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Release of initiation control by a mutational alteration in the R6K pi protein required for plasmid DNA replication
Abstract:
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.
Insights
A mutation in the pi protein of plasmid R6K (cos405) increases its copy number in E. coli. This suggests the pi protein regulates plasmid replication initiation frequency.
Area of Science:
- Molecular Biology
- Microbiology
- Genetics
Background:
- Plasmid R6K is a naturally occurring antibiotic resistance plasmid.
- Plasmid pRK419, a derivative of R6K, contains the pir gene encoding the pi initiation protein and R6K replication origins.
- The pir gene product is essential for R6K replication initiation.
Purpose of the Study:
- To investigate the function of the pi initiation protein in plasmid R6K replication.
- To characterize a mutation (cos405) affecting plasmid copy number.
Main Methods:
- Plasmid manipulation and propagation in Escherichia coli.
- Temperature shift experiments to assess copy number variations.
- Complementation assays to determine mutation location.
- Nucleotide sequencing to identify the specific mutation.
Main Results:
- The cos405 mutation in pRK419 leads to increased plasmid copy number in E. coli at 42°C, with a more significant increase at 30°C.
- The mutant phenotype was suppressed by providing wild-type pi protein in trans, confirming the mutation is in the pir gene.
- Sequence analysis revealed a single amino acid substitution (Gly81Asp) in the pi protein.
Conclusions:
- The cos405 mutation affects the pi protein's function.
- The pi protein is involved in the negative control of R6K replication initiation frequency, beyond its essential role in initiation.
- This finding provides new insights into the regulatory mechanisms of plasmid replication.