Figure 1.

Specific mutations in dnaA either bypass or suppress the inhibition of DNA replication initiation by Soj^G12V. (A) Pathway of the Soj activity cycle. (B) Point mutations in DnaA introduced by error-prone PCR were found to overcome the small colony phenotype characteristic of Soj^G12V overexpression. Strains were grown on NA plates in the presence or absence of 1% xylose to induce soj^G12V expression. Wild-type (HM524), DnaA^L294R (HM527), DnaA^V323D (HM528), DnaA^L337P (HM529), DnaA^A341V (HM530). (C) The oriC-to-terminus ratios of dnaA point mutations generated using PCR mutagenesis were determined using MFA in the presence and absence of Soj^G12V overexpression (1% xylose). Suppressor mutations (red) were found to be recalcitrant to Soj^G12V activity. Cells were grown in LB medium at 30°C. Values were normalized to the ori:ter ratio of the wild-type strain grown in the absence of xylose. DnaA^V121A (HM713), DnaA^A131T (HM714), DnaA^A132T (HM710), DnaA^G151R (HM705), DnaA^G154S (HM706), DnaA^H162Y (HM707), DnaA^R281G (HM708), DnaA^N311D (HM712), DnaA^N311T (HM709), DnaA^E314G (HM711). (D) The Soj^G12V suppressor mutations in dnaA perturb the formation of a Soj:DnaA–His₁₂ complex in vivo. Cells were grown in LB medium at 30°C, crosslinked with formaldehyde, and the DnaA–His₁₂ complexes were purified before the crosslinks were reversed and proteins were separated by SDS–PAGE. Soj and DnaA–His₁₂ were detected by western blot analysis. The top panel shows the amount of Soj protein in the cell lysate (Input) and the bottom panel shows the amount of Soj found in a complex with DnaA–His₁₂ following purification (Complex). DnaA–His₁₂ (HM657), DnaA^L294R–His₁₂ (HM716), DnaA^V323D–His₁₂ (HM555), DnaA^L337P–His₁₂ (HM658), DnaA^A341V–His₁₂ (HM725). (E) The amount of each DnaA^Sup–His₁₂ protein was determined by western blot analysis. DivIVA was used as a loading control.