Release of initiation control by a mutational alteration in the R6K pi protein required for plasmid DNA replication.

Plasmid pRK419, a derivative of the naturally occurring antibiotic resistance plasmid R6K, contains the pir gene that codes for the pi initiation protein and the beta and gamma replication origins of R6K. A mutation in plasmid pRK419, designated cos405, results in an elevated plasmid copy number in Escherichia coli growing at 42 degrees C and an even greater increase in copy number when the cells are shifted to 30 degrees C. This mutation was assigned to the structural gene for the pi protein on the basis of suppression of the mutant phenotype in E. coli when the wild-type pi protein is supplied in trans. Nucleotide sequence analysis of the cos405 mutant confirmed the pir gene location of the mutation and showed that this mutation results in a single amino acid substitution (glycine to aspartic acid) at the 81st position of the 305-amino acid pi protein. The properties of this mutant suggest that the pi protein plays a role in the negative control of the frequency of R6K initiation in addition to its requirement for the initiation of plasmid replication.