Studying Biomolecule Localization by Engineering Bacterial Cell Wall Curvature

<div><p>In this article we describe two techniques for exploring the relationship between bacterial cell shape and the intracellular organization of proteins. First, we created microchannels in a layer of agarose to reshape live bacterial cells and predictably control their mean cell wall curvature, and quantified the influence of curvature on the localization and distribution of proteins in vivo. Second, we used agarose microchambers to reshape bacteria whose cell wall had been chemically and enzymatically removed. By combining microstructures with different geometries and fluorescence microscopy, we determined the relationship between bacterial shape and the localization for two different membrane-associated proteins: i) the cell-shape related protein MreB of <i>Escherichia coli</i>, which is positioned along the long axis of the rod-shaped cell; and ii) the negative curvature-sensing cell division protein DivIVA of <i>Bacillus subtilis</i>, which is positioned primarily at cell division sites. Our studies of intracellular organization in live cells of <i>E. coli</i> and <i>B. subtilis</i> demonstrate that MreB is largely excluded from areas of high negative curvature, whereas DivIVA localizes preferentially to regions of high negative curvature. These studies highlight a unique approach for studying the relationship between cell shape and intracellular organization in intact, live bacteria.</p></div

Analysis of MreB distribution in artificially curved bacterial cells.

<p>(A) Composite, representative fluorescence and bright field images of filamented <i>E. coli</i> FB76 cells in V-shaped channels depicting the spatial distribution of MreB-RFP. Scale bar: 20 µm. (B) An image depicting the positive and negative mean cytoplasmic membrane curvature imposed on the cell. (C) A plot depicting the spacing between MreB foci as a function of mean cell wall curvature determined for cells isolated in V-shaped microchannels and imaged using fluorescence microscopy. The plot depicts the spacing between MreB foci along the positively and negatively curved cytoplasmic membranes in confined cells. The lines between the data points are provided as a guide. For each value of channel curvature, we measured n≥30 cells; error bars represent standard error of the mean.</p

Methodology to engineer curvature in bacterial cells.

<p>(A) A schematic diagram depicting the approach for engineering bacterial cell wall curvature by growing filamentous cells of bacteria in angled microchannels. We confined individual planktonic cells in microchannels printed into layers of agarose infused with LB nutrient media to physically impose cell curvature upon filamented bacterial cells. Bacteria were filamented using antibiotics that inhibit cell division. (B–C) Representative images of an experiment using <i>E. coli</i> FB76 cells comparing time points at (B) 0 min of growth and (C) 120 min of growth. In this experiment we filamented cells by infusing the agarose with 25 µg/mL of cephalexin. The values shown depict the angle that connects the two straight channel segments. Scale bars: 20 µm.</p

Analysis of DivIVA localization in spheroplasts from filamented cells of <i>E. coli</i> pKR196 and protoplasts from filamented cells of <i>B. subtilis</i> PE103.

<p>(A) A representative brightfield microscopy image of protoplasts from <i>B. subtilis</i> PE103 confined in agarose microchambers. (B) A fluorescence microscopy image of DivIVA-CFP in <i>B. subtilis</i> PE103 protoplasts confined in agarose microchambers. The images inset show the DivIVA-CFP fluorescence in two magnified protoplasts with imposed membrane curvature that is high and low negative. Scale bars: 5 µm. (C) A plot depicting the relative frequency of the DivIVA distribution in <i>E. coli</i> pKR196 spheroplasts (open squares) and <i>B. subtilis</i> PE103 protoplasts (shaded circles) and its relationship to microchamber curvature. Data for the relative frequency of MinD-YFP in spheroplasts of <i>E. coli</i> MG1655 pFX40 cells (shaded triangles) versus curvature for are provided for comparison to DivIVA-GFP and DivIVA-CFP.</p

List of bacterial strains used in this study.

<p>List of bacterial strains used in this study.</p

Model for the role of DivIVA during sporulation.

<p>A sporulating <i>B. subtilis</i> cell is depicted using the same color scheme as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004526#pgen-1004526-g001" target="_blank">Fig. 1A</a>. (A) FtsZ (red ring) initially assembles in the pre-divisional cell at mid-cell and recruits SpoIIE (blue ring); at this time, DivIVA (green arcs) pre-localizes as patches at the two cell poles to perform its role in chromosome anchoring. (B) FtsZ and SpoIIE redeploy to polar division sites. In addition to SpoIIE and the increased expression of <i>ftsZ</i>, this redeployment requires DivIVA in a manner that is independent of MinCD and RacA, but the molecular mechanism of which is unclear. (C) One of the polar FtsZ rings constricts, while the other is eventually disassembled. Sensing membrane invagination, DivIVA localizes to either side of the nascent asymmetric septum and then sequesters SpoIIE to the septum. Although DivIVA is initially on both sides of the forming polar septum, SpoIIE preferentially localizes to the forespore side. (D) Upon completion of polar septum formation, both DivIVA and SpoIIE persist preferentially on the forespore side of the polar septum. (E) SpoIIE is released into the forespore membrane. It is subsequently recaptured at the polar septum by a protein produced under the control of σ^F<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004526#pgen.1004526-Campo1" target="_blank">[29]</a>.</p

