ISOLATION AND CHARACTERIZATION OF A DNA-REPLICATION ORIGIN FROM THE 1,700-KILOBASE-PAIR SYMBIOTIC MEGAPLASMID PSYM-B OF RHIZOBIUM-MELILOTI

A 4-kb fragment active as an autonomously replicating sequence (ARS) f rom the Rhizobium meliloti symbiotic megaplasmid pSym-b was isolated b y selecting for sequences that allowed a normally nonreplicative pBR32 2 derivative to replicate in R. meliloti. The resulting Escherichia co li-R. meliloti shuttle plasmid (mini-pSym-b) containing the ARS also r eplicated in the closely related Agrobacterium tumefaciens, but only i n strains carrying pSym-b, suggesting that a megaplasmid-encoded trans -acting factor is required. The copy number of mini-pSym-b was approxi mately the same as that of the resident megaplasmid, and mini-pSym-b w as unstable in the absence of antibiotic selection. An 0.8-kb DNA subf ragment was sufficient for replication in both R. meliloti and A. tume faciens. The minimal ARS exhibited several sequence motifs common to o ther replication origins, such as an AT-rich region. three potential D naA binding sites, a potential 13-mer sequence. and several groups of short direct repeats. Hybridization experiments indicated that there m ay be a related ARS on the other megaplasmid, pSym-a. The pSym-b ARS w as mapped near exoA, within a region nonessential for pSym-b replicati on. These results suggest that the R. meliloti megaplasmids share cons erved replication origins and that pSym-b contains multiple replicatio n origins. Since the mini-pSym-b shuttle vector can coexist with IncP- 1 broad-host-range plasmids, it is also now possible to use two compat ible plasmids for cloning and genetic manipulation in R. meliloti.