REVERSIBILITY OF DNAA PROTEIN-ACTIVITY IN THE IRREVERSIBLE DNAA204 MUTANT OF ESCHERICHIA-COLI

The dnaA204 mutant, one of the so-called irreversible dnaA mutants whi ch cannot reinitiate chromosome replication upon a shift from non-perm issive to permissive growth temperature in the absence of protein synt hesis, was reinvestigated using flow cytometry and marker frequency an alysis, In a temperature downshift experiment and in the presence of p rotein synthesis the dnaA204 mutant reinitiates chromosome replication very fast, Using a lac promoter-controlled wild type or a dnaA204 mut ant gene carried on a plasmid, we have observed instantaneous initiati on of replication when synthesis of DnaA protein is induced in the dna A204 mutant at 42 degrees C, The data indicate that the dnaA204 mutant after a shift to 42 degrees C still contains functional DnaA protein, but that the activity level is below the initiation threshold. Thus, after synthesis of very small amounts of additional DnaA protein, init iation occurs very fast both after a shift to 30 degrees C, and after induction of DnaA protein synthesis at 42 degrees C. A model describin g the processing of DnaA protein in mutants and in the wild type is pr esented.