Figure 4. Experimental approach and separation of nucleoids after one round of replication, prior to production of FtsZ.

(A) Diagram of experimental approach (see Materials and Methods for complete details). Spores of the dnaB (ts) strains containing P_spac-ftsZ and P_xyl-ftsZ-yfp [strains SU671 (Min⁺, Noc⁺), SU678 (Min⁻), SU680 (Min⁻, Noc⁻), and SU683 (Noc⁻)] were germinated in GMD at 34°C and then transferred to 48°C to prevent re-initiation of DNA replication and allow separation of replicated nucleoids. IPTG (1 mM) and xylose (0.01%) were then added. Cells were removed and prepared for live visualization of Z rings and nucleoids. (B) Representative images of Z rings and nucleoids in the wild-type control strain SU492 (DnaB⁺). Spores of this strain were germinated in GMD with 0.01% xylose at 34°C for 105 min, transferred to 48°C for 30 min and then collected for analysis. These conditions differ from that of the test strains to allow visualization of Z rings in the first round of replication. (C to F) Representative images of cells of the dnaB (ts) strains containing two separated nucleoids, collected prior to induction of ftsZ expression with IPTG, when both MinCD and Noc are present (C), when both MinCD and Noc are absent (D), when MinCD is absent (E), when Noc is absent (F). Images are DAPI (nucleoid) pseudo-coloured in red (left), FtsZ-YFP pseudo-coloured in green (middle) and phase-contrast fluorescence overlay (right). Stars denote autofluorescent spore coats. Scale bars are 2 µm.