Fig 1.
(A) The putative three domain structure of SpoIIE (top) and a schematic of the SpoIIE construct used in this study (bottom), (B) SDS-PAGE of the sedimentation of ms-SpoIIEcyt. Ms-SpoIIEcyt was pre-incubated with 1 mM EDTA (lanes 1 and 2), followed by the incubation with 5 mM MnCl2 (lanes 3 and 4), followed by the incubation with 10 mM EDTA (lanes 5 and 6). Odd numbers below the image represent supernatant fractions and even numbers represent pellet fractions, (C) Electron microscopy of ms-SpoIIEcyt oligomers after co-purification with Fe2+ (i). Oligomers were not observed after incubation of the protein with 5 mM EDTA (ii). Arrows point to rod and circular structures of ms-SpoIIEcyt. Scale bar: 50 nm, (D, E) Fluorescence emission spectra of s-SpoIIEcyt-Cy5 titrated into a buffer in the absence (D) and presence (E) of Mn2+. Every spectrum represents a titration step with a 0.23 μM concentration increase of s-SpoIIEcyt-Cy5, with the bottom spectrum representing buffer without any added protein.
Table 1.
B. subtilis strains used in this study.
Table 2.
Metals copurifying with ms-SpoIIEcyt.
Metals associated with lyophilized ms-SpoIIEcyt were analyzed by ICP-OES. The mean and standard deviation from two independent determinations are shown.
Fig 2.
Binding to divalent cations reversibly enhances oligomerization of s-SpoIIEcyt.
(A, B) Light scattering measurement of 1,5 μM s-SpoIIEcyt (A) and MBP (B) in the presence of 10 mM of divalent cations added after 60 sec. (C) Light scattering signal of s-SpoIIEcyt, with 10 mM Mn2+ added after 60 sec and 20 mM EDTA or H2O (control) added after 600 sec.
Fig 3.
The absence of Mn2+ from the sporulation medium delays asymmetric Z-ring formation.
Pie-chart representation of sporulating B. subtilis cells in the presence (+) and absence (-) of Mn2+. Four different types of cell (representative images in the legend) were scored: cells without any Z-ring in the cell (no ring), cells with a Z-ring in the middle of the rod (mid-cell ring), cells with two rings assembled at the cell poles (two polar rings) and cells with only one polar ring (polar ring). Non-sporulating cells are marked in blue shades, while sporulating cells are marked in orange shades. Each pie chart is the result of two independent classification experiments in which at least 290 cells were classified per condition. Actual percentages and standard deviations are included in S1 Table.
Fig 4.
s-SpoIIEcyt interacts directly with FtsZ.
A) Light scattering signals of FtsZ (FtsZ/GTP) s-SpoIIEcyt (s-SpoIIEcyt/GTP) or FtsZ and s-SpoIIEcyt (FtsZ/GTP/s-SpoIIEcyt) in the presence of GTP, or FtsZ and s-SpoIIEcyt in the presence of GDP(FtsZ/GDP/s-SpoIIEcyt). B) Electron micrographs of a similar experiment as shown in A), samples were taken after 30 min incubation and placed on EM grids. Scale bar, 100 nm.