A vector for the site-directed, genomic integration of foreign DNA into soybean root-nodule bacteria

Abstract

A non-essential DNA region carrying two different repeated sequences (RSβ3 and RSα9) adjacent to a nitrogen fixation (nif) gene cluster has been identified previously in Bradyrhizobium japonicum strain 110. In closely related B. japonicum strains a similar genomic arrangement was found. We constructed a mobilizable plasmid vector carrying RSβ3 and RSα9, and a kanamycin resistance cassette (nptII gene) plus suitable cloning sites inserted between the two repeated sequences. Using this vector (pRJ1035), stable integration of a lacZ gene fusion into the B. japonicum genomic RS region was achieved. The resulting strain yielded more than 10-fold higher β-galactosidase activity in soybean root nodules as compared to a B. japonicum strain carrying the same lacZ fusion on a pRK290-based plasmid.