Suppressors of a temperature-sensitive copy-number mutation in plasmid NTP1

A temperature-sensitive high copy-number mutant of plasmid NTP1, first described by Grindley et al. (1978), is lethal to bacterial cells at the non-permissive temperature. This behavior has been used to select mutations in the plasmid replication origin region that suppress the over-replication phenotype. We have identified the site of the original ts lethal mutation and the positions of the reversion mutations. The ts mutation, designated orp, for over-replication, is a single nucleotide change 23 base-pairs upstream from the transcription start site for RNA I, the repressor of plasmid replication. This change simultaneously affects the promoter for RNA I and the precursor of the primer for plasmid replication, RNA II, which is also transcribed from this region. Fusions of the mutant promoter region with the galK gene indicate that transcription is not temperature sensitive. This result suggests that the mutation affects RNA II secondary structure. The reversion mutations are also located within the RNA II coding region more than 200 bp from the site of the original ts mutation. These mutations may also affect RNA II structure.