We have developed a procedure to directly clone large fragments from the genome of the soil bacterium
Sinorhizobium meliloti. Specific regions to be cloned are first flanked by parallel copies of an origin of transfer ( oriT) together with a plasmid replication origin capable of replicating large clones in Escherichia colibut not in the target organism. Supplying transfer genes in trans specifically transfers the oriT-flanked region, and in this process, site-specific recombination at the oriTsites results in a plasmid carrying the flanked region of interest that can replicate in E. colifrom the inserted origin of replication (in this case, the F origin carried on a BAC cloning vector). We have used this procedure with the oriTof the plasmid RK2 to clone contiguous fragments of 50, 60, 115, 140, 240, and 200 kb from the S. melilotipExo megaplasmid. Analysis of the 60-kb fragment allowed us to identify a 9-kb region capable of autonomous replication in the bacterium Agrobacterium tumefaciens. The nucleotide sequence of this fragment revealed a replicator region including homologs of the repA, repB, and repCgenes from other Rhizobiaceae, which encode proteins involved in replication and segregation of plasmids in many organisms.