CLONING OF RHIZOBIUM-MELILOTI GENES FOR EFFECTIVENESS OF SYMBIOSIS USING VECTOR PK18MOB

Wild-type copies of eff genes of Rhizobium meliloti, which determine t he effectiveness of symbiosis, were cloned. To this effect, DNA sequen ces marked with transposon Tn5 were cloned in four mutants of the stra in SKhM1-188 showing enhanced symbiotic effectiveness. Small-size DNA regions positioned at a distance of 1.5-4 kb from the site of transpos on integration were then subcloned into vector pK18mob to screen recom binant plasmids, capable of site-specific integration into the rhizobi al genome. The frequency of integrants formed upon introduction of the se plasmids into the strain SKhM1-188 varied from 5 x 10(-7) to 5 x 10 (-5), dependent on the length of cloned fragments. Total DNA of integr ants was digested by particular restriction enzymes that cut out the v ector together with a copy of the wild-type target gene. After ligatio n of total DNA digests, transformation of Escherichia coli cells, and selection of recombinants with respect to the Km(r) marker of the vect or, plasmids with insertions of genomic DNA were obtained. The fact th at all in vivo and in vitro manipulations allowed the isolation of DNA fragments with wild-type copies of eff genes was confirmed by means o f Southern blotting with mutant copies of these genes.