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The CloDF13 cop-l(Ts) mutant expresses a temperature-dependent plasmid copy number. At 42°C the mutant shows a "runaway" behavior, and cells harboring this plasmid are killed. The cop-l(Ts) mutation is a G-to-A transition that disturbs one of the two methylation sites which are located opposite in the stem-loop structure within a region involved in both the initiation of primer synthesis for DNA replication and the termination of the cloacin operon transcript. We demonstrate that the mutation results in an increased primer (RNA II) synthesis resulting from nonconditional enhanced RNA II promoter activity, which at 42°C causes a decrease in the amount of active replication repressor molecules (RNA I) synthesized from the opposite strand. We found that the absence of Dam methylation abolishes the mutant phenotype and that under this condition the high mutant level of RNA H synthesis is reduced, which is accompanied by a restoration of the regulation by RNA I. The role of methylation in the regulation of plasmid replication is discussed.

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Mobilization of the non-conjugative plasmid Clo DF13 requires both gene products of a conjugative plasmid and Clo DF13 encoded proteins as well as a cis-acting Clo DF13 DNA region, termed bom (basis of mobility). The bom region was located within a 264 bp fragment around the unique HpaI site. Comparison with the corresponding ColE1 and pBR322 sequences showed similarities with respect to the secondary structure. With respect to a possible relationship between the origin of vegetative replication (oriV) and the origin of transfer (oriT), we found that neither distance nor orientation of the Clo DF13 bom region with respect to oriV had any significant influence on the mobilization frequency. Surprisingly, after cloning of the 264 bp HpaII fragment in a bom- vector, restoration of the bom+ phenotype was only observed in one orientation. From these observations and from the sequence analysis of the bom region we suggest that transcription into this cloned bom fragment is essential for effective mobilization of the plasmid.

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