MOLECULAR ANALYSIS AND CHARACTERIZATION OF A BROAD-HOST-RANGE PLASMID, PEP2

Plasmid pEP2 was found to encode a protein, RepA, which is essential a nd rate limiting for its replication in Escherichia coil and Corynebac terium pseunotuberculosis. Mutations which altered the rate of synthes is of this protein in E. coil affected the copy number and segregation al stability of pEP2 in the two hosts. RepA contains 483 amino acid re sidues and has the calculated molecular weight of 53,925. It shows 45% amino acid residue identity with open reading frame ORF2 of pSR1, a p lasmid isolated from Corynebacterium glutamicum (J. A. C. Archer and A . J. Sinskey, J. Gen. Microbiol. 139:1753-1759, 1993). Plasmid pEP2 wa s shown to accumulate single-stranded DNA corresponding to the RepA co ding strand during its replication in E. coli and C. pseudotuberculosi s, suggesting that it may replicate by a rolling circle mechanism. How ever, RepA has no significant sequence homology with the replication i nitiator proteins of plasmids known to use this mode of replication.