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. 2015 May 7;4:e06474. doi: 10.7554/eLife.06474

Figure 4. Direct visualization of two SpoIIIE clusters at the septal midpoint.

(A) PALM image of single focus and a rare dual focus of SpoIIIE-Dendra2 in wild type B. subtilis (TCF25), with FM5-95 stained membranes (white). The bottom PALM images are zoomed in from the yellow boxes. Bar is 500 nm and 50 nm for overlaid and PALM images, respectively. Membrane is diffraction-limited image. (B) Schematic diagram of septal thinning. (I) After septation, septal peptidoglycan is degraded by a complex containing the SpoIID, SpoIIM and SpoIIP proteins (pacman) and the second potential division site is blocked. (II) Elimination of SpoIIDMP in the ΔspoIIDMP (spoIID, spoIIM, spoIIP) strain inhibits septal thinning without impairing DNA segregation. (III) Elimination of σE in the ΔσE (spoIIGB) strain inhibits septal thinning and produces disporic cells without impairing DNA segregation. (C) PALM images of SpoIIIE-tdEos in ΔσE strain (JS03). Classification of PALM images as dual foci or single foci were defined according to the parameters of our cluster analysis. Scale bar is 500 nm and 50 nm for overlaid and PALM images, respectively. Membrane is diffraction-limited image. (D) PALM images of SpoIIIE-tdEos in ΔSpoIIDMP strain (JLG571). The diffraction-limited images of the membranes (white) and the DNA (green) were obtained by staining with FM5-95 and DAPI, respectively. The relative forespore DAPI intensity was ∼25% in both sporangia. SpoIIIE dual foci are shown in the left panels. In-gel fluorescence and western blots of the different SpoIIIE fusion proteins used here can be found in Figure 4—figure supplement 7.

DOI: http://dx.doi.org/10.7554/eLife.06474.008

Figure 4.

Figure 4—figure supplement 1. Classification of cells in vegetative, monosporic and disporic cells at t1.75.

Figure 4—figure supplement 1.

Mutation in σE produces a much higher percentage of disporic cells compared to the σE+ strain PY79. More than 700 cells were classified per strain. The strains used were KP161, TCF25 and JS03.
DOI: http://dx.doi.org/10.7554/eLife.06474.009
Figure 4—figure supplement 2. SpoIIIE segregates the chromosomes in the ΔσE strain.

Figure 4—figure supplement 2.

(A) Mutation in σE does not impair DNA translocation. Forespores show brighter DNA signal (single arrowhead) at t2.5 compared to t1.75 of sporulation time showing chromosome translocation from the mother cell into the forespore. ΔσE cells expressing SpoIIIE fused to tdEos (JS03) were stained with DAPI and FM5-95 to visualize DNA (Green) and membrane (red), respectively. DAPI signal was false-colored green for a better contrast. (B) Quantification of DNA translocation at different sporulation times. The amount of DNA in the forespore increases as sporulation progresses (from ∼60–100%, inset). Our PALM experiments using ΔσE strains (Figures 4, 5) were done at t1.75 to maximize the percentage of cells are actively translocating DNA. Error bar represents standard error of the mean from 20 cells for each of the time points. (C) Cells were sporulated at 37°C and samples were taken at indicated times and stained with DAPI and FM5-95 to visualize DNA and membrane (red), respectively. The amount of DAPI (labeled-DNA) in the forespore increased from t1.5 to t2 in ΔσE sporangia expressing wild type SpoIIIE (SpoIIIEWT) fused to tdEos (JYS03), indicating that the forespore received the chromosomal complement from the mother cell. In contrast, ΔσE cells expressing SpoIIIE ATPase mutant (SpoIIIEATP−) fused to tdEos (JYS04) did not translocate DNA. SpoIIIEWT fused to Dendra2 (JYS00) also translocated DNA in ΔσE strain. Altogether, these results indicate that SpoIIIE translocates chromosome during sporulation in ΔσE strain and the fusion protein does not impair the ability of this translocation.
DOI: http://dx.doi.org/10.7554/eLife.06474.010
Figure 4—figure supplement 3. Examples of single foci in the ΔσE strain when SpoIIIEWT is fused to tdEos.

Figure 4—figure supplement 3.

