CHARACTERIZATION OF A REGION OF PLASMID PBL1 OF BREVIBACTERIUM-LACTOFERMENTUM INVOLVED IN REPLICATION VIA THE ROLLING CIRCLE MODEL

The minimal region for autonomous replication of pBL1, a 4.5-kb crypti c plasmid of Brevibacterium lactofermentum ATCC 13869 that has been us ed to construct a variety of corynebacterium vectors, was shown to be contained on a 1.8-kb HindII-SphI DNA fragment. This region contains t wo open reading frames (ORFs) (ORF1 and ORF5) which are essential for pBL1 replication in B. lactofermentum, Accumulation of single-strand i ntermediates in some of the constructions indicates that plasmid pBL1 replicates via the rolling circle replication model; its plus strand a nd minus strand were identified by hybridization with two synthetic ol igonucleotide probes complementary to each pBL1 strand. ORF1 seems to encode the Rep protein and showed partial homology with sequences for Rep proteins from Streptomyces plasmids which replicate via rolling ci rcle replication such as pIJ101, pSB24, and pJV1.