Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants

Abstract We have constructed a cosmid derivative of the low copy-number broad host-range cloning vector pRK.290 (Ditta et al., 1980) by inserting a 1.6-kb Bgl II fragment containing λ cos into the unique Bgl II site in pRK.290. The new vector, pLAFRl, is 21.6 kb long, confers tetracycline resistance, contains a unique Eco RI site, and can be mobilized into and stably replicates within many Gram-negative hosts. We constructed a clone bank of Rhizobium meliloti DNA in pLAFRl using a partial Eco RI digest. The mean insert size was 23.1 kb. When the clone bank was mated (en masse) from Escherichia coli to various R. meliloti auxotrophic mutants, tetracycline-resistant (Tc r ) transconjugants were obtained at frequencies ranging from 0.1 to 0.8, and among these, prototrophic colonies were obtained at frequencies ranging from 0.001 to 0.007. pLAFRl cosmids were mobilized from R. meliloti prototrophic colonies into E. coli and then reintroduced into R. meliloti auxotrophs. In most cases, 100% of these latter Tc r transconjugants were prototrophic.