Mutations in the E. coli Rep helicase increase the amount of DNA per cell

The mbrA4 mutation confers camphor resistance, severe growth defects and up to a two-fold increase in the amount of chromosomal DNA per cell. The extra DNA is replicated from oriC in a synchronous fashion. Cells containing mbrA4 are more resistant to X-rays, indicating that the extra DNA represents complete or nearly complete chromosomes. I report here that mbrA4 is an unusual allele of the leading strand DNA helicase, Rep. Eight independently isolated alleles of rep(mbrA) contain the same three changes in the rep gene: a G to A at position -44 from the start of the mRNA (+1); an opal stop at codon 142; and a glycine to serine at codon 414 (G414S). My data indicate that rep(mbrA4) is not a null mutation and that the third mutation, G414S, is necessary for camphor resistance, the phenotype associated with increased DNA content per cell. I also show that increase in DNA content does not lead to independently segregating chromosomes.