Plasmid pL32 from the Natto strain of Bacillus subtilis belongs to a group of low-copy-number plasmids in gram-positive bacteria that replicate via a theta mechanism of replication. We studied the DNA region encoding the replication protein, RepN, of pLS32, and obtained the following results. Transcription of the repN gene starts 167 nucleotides upstream from the translational start site of repN. The copy number of repN-coding plasmid pHDCS2, in which the repN gene was placed downstream of the IPTG (isopropyl-1-thio--D-galactopyranoside)-inducible Pspac promoter, was increased 100 fold by the addition of IPTG. Histidine-tagged RepN bound to a specific region in the repN gene containing five 22-bp tandem repeats (iterons) with partial mismatches, as shown by gel retardation and foot printing analyses. Sequence alterations in the first three iterons resulted in an increase in plasmid copy number, whereas those in either the forth or fifth iteron resulted in the failure of plasmid replication. The iterons expressed various degrees of incompatibility with an incoming repN-driven replicon pSEQ243, with the first three showing the strongest incompatibility. Finally, by using a plasmid, pHDMAEC21, carrying the sequence alterations in all the five iterons in repN and thus unable to replicate but encoding intact RepN, the region necessary for replication was confined to a 96-bp sequence spanning the 3-terminal half of the fourth iteron to an A؉T-rich region located downstream of the fifth iteron. From these results, we conclude that the iterons in repN are involved in both the control of plasmid copy number and incompatibility, and we suggest that the binding of RepN to the last two iterons triggers replication by melting the A؉T-rich DNA sequence.Circular plasmids can be grouped into two classes by the mode of replication. One group replicates via a rolling-circle intermediate, while the other uses the theta-type intermediate. In the case of plasmids from natural isolates of Bacillus subtilis, it has been demonstrated that the sizes of the plasmids belonging to the first group are small, while those belonging to the second group are large (37). The small plasmids of B. subtilis are further grouped into seven classes based on their sizes and restriction patterns and show a replication function related to that of pC194 derived from Staphylococcus aureus (26). It remains unknown why the only pC194-type replicon is found in B. subtilis among rolling-circle replicons with different types of replication functions (13,18,20). On the other hand, plasmids that replicate via a theta mechanism of replication are classified into six groups (36); in addition, a recently reported plasmid, pBS72, carries a replicon of a new type (37). The large plasmids found in natural isolates of B. subtilis show diverse modes of theta replication, as exemplified by pLS20 (25), pLS32 (36), and pBS72 (37). Whereas the replication functions are unique for pLS20 and pBS72, several plasmids, including pLS32 and their relatives (see below), show amino a...