Figure 3.

The MreB^D285A variant inhibits cell division but not cell elongation. (A) Representative brightfield micrographs showing the MC1000 MreBCD depletion strain (MC1000 Δmre, pKG339, pTK554). Images show cells without MreB depletion ‘un-induced’, depleted of MreB ‘depleted’ or complemented with either mreBCD or mreB^D285ACD expressed from pAKF128 (P_BAD::mreBCD) or pAKF129 (P_BAD::mreB^D285ACD) ‘replaced’. Cells were grown in LB +tetracycline (20 μM), 2 mM IPTG and 0.2% arabinose for 3 h at 30°C prior to imaging. See Supplementary Figure S4A for diagram of depletion system. (B) MC1000 cells containing pAKF128 or pAKF129. Cells were grown in M9 medium with 0.000,05% arabinose inducer at 30°C for 5 h. (i) Representative bright field micrographs of cells with and without induction (ii) Box and Whisker plot showing cell length measurements with mreBCD and mreB^D285ACD ‘overexpression’ compared to an empty vector control (pBAD24). Central line=mean length, box=± s.d. and whiskers=maximum/minimum lengths observed. *t-test=P>0.01, ^o=no significant difference between populations. 670 cells were sampled for each condition, n=3. For cell width measurements and a full series of control images, see Supplementary Figure S4C and D. (C) Representative brightfield and fluorescent micrographs of MC1000 carrying pAKF131 (P_BAD::mypet–mreBCD; left panel) or pAKF132 (P_BAD::mypet–mreB^D285ACD; right panel), n=3. Note the septal localisation of the mYpet–MreB versus the punctate localisation pattern of mYpet–MreB^D285A. (D) mYpet–MreB (pAKF131) and mYpet–MreB^D285A (pAKF132) colocalise with the MreB–RFP^SW in the FB76 strain. Arrows have been used to highlight selected colocalising foci, n=3. Scale bars=5 μm (A, B) and 2 μm (C, D).