Dynamic patterns of subcellular protein localization during spore coat morphogenesis in Bacillus subtilis

Abstract

Endospores of Bacillus subtilis are encased in a thick, proteinaceous shell known as the coat, which is composed of a large number of different proteins. Here we report the identification of three previously uncharacterized coat-associated proteins, YabP, YheD, and YutH, and their patterns of subcellular localization during the process of sporulation, obtained by using fusions of the proteins to the green fluorescent protein (GFP). YabP-GFP was found to form both a shell and a ring around the center of the forespore across the short axis of the sporangium. YheD-GFP, in contrast, formed two rings around the forespore that were offset from its midpoint, before it eventually redistributed to form a shell around the developing spore. Finally, YutH-GFP initially localized to a focus at one end of the forespore, which then underwent transformation into a ring that was located adjacent to the forespore. Next, the ring became a cap at the mother cell pole of the forespore that eventually spread around the entire developing spore. Thus, each protein exhibited its own distinct pattern of subcellular localization during the course of coat morphogenesis. We concluded that spore coat assembly is a dynamic process involving diverse patterns of protein assembly and localization.

FIG. 1.

Subcellular localization of YabP-GFP and YabQ-GFP. (A) Localization of YabP-GFP. Cells of strain CVO1084 (amyE::yabP-gfp) were induced to sporulate, stained with the membrane dye FM4-64, and prepared for microscopy at the times (in hours) indicated on the left as described in Materials and Methods. YabP-GFP fluorescence is shown in the left panels, and the membrane staining is shown in the right panels. The arrows and arrowheads indicate corresponding cells in adjacent panels that have almost completed engulfment and are in the process of engulfing the forespore, respectively. The asterisks indicate a cell in which engulfment of the forespore is in a very early stage but in which YabP-GFP is nonetheless already concentrated on the sporulation septum. (B) Localization of YabQ-GFP. Cells of strain CVO1088 (amyE::yabPQ-gfp) were induced to sporulate and observed at the times (in hours) indicated on the left. The cells viewed after 3 h of sporulation were additionally stained with the membrane dye FM4-64. The panels on the left show the YabQ-GFP fluorescence. The panels on the right show the membrane dye fluorescence (top) or phase microscopy (bottom) of corresponding cells. The arrows indicate corresponding cells in the left and right panels. YabQ-GFP was concentrated on the outer forespore membrane in all cells examined. (C) Localization of YabP-GFP in the absence of YabQ. Cells of strain CVO1202 (yabQ::tet amyE::yabP-gfp) were induced to sporulate and observed after 3 h. Note the bright staining of the dots and the greatly decreased staining of the outer forespore membrane compared to the staining of YabP-GFP in a wild-type background (as shown in panel A).

FIG. 2.

Deconvolution microscopy of sporulating cells producing YabP-GFP. Cells of strain CVO1084 (amyE::yabP-gfp) were prepared for deconvolution microscopy as described in Materials and Methods. Panel 1 shows the focal plane closest to the slide, and each consecutive image is ∼0.1 μm farther from the slide. Note the increase in the distance between the dots of YabP-GFP fluorescence as the focal plane moved up through the first five images. The last three images show that the distance between the dots decreased until they combined to form a single dot in panel 8, which shows the region of the cell farthest from the slide.

FIG. 3.

Subcellular localization of YabP-GFP requires σ^E-directed gene expression. Transcription of yabP-gfp was induced by addition of IPTG in strains carrying the P_hyperspank-yabP-gfp translational fusion in backgrounds with and without σ^E activity (strains CVO1219 and CVO1233, respectively). Cells were prepared for microscopy 3 h after the induction of sporulation, as described in the legend to Fig. 1. The YabP-GFP fusion was recruited to the outer forespore membrane normally when it was expressed in the wild-type (WT) background from the inducible promoter (top panels). In the absence of σ^E, YabP was not recruited to the sporulation septum, and the protein was detected throughout the cytoplasm (bottom panels).

FIG. 4.

Subcellular localization of YheD-GFP. Cells of strain CVO1728 (yheD-gfp) were induced to sporulate, stained with the membrane dye FM4-64, and prepared for microscopy as described in Materials and Methods. GFP fluorescence is shown in the left panels, and membrane staining is shown in the right panels. Note the distinct subcellular localization pattern of YheD-GFP around the outer forespore membrane at 3 h after induction of sporulation (top panels). Engulfment had been completed in these cells, which greatly decreased the staining of the forespore. At 6.5 h after the induction of sporulation (bottom panels), YheD-GFP was redistributed to form a complete shell around the outer forespore membrane. The arrows indicate corresponding cells in the left and right panels.

FIG. 5.

Subcellular localization of YheD-GFP depends on SpoIVA. Cells harboring a yheD-gfp fusion were grown for 18 h in Difco sporulation medium at 37°C, stained with the membrane dye FM4-64, and prepared for microscopy as described in Materials and Methods. YheD-GFP was visualized in a otherwise wild-type derivative of strain PY79 (strain CVO1728), in a spoIVA mutant (PE556), and in a cotE mutant (PE557), as indicated on the left. Representative sporangia are shown. Staining with the membrane dye FM4-64 is shown in the right panels, and GFP fluorescence is shown in the left panels.

FIG. 6.

Subcellular localization of YutH-GFP. Cells harboring a yutHΩyutH-gfp fusion were induced to sporulate, stained with the membrane dye FM4-64, and prepared for microscopy as described in Materials and Methods. (A) Fluorescence micrographs of representative cells producing YutH-GFP after growth for 18 h in Difco sporulation medium at 37°C. YutH-GFP was visualized in an otherwise wild-type derivative of strain PY79 (strain PE479), in a spoIVA mutant (PE547), and in a cotE mutant (PE500), as indicated on the left. Staining with the membrane dye FM4-64 is shown in the right panels, and GFP fluorescence is shown in the left panels. (B) Time course of YutH-GFP localization during sporulation. Cells of strain PE479 were sporulated by suspension in Sterlini-Mandelstam medium at 37°C, and samples were collected at the times indicated and analyzed by fluorescence microscopy. Only representative cells are shown. Staining with the membrane dye FM4-64 is shown in the right panels, and GFP fluorescence is shown in the left panels.

FIG. 7.

Model of localization of YabP, YheD, and YutH during sporulation. The cartoons show the following progressive stages of sporulation (from left to right): septation, engulfment in progress, engulfment completed, and maturing forespore. YabP initially associates with the sporulation septum and then migrates with the mother cell membrane that is engulfing the forespore, where it is concentrated at its leading edge. After engulfment is complete, YabP is concentrated in a ring that surrounds the forespore at its widest point but is also present as a shell around the outer forespore membrane. The latter pattern of localization is dependent on YabQ, as shown in the cartoon depicting the YabP localization in a yabQ mutant cell. At later stages of sporulation YabP is found primarily in the cytosol of the mother cell and occasionally as a small dot on the mother cell side of the forespore. YheD is detected initially in two caps at the poles of the forespore. Ultimately, it forms a complete shell around the forespore. Recruitment of YheD to the outer forespore depends on SpoIVA. YutH is initially detected as a thin cap at the mother cell side of the forespore, which gradually covers the outer forespore membrane as sporulation progresses. Localization of YutH on the outer forespore membrane is dependent on SpoIVA. Proteins are indicated by grey shading, and membranes are indicated by black lines; the intensity of shading indicates the relative amount of protein as detected by fluorescence microscopy.