-XnZ plasmids contained mutated exoP genes carrying single base pair substitutions causing the replacement of amino acid residue X in position n of the ExoP protein for residue Z (Fig. 2D).