Characterization of dnaC2 and dnaC28 Mutants by Flow Cytometry

Escherichia coli strains containing thermosensitive dnaC alleles were studied by flow cytometry. Strains containing either the dnaC2 or dnaC28 allele were shifted between different temperatures, and DNA content distributions were gathered. Inhibition of initiation of chromosome replication at nonpermissive temperature, as well as reinitiation of replication at permissive temperature, were found to be affected by a number of parameters. These included the choice of permissive and nonpermissive temperatures, the length of the time of incubation at the nonpermissive temperature, the growth medium, the type of temperature shift used for reinitiation of replication (transient or nontransient), the genetic background of the host cell, and the cell concentration. Reinitiation of replication required neither transcription nor translation, whereas the elongation stage of replication was dependent upon ongoing protein synthesis in the mutants. Efficient use of dnaC mutants for cell cycle studies is discussed.