Reversibility of DnaA protein activity in the‘irreversible’dnaA204 mutant of Escherichia coli

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Abstract
The dnaA204 mutant, one of the so-called irreversible dnaA mutants which cannot reinitiate chromosome replication upon a shift from non-permissive to permissive growth temperature in the absence of protein synthesis, was reinvestigated using flow cytometry and marker frequency analysis. In a temperature down-shift experiment and in the presence of protein synthesis the dnaA204 mutant reinitiates chromosome replication very fast. Using a lac promoter-controlled wild type or a dnaA204 mutant gene carried on a plasmid, we have observed instantaneous initiation of replication when synthesis of DnaA protein is induced in the dnaA204 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, initiation occurs very fast both after a shift to 30 degrees C, and after induction of DnaA protein synthesis at 42 degrees C. A model describing the processing of DnaA protein in mutants and in the wild type is presented.