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) from the Rhizobium meliloti symbiotic megaplasmid pSym-b was isolated by selecting for sequences that allowed a normally nonreplicative pBR322 derivative to replicate in R. meliloti. The resulting Escherichia coli-R. meliloti shuttle plasmid (mini-pSym-b) containing the ARS also replicated in the closely related Agrobacterium tumefaciens, but only in strains carrying pSym-b, suggesting that a megaplasmid-encoded trans-acting factor is required. The copy number of mini-pSym-b was approximately the same as that of the resident megaplasmid, and mini-pSym-b was unstable in the absence of antibiotic selection. An 0.8-kb DNA subfragment was sufficient for replication in both R. meliloti and A. tumefaciens. The minimal ARS exhibited several sequence motifs common to other replication origins, such as an AT-rich region, three potential DnA binding sites, a potential 13-mer sequence, and several groups of short direct repeats. Hybridization experiments indicated that there may be a related ARS on the other megaplasmid, pSym-a. The pSym-b ARS was mapped near exoA, within a region nonessential for pSym-b replication. These results suggest that the R. meliloti megaplasmids share conserved replication origins and that pSym-b contains multiple replication 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 compatible plasmids for cloning and genetic manipulation in R. meliloti.
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Department of Biological Sciences, Stanford University, California 94305-5020.
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D016960:Agrobacterium tumefaciens / Q000235:genetics; D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics; D008040:Genetic Linkage; D008969:Molecular Sequence Data; D010957:Plasmids; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D016962:Sinorhizobium meliloti / Q000235:genetics*
D004269:DNA, Bacterial