Figure 2.

MreB interacts directly with FtsZ and this interaction is abolished in the MreB^D285A variant. (A) Bacterial two-hybrid (BTH) screen of E. coli cell division factors against MreB. FtsZ has been labelled in both the N-terminus (N) and C-terminus (c). An MreB variant with a 9 aa N-terminal truncation (ΔN) is also included in this screen. Both T25- and T18-fusion orientations are shown for each BTH combination (i) and (ii). (B) BTH plate showing full-length T18-labelled MreB and MreB^D285A against T25-labelled FtsZ (c) and known MreB interaction partners essential for cell elongation. The BTH101 strain was used for all BTH cloning, a full list of plasmid vectors used for this screen can be found in Supplementary Table SI. Cells were spotted onto NA plates containing ampicillin (50 μg/ml), kanamycin (25 μg/ml) and X-gal (40 μg/ml). Plates were incubated for 48 h at 30°C prior to imaging. (C) BTH β-galactosidase activity assays showing T18-labelled MreB and D285A variant against T25-labelled FtsZ and MreB^wt. Error bars=s.d., n=4. In all cases, BTH −ve control strains contained empty pKNT25 and pUT18 vectors and +ve control strains contained the pKT25-zip and pUT18C-zip fusions. (D) Western blots of His-tagged MreB and MreB^D285A formaldehyde crosslink and pull-down preparations, using both anti-FtsZ and anti-MreB primary antibodies. MC1000 cells containing either pTK500 (P_lac::His₈–mreB) or pAKF126 (P_lac::His₈–mreB^D285A) were grown to an OD₆₀₀ of ≈0.3 without induction, crosslinked with 1% formaldehyde and quenched after 10 min using 150 mM glycine. Resulting crosslinked complexes were affinity purified using the MagneHis^TM suspension, crosslinks were reversed by incubation at 95°C for 1 h and complexes analysed. Equal volumes of protein complex preparations, un-crosslinked His–MreB and His–MreB^D285A preparations and purified His–MreB (200 ng) and native FtsZ (100 ng) proteins were used as controls, n=4. MreB and FtsZ antibody affinity control western blots are shown in Supplementary Figure S3B.