Selection of EPS-deficient mutants (Exo-) from Rhizobiun huakuii 107 by region-directed Tn5 mutagenesis.

pJB-B5 was a recombinant plasmid of pRK415 containing the 5.9 kb B5 fragment from exoR'-11. After pJB-B5 was mutagenized by MT614 (mal::Tn5), 10 plasmids TN1-1, TN1-12, TN2-2, TN2-3, TN3-1, TN4-1, TN9-1, TN10-1, TN13-1, and TN14-1 with different Tn5 insertions in the B5 fragment were constructed. By conjugation of TN1-1, etc., into Rhizobium huakuii 107 containing the P-group plasmid pPH1JI which is incompatible with pRK415, and simultaneous selection for Rifr (conferred by strain 107), Gm(r) (conferred by pPH1JI), and Nmr (retention of Tn5), R. huakuii 107 transconjugant yields a strain in which Tn5 has recombined into the R. huakuii 107 genome. Three EPS-deficient (Exo-) mutants 107 (TN2-2), 107 (TN10-1), and 107 (TN13-1) were isolated and their inserted Tn5 was certified as the result of a double homologous recombinant event by Southern hybridization analysis. This result showed the Tn5 region-directed mutagenesis is an efficient way to select Exo- mutant in R. huakuii.