Asymmetric Division and Differential Gene Expression during a Bacterial Developmental Program Requires DivIVA

<div><p>Sporulation in the bacterium <i>Bacillus subtilis</i> is a developmental program in which a progenitor cell differentiates into two different cell types, the smaller of which eventually becomes a dormant cell called a spore. The process begins with an asymmetric cell division event, followed by the activation of a transcription factor, σ^F, specifically in the smaller cell. Here, we show that the structural protein DivIVA localizes to the polar septum during sporulation and is required for asymmetric division and the compartment-specific activation of σ^F. Both events are known to require a protein called SpoIIE, which also localizes to the polar septum. We show that DivIVA copurifies with SpoIIE and that DivIVA may anchor SpoIIE briefly to the assembling polar septum before SpoIIE is subsequently released into the forespore membrane and recaptured at the polar septum. Finally, using super-resolution microscopy, we demonstrate that DivIVA and SpoIIE ultimately display a biased localization on the side of the polar septum that faces the smaller compartment in which σ^F is activated.</p></div

DivIVA and SpoIIE preferentially localize to the forespore side of the polar septum.

<p>Subcellular localization of DivIVA-GFP or SpoIIE-GFP was monitored using (A–F) structured illumination microscopy (SIM), (G–J) multifocal structured illumination microscopy (MSIM), or (K–O) instant structured illumination microscopy (ISIM). (A–C) Localization of DivIVA-GFP in sporulating cells (strain KR604) displaying (A) nascent, (B) mature, or (C) slightly curved polar septa, using SIM. (D–F) Localization of SpoIIE-GFP in sporulating cells (strain PE118) displaying (D) nascent or (E) mature septa. (F) Localization of SpoIIE after release into the membrane surrounding the forespore <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004526#pgen.1004526-Campo1" target="_blank">[29]</a>, using SIM. Localization of DivIVA-GFP in sporulating Δ<i>spoIID</i> Δ<i>spoIIM</i> cells (strain PE275) displaying (G) nascent, or (H) mature polar septa using MSIM. Localization of SpoIIE-GFP in sporulating Δ<i>spoIID</i> Δ<i>spoIIM</i> cells (strain PE274) displaying (I) nascent, or (J) mature polar septa using MSIM. Localization of SpoIIE-GFP in sporulating Δ<i>spoIID</i> Δ<i>spoIIM</i> cells (strain PE274) displaying (K) nascent, or (L) mature polar septa using ISIM. Localization of SpoIIE-GFP in sporulating Δ<i>spoIID</i> Δ<i>spoIIM</i> Δ<i>spoIIQ</i> cells (strain PE368) displaying (M) nascent, (N) mature polar septa using ISIM. (O) Localization of SpoIIE after release into the membrane surrounding the forespore <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004526#pgen.1004526-Campo1" target="_blank">[29]</a>, using ISIM. All white arrows indicate the localization of DivIVA-GFP or SpoIIE-GFP relative to the polar septum. Linescan analyses of normalized fluorescence intensity along the axis of the dashed line (along the entire width of the bacterium) in both channels at the selected polar septa are shown at the right; green arrows indicate the fluorescence from the GFP signal indicated with white arrows in the micrographs. Scale bars: (A–F), 1 µm; (G–O), 0.5 µm.</p

SpoIIE copurifies with DivIVA and proper SpoIIE localization requires DivIVA.

