Data are presented suggesting that the most critical factor determining whether an Hfr dnaAts strain can synthesize deoxyribonucleic acid and form colonies at temperatures that are nonpermissive for corresponding F- strains is neither the site of insertion of F nor the presence of additional mutations in the F particle or the chromosome; it is whether the particle is capable of autonomous replication at the temperature used. Consequently, suppression of the DnaA phenotype in Hfr strains occurs at 40 C but not, in most of them, at 42 C without the occurrence of additional mutations. The site of insertion of F may also be important since it is shown that in one Hfr dnaA strain partial suppression does occur at 42 C. In addition, it is shown that strains exhibiting suppression by integration of F at 40 C on minimal agar plates do not do so at this temperature on nutrient agar plates.

The pattern of segregation of DNA in Escherichia coli K-12 was analyzed by labeling replicating DNA with 5-bromodeoxyuridine followed by differential staining of nucleoids. Three types of visible arrangement were found in four-nucleoid groups derived from a native nucleoid after two replication rounds. Type A, segregation of both old strands toward cell poles, appeared with the highest frequency (0.6 to 0.8). Type B, segregation of one old strand toward the cell pole and the other toward the cell center, was twice as frequent as type C, segregation of both old strands toward the cell center. These results confirm previous data showing that DNA segregation in E. coli is nonrandom while presenting a certain degree of randomness. The proportions of the three indicated types of arrangement suggest a new probabilistic model to explain the observed segregation pattern. It is proposed that DNA strands segregate either nonrandomly, with a probability of between 0 and 1, or randomly. In nonrandom segregation, both old strands are always directed toward cell poles. Experimental data reported here or by other authors fit better with the predictions of this model than with those of other previously proposed deterministic or probabilistic models.

Cell size and DNA concentration were measured in Escherichia coli K-12 ET64. This strain carries a dnaA(Ts) mutation that has been suppressed by the insertion of the F plasmid into the chrontosome. ET64 can grow in a balanced steady state of exponential growth at the restrictive temperature for its dnaA allele (39°C), in which chromosome replication is controlled by the F plasmid, and at the permissive temperature (30°C), in which chromosome replication is controlled by dnaA-oriC. When cells grown at the indicated temperatures were compared, it was observed that at 39°C, the cell mass increased and the amount of cellular DNA decreased slightly; therefore, the DNA concentration was strongly'reduced. These changes can neither be explained by the reduction of the generation time (which is only 10-15%) nor from observed changes in the replication time and in the time between DNA synthesis termination and cell division. Variations were mainly due to the increase in cell mass per origin of replication, at initiation, in cells grown at 39°C. Control of chromosome replication by the F plasmid appears to be the reason for the increase in the initiation mass. Other possible causes, such as the modification of growth temperature, the generation time, or both, were discarded. These observations suggest that at one growth rate, the F plasmid replicates at a particular cell mass to F particle pumber ratio, and that this ratio is higher than the cell mass to oriC ratio at the initiation of chromosome replication. This fact might be significant to coordinate the replication of two different replicons in the same cell.Replication of the chromosome of Escherichia coli is regulated at the step of initiation. Replication is normally initiated at a fixed origin, oriC (2), and proceeds bidirectionally to the terminus region of the chromosome. Dependence of initiation at oriC on dnaA product is absolute (8,22). However, the dnaA mutation can be suppressed by the insertion of a plasmid into the bacterial chromosome (11,13,21). There is evidence that such suppressed strains initiate replication at the plasmid site (3, 11, 13), which is in apparent contradiction with the failure of initiation at the plasmid site when integrated in dnaA+ strains. A model for the control of replication that explains these facts has been proposed by Pritchard et al. (17,19). This model states that replicons are controlled by their own negatively acting gene products. Initiation of replication raises the inhibitor concentration, reducing the probability of further initiations; growth of the host cell between two rounds of replication progressively reduces inhibitor concentration, therefore increasing the probability of initiation. A mechanism of this type has been demonstrated to control the replication of several bacterial plasmids, e.g., ColEl and Ri (10,12,18 Kingdom). Samples (1 ml) were added to 1 ml of 10% trichloroacetic acid and kept on ice for at least 1 h. The precipitates were collected on Whatman GF/F filters, washed with hot water, air dried, a...