PMC

The growth twist of E. coli depends on the MreB cytoskeleton. (A) DIC images of an FtsZ-depleted cell grown at room temperature with A22. Scale bar represents 5 μm. (B) The circumferential and longitudinal distance between the beads are linearly related during a phase of twisting elongation until twisting ceases and circumferential distance remains constant as the cell elongates during straight elongation. (A, B) Strain, WX7 (ΔftsZ)/λGL100(Plac∷ftsZ). (C) The growth angle (mean and standard error) of E. coli cells with and without A22 treatment. A22 treatment, which causes rapid depolymerization of MreB, results in a slow decrease in the growth angle over a timescale of one doubling time. YS34: without A22 (N = 10). A22 (short): cells were incubated with A22 for 3 min before observation, and imaged for 45 min at room temperature in the presence of A22 (N = 12). A22 (long): cells were incubated with A22 for 30 min at 37 °C before observation, and then imaged at room temperature in the presence of A22 (N = 10). WA220 (parental strain for A22-resistant strain WA221): without A22 (N = 5) and with A22 (long) (N = 6). WA221 (A22-resistant strain): without A22 (N = 5) and with A22 (long) (N = 5). Double asterisks (**) indicate significant difference from zero growth angle in t test, with p < 0.01.

A22 does not block origin segregation. (A) ori segregation in exponential culture before and after A22 treatment. Cells growing exponentially were harvested at different time points before and after A22 (10 μg/ml) treatment. The number of ori1 (lacO) foci was analyzed and plotted. More than 500 cells were analyzed at each time point. (B) A22 does not block origin segregation after synchronization using dnaC(Ts) mutant cells. Cells were grown exponentially at 30°C and then shifted to 37°C for 1 h for synchronization. The culture was then split in two, one with and one without A22 treatment, and grown at 30°C for 1 h. Cells were harvested at each time point, and the ori1 (lacO) foci in each cell were counted. The proportions of cells with different numbers of origins are shown. More than 500 cells were counted at each time point. (C) Time-lapse progression of cells treated with A22. Exponentially growing cells were treated with A22 for 1 min in liquid culture and subsequently mounted on an agarose slide containing medium and A22. Images were taken at 5 min, 1 h, 2 h, and 3 h after A22 treatment. ori1 (lacO) was labeled green.

Morphological effects of inhibiting FtsZ, MreB, or PBP 2. E. coli MG1655 or AV23-1 was treated to inhibit one or more of the following proteins: FtsZ was inhibited by inducing SulA from pFAD38 by adding 0.2% arabinose, MreB was inhibited by adding A22 at the indicated concentrations, and PBP 2 was inhibited by adding amdinocillin (2 μg/ml). (A) AV23-1, with inhibition of both FtsZ and MreB (A22, 5 μg/ml). (B) AV23-1. Conditions were as described above (A), but A22 was removed before a shift to growth at 42°C. (C) MG1655, with inhibition of MreB (A22, 5 μg/ml). (D) AV23-1, with inhibition of MreB (A22, 5 μg/ml). (E) AV23-1, with inhibition of both FtsZ and MreB (A22, 2 μg/ml). (F) AV23-1, with inhibition of FtsZ and PBP 2.

Effect of A22 on cell shape, origin positions, and nucleoid segregation, as determined by fluorescence microscopy. (A) Origins were visualized by tagging with the P1parS-GFP-ParB system. The oriI foci are shown in white, and oriII foci in red. In panels showing cells treated with A22 (+A22), positions of origin foci were variable but not necessarily random, as they sometimes appeared in straight lines or symmetrically disposed with respect to each other (selected examples). −A22, not treated with A22. (B) Formation of anucleate cells upon exposure to A22. The cells were analyzed after staining with DAPI. Cells with DNA are seen as blue, and cells without DNA as black (arrow).