<p>(A) Localization of SpoIIE-GFP in sporulating cells (strain PE118). (B) Localization of DivIVA-GFP in sporulating cells in the absence of SpoIIE (strain PE266). First panel: membranes visualized using FM4-64; second panel: GFP fluorescence; third panel: chromosomes visualized using DAPI; fourth panel: overlay of membrane and GFP; fifth panel: overlay of membrane, GFP, and DNA. Polar division sites are indicated with arrows. (C) Top: localization of FtsZ-mCherry and SpoIIE-GFP at polar septa of sporulating cells (strain PE369). First panel: DIC; second panel: mCherry fluorescence; third panel: GFP fluorescence; fourth panel: overlay of mCherry and GFP; fifth panel: overlay of DIC, mCherry, and GFP. Bottom: GFP and mCherry fluorescence represented as a three-dimensional surface. Arrowhead indicates nascent polar septum before FtsZ constriction; arrow indicates a polar division site at which FtsZ is constricting. (D) Co-immunoprecipitation experiment of DivIVA with SpoIIE. Total detergent-solublized extracts (T) prepared from sporulating cells producing DivIVA-FLAG and SpoIIE-GFP (top left, strain PE148), only SpoIIE-GFP (top right, strain PE118), DivIVA-FLAG and SpoVM-GFP (middle, strain PE388), SpoIIE-FLAG (bottom left, strain PE375), or SpoIIE-GFP (bottom right, strain PE130) were incubated with resin covalently attached to anti-FLAG antibodies. Unbound (UB) material was removed, the resin was washed extensively (W₁–W₃), and bound material was eluted (E) using excess FLAG peptides. Fractions were analyzed by immunoblotting using antisera specific to GFP, DivIVA, or σ^A. (E–H) Localization of SpoIIE-GFP produced under control of its native promoter in cells harvested 90 min after the induction of sporulation: (E) Otherwise wild type (strain PE118), (F) Δ<i>divIVA</i> (strain PE122), (G) Δ<i>minCD</i> (strain PE138), or (H) Δ<i>divIVA</i> Δ<i>minCD</i> (strain PE141). Fraction of cells in which SpoIIE-GFP was at a polar division site, at mid-cell, or adjacent to mid-cell are indicated to the right. First panel: membranes visualized using FM4-64; second panel: chromosomes visualized using DAPI; third panel: GFP fluorescence; fourth panel: overlay of membrane and GFP; fifth panel: overlay of membrane, GFP, and DNA. Arrows indicate forespores; white arrowheads indicate SpoIIE-GFP at a nascent cell division site before elaboration of a septum; gray arrowheads indicate the absence of SpoIIE-GFP at a mature septum. (I–L) Localization of SpoIIE-GFP produced under control of an inducible promoter in vegetatively growing cells: (I) otherwise wild type (strain PE130), (J) Δ<i>divIVA</i> (strain PE133), (K) Δ<i>minCD</i> (strain PE224), or (L) Δ<i>divIVA</i> Δ<i>minCD</i> (strain PE225). First panel: membranes visualized using FM4-64; second panel: GFP fluorescence; third panel: chromosomes visualized using DAPI; fourth panel: overlay of membrane and GFP; fifth panel: overlay of membrane, GFP, and DNA. Arrows indicate SpoIIE at mature septa; white arrowheads indicate SpoIIE-GFP at a nascent cell division site before elaboration of a septum; gray arrowheads indicate the absence of SpoIIE-GFP at a mature septum. (M) β-galactosidase accumulation was measured at different time points after the induction of sporulation in cells harboring a P<i>_spoIIE-lacZ</i> reporter fusion in otherwise wild type cells (•; strain PE301), Δ<i>minCD</i> (▪; strain PE323), Δ<i>divIVA</i> (▴; strain PE324), or Δ<i>divIVA</i> Δ<i>minCD</i> (▾; strain PE328). Scale bars: 2 µm.</p

DivIVA assembles into a ring-like structure at the polar septum during sporulation.

<p>(A) Schematic representation of sporulation in <i>Bacillus subtilis</i>. Depicted is a progenitor rod-shaped cell (above), the onset of asymmetric septation (middle), and formation of the polar septum (below), where the mother cell (MC) and forespore (FS) are labeled; activation of σ^F exclusively in the forespore is also depicted. (B) Immunoblot analysis of wild type cells (strain PY79; for genotypes, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004526#pgen.1004526.s011" target="_blank">Table S1</a>), induced to sporulate and harvested at the times indicated above, using antisera specific to DivIVA or σ^A (an abundant unrelated protein that served as a loading control). Fraction of cells that had entered sporulation, as determined by epifluorescence microscopy, is indicated below. (C) Localization of DivIVA-GFP in strain KR604 induced to sporulate for 1.5 h. First panel: membranes were visualized using the fluorescent dye FM4-64; second panel: fluorescence from GFP; third panel: chromosomes were visualized using DAPI; fourth panel: overlay of membrane and GFP; fifth panel: overlay of membrane, GFP, and DNA. Cell #1 is a pre-divisional cell; cell #2 has elaborated a polar septum; cell #3 has just initiated asymmetric division, as evidenced by an increase in membrane staining at the future site of septation. Arrows indicate the sites of asymmetric division. (D) Top: rotation of the region of cell #3 indicated in (C), second panel, around the x- and y-axes. Degrees rotated are indicated below each panel; arrowheads indicate DivIVA patches. Bottom: GFP and DAPI fluorescence from cell #3 (C) represented as a three-dimensional surface. Arrow indicates DivIVA ring at the polar septum; arrowheads indicate the same DivIVA patches at poles. (E) Top: Localization of DivIVA-CFP (shown in green) and FtsZ-YFP (shown in red) in strain PE177. First panel: differential interference contrast (DIC); second panel: fluorescence from YFP; third panel: fluorescence from CFP; fourth panel: overlay of YFP and CFP; fifth panel: overlay of YFP, CFP, and DIC. Bottom: YFP and CFP fluorescence from the cell shown above represented as a three-dimensional surface and rotated to view at different angles. Panels 1–3: overlay of YFP and CFP. Arrow indicates DivIVA-CFP ring or constricting FtsZ-YFP at the polar division site; arrowheads indicate DivIVA-CFP patches at the extreme poles of the cell. Scale bars: 2 µm.</p