The sequence requirements for a functional Escherichia coli replication origin are different for the chromosome and a minichromosome

We have developed a simple three-step method for transferring oriC mutation s from plasmids to the Escherichia coli chromosome. Ten oriC mutations were used to replace the wild-type chromosomal origin of a recBCsbcB host by re combination. The mutations were subsequently transferred to a wild-type hos t by transduction, oriC mutants with a mutated DnaA box R1 were not obtaine d, suggesting that R1 is essential for chromosomal origin function. The oth er mutant strains showed the same growth rates, DNA contents and cell mass as wild-type cells. Mutations in the left half of oriC, in DnaA boxes M, R2 or R3 or in the Fis or IHF binding sites caused moderate asynchrony of the initiation of chromosome replication, as measured by flow cytometry, In mu tants with a scrambled DnaA box R4 or with a modified distance between DnaA boxes R3 and R4, initiations were severely asynchronous. Except for oriC14 and oriC21, mutated oriCs could not, or could only poorly, support minichr omosome replication, whereas most of them supported chromosome replication, showing that the classical definition of a minimal oriC is not valid for c hromosome replication. We present evidence that the functionality of certai n mutated oriCs is far better on the chromosome than on a minichromosome.