Figure 2.

DnaE is required at the replication fork to synthesize nascent DNA. (a) Schematic representation of the tested strains. Strains DGRM821, 824–825 and 827 encode the DnaE2.6 thermosensitive form of DnaE from the natural locus and a WT or inactive (D > A) form of DnaE from the IPTG-inducible promoter Pspank at the amyE locus. Star: Ts mutation; dotted boxes: 5′ and 3′ regions of the inactivated amyE locus. (b) Complementation assay. Strains were grown in LB at 30°C and plated on a solid broth in the presence and absence of 1 mM IPTG, as indicated. The concentration of cell forming unit (CFU ml⁻¹) was determined at various temperatures, as indicated. (c) The ectopic copy of dnaE was fused to a tag (SPA) at the C-terminus (DGRM830-833) for Western blot detection. Expression of the DnaED > A protein was examined by Western blot, 2 h after IPTG addition (ii), and total proteins were visualized using Sypro red staining (i). (iii) IPTG dependent accumulation DnaED > A and its effect on cell growth at 30°C. Cells were grown exponentially in LB at 30°C. At OD_{600 nm} of approximately 0.002, IPTG (0, 25, 200 and 1,000 µM) was added and cell growth was followed spectrophotometrically. The three DnaED > A mutated strains gave similar results (presented data are with the dnaED382A allele).