DOI: http://dx.doi.org/10.7554/eLife.06474.011
Figure 4—figure supplement 4. Distribution of single and double foci in monosporic and disporic sporangia.

Figure 4—figure supplement 4.

Classification of monosporic and disporic sporangia in relation to the presence of either single focus or dual foci at the sporulation septum of cells expressing either SpoIIIE fused to tdEos (JS03) or Dendra2 (JS00) in the ΔσE strain.
DOI: http://dx.doi.org/10.7554/eLife.06474.012
Figure 4—figure supplement 5. SpoIIIE ATPase mutant (SpoIIIEATP−) fused to tdEos in ΔσE strain also organizes into dual foci.

Figure 4—figure supplement 5.

To demonstrate that SpoIIIEWT dual foci (Figure 4) are observed when the chromosome is trapped in the septum and discard that are only at septa where chromosome transport is complete we imaged SpoIIIEATP− fused to tdEos by PALM. Single mutation G467S in the conserved region of the SpoIIIE ATPase motor domain impairs DNA translocation without affecting SpoIIIE foci assembly, and the forespore chromosome remains trapped in the septum (Sharp and Pogliano, 1999; Fleming et al., 2010). qPALM shows that SpoIIIEATP− also assembles into dual foci (PALM: left panels and, first and second right panels) and single foci (third right panel) similar to the SpoIIIEWT (Figure 4). As expected sporangia expressing SpoIIIEATP− did not translocate DNA (Figure 4—figure supplement 2C).
DOI: http://dx.doi.org/10.7554/eLife.06474.013
Figure 4—figure supplement 6. SpoIIIE organizes into dual foci at the septum of sporangia where chromosome transport is incomplete.

Figure 4—figure supplement 6.

To demonstrate that dual foci represent SpoIIIE complexes that are actively translocating DNA, we imaged SpoIIIEWT in a different Bacillus strain, spoIIDMP triple mutant strain, which mantains a thick sporulation septum due to the inhibition of septal thinning (Pogliano et al., 1999; Abanes De Mello et al., 2002; Chastanet and Losick, 2007; Gutierrez et al., 2010). In contrast to the ΔσE mutant, the completion of the second polar septum is inhibited in the spoIIDMP triple mutant, leading to the formation of fewer disporic sporangia. This feature allows the estimation of the degree of chromosome translocation using DNA-specific dyes and measuring the dye-intensity in the forespore vs the total intensity (forespore plus mother cell), a standard procedure in the field (Becker and Pogliano, 2007; Ptacin et al., 2008). The relative forespore DAPI intensity of sporangia showing dual foci in the spoIIDMP mutant ranged between 0.1 and 0.32, suggesting that they were actively translocating DNA. These observations suggest that dual foci represent the organization of the SpoIIIE complex during the process of DNA translocation. Criteria for complete and incomplete DNA translocation is described in the chromosome translocation section of the results and in Figure 7—figure supplement 1.
DOI: http://dx.doi.org/10.7554/eLife.06474.014
Figure 4—figure supplement 7. In-gel fluorescence and western blot of the SpoIIIE fusion proteins used in this study.

Figure 4—figure supplement 7.

Our single SpoIIIE molecule quantification relies on the fluorescent protein fused to SpoIIIE. Therefore, it is crucial that all SpoIIIE molecules are fused to the fluorescent protein and also all the fluorescent proteins fused to SpoIIIE. (A) Fluorescent proteins (GFP, Dendra2 and tdEos) fused to SpoIIIE migrate as a single band in 7% semi-denaturing in-gel fluorescence PAGE (Drew et al., 2006). As expected the tandem dimer Eos (tdEos) fusion proteins migrate slightly higher than the GFP and the Dendra2 fusion proteins. (B) SpoIIIE fused to fluorescent proteins migrates as a higher molecular weight compared to the native SpoIIIE (lanes 2 and 3), indicating that all the SpoIIIE molecules are fused to the fluorescent proteins. The molecular weight of the fusion proteins corresponded to the in-gel fluorescence. The faint band at ∼130 kDa is unspecific. (C) Total protein visualized by Coomassie-Blue staining.
DOI: http://dx.doi.org/10.7554/eLife.06474.015