Mol Gen Genet (1991) 229:175-180 002689259100292C © Springer-Verlag 1991 Studies on the alteration of chromosome copy number and cell division potential in a dnaA mutant of Escherichia coli Joe A. Fralick Dept. Microbiology,TexasTech UniversityHealth SciencesCenter, Lubbock,TX 79430, USA Received January 21, 1991 / April 23, 1991 Summary. The dnaA167 mutant of Escherichia coli, N167, maintains, on the average, two replicating chro- mosomes per cell at the perimissive growth temperature of 30° C and only one per cell at the higher permissive growth temperature of 38° C. When the growth tempera- ture of this mutant is changed from 30° to 38° C the cells rapidly readjust their chromosome copy number from two to one. I have examined the kinetics of this transition with reference to DNA replication and cell division. My results indicate that this mutant uncouples cell division from chromosome duplication to achieve the appropriate copy number, suggesting that the dnaA gene product may be involved in the coordination be- tween these two cellular events. Key words: Escherich& coli- Chromosome copy number Introduction The means by which bacteria regulate their chromosome copy number is unknown. However, it seems likely that this parameter is influenced by the regulation of the initi- ation of chromosome replication. In a previous study, using a temperature-sensitive, initiation-defective, dnaA mutant of Escherichia coli, it was shown that chromo- some copy number could be altered in the mutant by growth temperature (Fralick 1978). In that study it was demonstrated that under balanced growth conditions the dnaA167 mutant contained two independently replicat- ing chromosomes per cell when grown in a glucose-Casa- mino acids medium at 30° C and only one per cell when grown in the same medium at 38° C. At both growth temperatures each chromosome contained a single pair of replication forks and the time interval between succes- sive initiation events (initiation pace) was normal as compared with the dnaA + parental strain. The effect of growth temperature on copy number was attributed to a decreased activity or availability of the labile dnaA gene product of this mutant at the higher "permissive" growth temperatures (35°-38 ° C). These results were in- terpreted to indicate that the dnaA gene product is some- how involved in the structure and/or assembly of the "initiation-replication aparatus" and as such may influ- ence the stoichiometry of chromosome replication (i.e. chromosome copy number capacity). Since that time it has been demonstrated that the DnaA protein binds co- operatively to several sites within oriC, resulting in the successive opening and entry of the DnaB-DnaC pre- priming complex into the replicative origin of E. coli (Bramhill and Kornberg 1988). The dnaA gene product has also been shown to be involved in the regulation of numerous genes including the ftsA and ftsQ genes whose products are involved in cell division (Aldea et al. 1990). In the present study I have examined the means by which the dnaA167 mutant regulates its chromosome copy number. It has been found that this mutant can rapidly readjust its copy number from two to one, fol- lowing a shift in growth temperature from 30°C to 38° C. The kinetics of this change in chromosome con- tent have been followed with respect to DNA replication and cell division. The results indicate that the alteration in copy number is brought about via a temporal halt in initiation of chromosome replication and an uncou- pling of chromosome duplication from cell division. Materials and methods Bacterial strains and growth conditions. Th E. coli K12 strain used, N167, was generously provided by Dr. J. Tomizawa and has been well characterized as a dnaA temperature-sensitive, conditionally lethal mutant un- able to initiate new rounds of chromosome replication at the nonpermissive temperature (Abe and Tomizawa 1971 ; Hiraga and Saitoh 1974). In our hands the restric- tive temperature lies between 42° and 43°C (Fralick 1978). Cells were routinely grown in a New Brunswick gyra- tory water bath (Aquatherm G86) at 200 rev/min. The