6E), SpoIIE localized to the forespore side of the septum, and finally, as reported previously, SpoIIE was released from the polar septum and localized uniformly in the membrane surrounding the forespore (Fig. cells in the population that were minicells. (D) Sporulation efficiencies of strains harboring various alleles of as measured by heat resistance (relative to WT). First column: WT (strain PY79), (strain KR610), (strain PE180), (strain PE390) as the only copy of cell producing DivIVA-GFP (strain KR541) while elaborating a polar septum. Arrows indicate polar septa; time (min) is indicated on the left.(TIF) pgen.1004526.s002.tif (250K) GUID:?EA3DAB08-9DD6-44BB-B6AE-4DB708AB5EAA Figure S3: Degradation of DivIVA-SsrAEc by Lyn-IN-1 IPTG-induced production of SspB. Immunoblot analysis of cells induced to sporulate and harvested at the times indicated above, producing DivIVA-SsrAEc (left; strain PE304), or DivIVA-SsrAEc and SspB (strain PE330) in the absence (center) or presence (right) of IPTG added at 45 min to induce expression of cell LAG3 extracts prepared at the times indicated (h) after the induction of sporulation. Shown are three independent trials (numbered on the right) from independent sporulating cultures of the following strains: WT (PY79); (PE362); (KR620); (KR543);(PE308). (B) Localization of ZapA-GFP (top; strain PE290) in cells either 60 min or 120 min after the induction of sporulation, as indicated. Arrows indicate ZapA-GFP signal at polar division sites.(TIF) pgen.1004526.s004.tif (278K) GUID:?D5F666F0-2702-41B0-B5B4-14D6C1398A57 Figure S5: SpoIIE-GFP is solubilized by the nonionic detergent Triton X-100. Immunoblot analysis, using antisera specific to GFP, DivIVA, or A, of cell extracts (strain PE130), which overproduces SpoIIE-GFP, prepared 1.5 h after the induction of sporulation and separated into soluble supernatant (S) and insoluble pellet (P) fractions either without (?TX-100) or with (+TX-100) extraction with the nonionic detergent Triton X-100 in lysis buffer (see Materials and Methods for buffer components). Asterisk indicates a soluble GFP-tagged species that is likely a truncated form of SpoIIE-GFP.(TIF) pgen.1004526.s005.tif (85K) GUID:?D7CC0916-2513-48D7-BA05-05FFE9BFB14F Figure S6: Premature activation of F is not responsible for the asymmetric septation defect in the absence of DivIVA. (A) -galactosidase accumulation was measured at different time points after the induction of sporulation in cells harboring a F-dependent reporter fusion in otherwise wild type cells (?; strain PE300), (?; strain PE321), (?; strain PE322), or (?; strain PE327). (BCF) Polar septum formation was monitored using the fluorescent membrane dye FM4-64 in cells that had initiated sporulation for 2 h in (B) wild type cells (strain PE80), (C) (strain RL1275); (D) (strain PE196); (E) (strain PE199); (F) (strain PE198). First panel: membranes visualized using FM4-64; second panel: chromosomes visualized using DAPI; third panel: overlay of membranes and DNA. Fraction of cells elaborating a polar septum is indicated to the right (ND, none detected).(TIF) pgen.1004526.s006.tif (332K) GUID:?8C7A8ABE-C313-4467-9D0C-E4DAA26ABA6C Figure S7: Super-resolution micrographs of sporulating cells. (A) Examples of types of deformation to the polar septum that were routinely observed using the lowest laser power available when viewing the cells using several commercial SIM setups: DeltaVision OMX Blaze (top row), Nikon N-SIM (middle row), or Zeiss Elyra (bottom row) at either nascent (left column) or mature (right column) polar septa. Arrows indicate the site of deformation. (B) Localization of SpoIIE-GFP in sporulating cells (strain PE274) observed using MSIM. Internal calibration of fluorescence Lyn-IN-1 from red and green channels using a bead that fluoresces in both channels (arrowhead) as viewed at (top) a Lyn-IN-1 plane close to the coverslip or Lyn-IN-1 at (bottom) an intermediate plane. Scale bar: 0.5 m.(TIF) pgen.1004526.s007.tif (221K) GUID:?BA732CBF-72B0-4432-99CC-07E48F4A6E8F Figure S8: Localization of SpoIIE-GFP in the absence of SpoIIQ and engulfment. Subcellular localization of SpoIIE-GFP in mutant cells arrested at the flat septum stage before the onset of engulfment, 1.5 h after the induction of sporulation, in the presence (above, strain PE274) or absence (below, strain PE368) of SpoIIQ.(TIF) pgen.1004526.s008.tif (198K) GUID:?ED6D92E1-51F7-429C-B102-76358DD4B44A Figure S9: Gallery of sporulating cells displaying forespore-biased localization of SpoIIE-GFP. Subcellular localization of SpoIIE-GFP in mutant cells arrested at the flat septum stage before the onset of engulfment, 1.5 h after the induction of sporulation, in the presence (left, strain PE274) or absence (right, strain PE368) of SpoIIQ, visualized using either the MSIM or ISIM super-resolution technique. Arrows indicate fluorescent beads that were visible.