Initiation of chromosomal DNA replication which is stimulated without oversupply of DnaA protein in Escherichia coli

Abstract

The temperature-sensitive dnaA46 mutation in Escherichia coli can be phenotypically suppressed at 42 degrees C by oversupply of GroELS proteins, and the suppressed cells grow extremely slowly at 30 degrees C. We found that the phenotype of dnaA46 showing this cold sensitivity was dominant over the phenotype of dnaA+, and could not be rescued by introduction of oriC-independent replication systems. These results suggest that the cold sensitivity was not caused by a simple defect in replication. When a growing culture of a dnaA46 strain with a GroELS-overproducing plasmid was shifted from 42 degrees to 30 degrees C in the presence of chloramphenicol, the chromosomal DNA replicated excessively. Initiation of replication occurred at the site of oriC repeatedly four or five times during a 4 h incubation period without concomitant protein synthesis, indicating an excessive capacity for initiation. Such overreplication did not take place at 42 degrees C in the suppressed dnaA46 strain, or at either temperature in GroELS-oversupplied dnaA+ cells. No significant difference was detected between the cellular content of DnaA protein in suppressed cells where the initiation capacity was abnormally high, and that in wild-type cells in which the initiation capacity was normal. Thus, DnaA protein might function in vivo through some phase control mechanism for initiation, apart from a simple regulation by its total amount. A possible mechanism is proposed based on the participation of GroELS proteins in protein folding.