Characterization of the replication region of plasmid pLS32 from the Natto strain of Bacillus subtilis.

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-beta-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.