Development of a Tn10 Vehicle for Insertion Mutagenesis in Rhizobium

A transposon Tn10 vehicle was developed using a self transmissible (Tra+) plasmid pRK2013 having narrow host range orl of replication (ColEI). The construct pSA10-3 carrying Tn10 was useful in efficiently transferring transposon Tn10 from E. coli Into various rhizobia. The ColEI replicon conferred suicidal property to vector in Rhizobium background where it falls to replicate stably. Thus this plasmid can be employed to cause Independent insertion mutations in rhizobla by Tn 10 transposition. The frequency of tetracycline resistant colonies of Rhizobium (Tn10 mutants) was approximately 10⁵ folds higher than the spontaneous Tet^R mutants. Reversion frequency of these mutants was less than 10⁻⁹ indicating adequate stability of Tn10 mutations.