Evidence that the SpoIIIE DNA translocase participates in membrane fusion during cytokinesis and engulfment

Abstract

SummaryDuring Bacillus subtilis sporulation, SpoIIIE is required for translocation of the trapped forespore chromosome across the sporulation septum, for compartmentalization of cell-specific gene expression, and for membrane fusion after engulfment. We isolated mutations within the SpoIIIE membrane domain that block localization and function. One mutant protein initially localizes normally and completes DNA translocation, but shows reduced membrane fusion after engulfment. Fluorescence recovery after photobleaching experiments demonstrate that in this mutant the sporulation septum remains open, allowing cytoplasmic contents to diffuse between daughter cells, suggesting that it blocks membrane fusion after cytokinesis as well as after engulfment. We propose that SpoIIIE catalyses these topologically opposite fusion events by assembling or disassembling a proteinaceous fusion pore. Mutants defective in SpoIIIE assembly also demonstrate that the ability of SpoIIIE to provide a diffusion barrier is directly proportional to its ability to assemble a focus at the septal midpoint during DNA translocation. Thus, SpoIIIE mediates compartmentalization by two distinct mechanisms: the SpoIIIE focus first provides a temporary diffusion barrier during DNA translocation, and then mediates the completion of membrane fusion after division to provide a permanent diffusion barrier. SpoIIIE-like proteins might therefore serve to couple the final step in cytokinesis, septal membrane fusion, to the completion of chromosome segregation.

Visualization of pinholin lesions in vivo

“…Except the superresolution experiments, fluorescence microscopy was performed as described previously (32). For endpoint experiments, cultures were grown to A550 = 0.4 and induced thermally or with IPTG/arabinose; 20 μL each sample were taken out and mixed with 0.5 μL FM4-64 (1 mg/mL) at the time of pinholin triggering or 30 min after induction for irsS 21 68-GFP, and 10 μL mixture were then put onto an agarose pad [1.2% (wt/vol) agarose, tryptone broth, 1 μg/mL FM 4-64].…”

FisB mediates membrane fission during sporulation in Bacillus subtilis

“…Based on these observations, we favor the idea that SpoIIIE is only indirectly involved in the fission reaction, impacting membrane migration and FisB abundance and/or localization. Interestingly, it has also been proposed that SpoIIIE participates in fission during asymmetric division in sporulating cells (Liu et al 2006;Fleming et al 2010). This membrane remodeling event, wherein cytosol fills the gap inside the closing membrane annulus, has the opposite topology to the fission at the end of engulfment, where the extracellular medium fills the gap to be closed by the engulfing membrane (Supplemental Fig.…”

“…It has been proposed that protein components of the cell division machinery could catalyze fission (Sharp and Pogliano 1999;Liu et al 2006;de Boer 2010;Fleming et al 2010). Alternatively, it has been suggested that cell wall synthesis on the outside of the cell could force the constricting membranes into close proximity, ultimately leading to fission (Weiss 2004;Judd et al 2005;Meyer et al 2010).…”

Impact of Membrane Fusion and Proteolysis on SpoIIQ Dynamics and Interaction with SpoIIIAH

“…To address this question, we made use of a spoIIIE mutant that completes membrane migration but not membrane fusion (and which translocates DNA (33)). The membrane fusion-defective mutant abolished degradation of native SpoIIQ (Fig.…”

“…Images were collected with an Applied Precision Spectris microscope with a QLM laser module (described in Ref. 33). Photobleaching was performed and quantified as described (22), using a 0.02-to 0.05-s pulse of a 488 nm argon laser at 50% power.…”