Construction of a single-transposon-insertion mutant in Rhizobium sp. strain TAL1145 from a double-insertion mutant

A method for developing a single-transposon-insertion mutant from a double-insertion mutant in Rhizobium is described. An exopolysaccharide (EPS)-defective mutant containing two Tn5-lacZ insertions was complemented with cloned wild-type DNA for EPS synthesis. One of the Tn5-lacZ insertions from the mutant was transferred to the complementing plasmid by homologous recombination. The plasmid containing the Tn5-lacZ insertion in the gene involved in EPS synthesis was transferred into the wild-type strain and the Tn5-lacZ was homogenized to obtain an EPS-defective mutant with a single Tn5-lacZ insertion.