CHARACTERIZATION OF THE REPLICATION REGION OF THE BACILLUS-SUBTILIS PLASMID PLS20 - A NOVEL TYPE OF REPLICON

A 3.1 kb fragment of the large (similar to 55 kb) Bacillus subtilis pl asmid pLS20 containing all the information for autonomous replication was cloned and sequenced, In contrast to the parental plasmid, derived minireplicons were unstably maintained, Using deletion analysis the f ragment essential and sufficient for replication was delineated to 1.1 kb. This 1.1 kb fragment is located between two divergently transcrib ed genes, denoted orfA and orfB, neither of which is required for repl ication. orfA shows homology to the B.subtilis chromosomal genes rapA (spoOL, gsiA) and rapB (spoOP), The 1.1 kb fragment, which is characte rized by the presence of several regions of dyed symmetry, contains no open reading frames of more than 85 codons and shows no similarity wi th other known plasmid replicons. The structural organization of the p LS20 minimal replicon is entirely different from that of typical rolli ng circle plasmids from Gram-positive bacteria, The pLS20 minireplicon s replicate in polA5 and recA4 B.subtilis strains, Taken together, the se results strongly suggest that pLS20 belongs to a new class of theta replicons.