CONSTRUCTION AND CHARACTERIZATION OF A RHIZOBIUM-LEGUMINOSARUM BIOVARVICIAE STRAIN DESIGNED TO ASSESS HORIZONTAL GENE-TRANSFER IN THE ENVIRONMENT

An integration vector was developed which inserts cloned DNA in a non- essential site of the Rhizobium leguminosarum biovar viciae chromosome . The expression of integrated genes is under the control of the const itutive neomycin phosphotransferase II (nptII) promotor of transposon Tn5. The design of the vector ensures that loss of vector sequences ca n be detected, enabling selection of progeny containing only the requi site DNA. The newly constructed vector was employed to insert the Esch erichia coli gusA gene conferring GUS activity into R. leguminosarum b y. viciae strain LRS39401 which is cured of its symbiotic plasmid (pSy m). One GUS-positive transconjugant, strain CT0370, was shown to have lost all vector sequences. Conjugal transfer of pSym2004 (a Tn5-tagged derivative of symbiotic plasmid pRL1JI, which specifies pea nodulatio n and symbiotic nitrogen fixation) to CT0370, restored the GUS-positiv e strain's symbiotic proficiency. Strain CT0370 is presently being use d in a field release experiment in order to assess the extent of pSym transfer in a natural R. leguminosarum by. viciae population under env ironmental conditions.