Bacterial cell curvature through mechanical control of cell growth

Abstract

The cytoskeleton is a key regulator of cell morphogenesis. Crescentin, a bacterial intermediate filament-like protein, is required for the curved shape of Caulobacter crescentus and localizes to the inner cell curvature. Here, we show that crescentin forms a single filamentous structure that collapses into a helix when detached from the cell membrane, suggesting that it is normally maintained in a stretched configuration. Crescentin causes an elongation rate gradient around the circumference of the sidewall, creating a longitudinal cell length differential and hence curvature. Such curvature can be produced by physical force alone when cells are grown in circular microchambers. Production of crescentin in Escherichia coli is sufficient to generate cell curvature. Our data argue for a model in which physical strain borne by the crescentin structure anisotropically alters the kinetics of cell wall insertion to produce curved growth. Our study suggests that bacteria may use the cytoskeleton for mechanical control of growth to alter morphology.

Figure 1

Crescentin forms a single filamentous structure that collapses into a helical structure when released from the membrane. (A) Time lapse of log-phase CJW2203 cells (CB15N Δbla creS∷pBGST18creS-gfp∷pBGENTcreS) grown on an agarose-M2G⁺ pad containing 40 μg/ml mecillinam. Asterisk represents cell lacking crescentin-GFP signal after retraction of the crescentin structure into only one daughter cell. DIC, differential interference contrast. (B) CJW2995 cells (CB15N Δbla/pMR20creS-tc) treated in liquid with 40 μg/ml mecillinam for 4.5 h and stained with FlAsH dye. Dashed lines indicate cell outlines. (C) CJW2592 cells (CB15N Δbla ftsZ∷pBGENTPxylftsZ creS∷pBGST18creS-gfp∷pBGENTcreS) depleted for FtsZ for 2.5 h, then grown in liquid for 4.3 h after the addition of 10 μg/ml mecillinam to induce detachment of crescentin structures. Some crescentin structures displayed partial detachment (arrow). (D) Two optical sections of a detached crescentin structure, showing left-handed helicity. CJW2592 cells were depleted of FtsZ as in panel C for 3 h, then grown for another 4 h with 5 μg/ml mecillinam in liquid culture. Bars, 2 μm.

Figure 2

Cell straightening upon dominant-negative crescentin_ΔL1 production is gradual and growth dependent. (A) Crescentin domain organization. Amino acid positions are shown at the bottom. Green bars indicate coiled-coil forming regions. The N-terminal head, C-terminal tail, linkers, stutter (interruption in coiled-coil repeat) and N27 region are marked. (B) FlAsH-stained cells expressing crescentin_ΔL1-TC in a ΔcreS background (CJW1521; CB15N ΔcreS pMR20creSΔL1-tc). (C) FlAsH-stained cells (CJW2788; CB15N ΔcreS xylX∷pHL23PxylcreSΔL1/pMR20creS-tc) expressing wild-type crescentin-TC (red), without (left) or with (right) co-expression of untagged crescentin_ΔL1 for 10 h. (D) Graphical representation of cell curvature loss and crescentin localization during crescentin structure disruption. Coloured bars show the fraction of cells (left axis) with each crescentin localization pattern at each time point. The black line shows mean cell curvature in μm⁻¹ for a population of cells (713<n<1919) at each time point (right axis); error bars indicate standard deviation. (E) Representative FlAsH-stained cells of CJW2788 strain taken at the noted intervals after addition of the xylose inducer of crescentin_ΔL1 synthesis. Only wild-type crescentin-TC is visualized with FlAsH. White arrows denote cells in which crescentin displays punctate localization pattern but that still retain some curvature. (F) CJW2778 cells preincubated in M2G⁺ with 0.3% xylose for 2 h, then mounted on M2G⁺ agarose pads with 0.3% xylose and imaged at the indicated time intervals. Arrows indicate examples of straight daughter cells produced by growth in the absence of an intact crescentin structure. (G) Experiment as in (F), but with 10 μg/ml chloramphenicol added to the agarose pad to halt cell growth. Bars, 2 μm.

Figure 3

Cell curvature is the result of a peptidoglycan growth differential around the cell circumference. (A) Strains lacking crescentin (CB15N ΔcreS), with wild-type levels of crescentin (CB15N), or overproducing crescentin (CB15N/pJS14creS). Bar, 2 μm. (B) DAPI staining (blue) overlaid with anti-crescentin immunofluorescence (red) of wild-type CB15N cells and cells overproducing crescentin (CB15N/pJS14creS). The line scan gives the fluorescence values along the yellow lines in the micrographs. Bar, 2 μm. (C) Transmission EM of uranyl acetate-contrasted peptidoglycan sacculi isolated from the three differently curved strains in panel A. As observed earlier (Poindexter and Hagenzieker, 1982), the isolated sacculi contained polyhydroxybutyrate (PHB) granules (arrow). Bar, 1 μm. (D) After digestion of the sacculi, muropeptides were subjected to HPLC to determine their relative composition. For the hypercurved CB15N/pJS14creS strain (CJW1430), which carries a plasmid encoding chloramphenicol resistance, we used a strain containing the empty vector (CB15N/pJS14; CJW1534) as a control; this strain exhibits wild-type curvature (data not shown). Major peaks 1–6 are proposed to have the following structures based on elution similarity to known E. coli muropeptides and to the published muropeptide profile from C. crescentus (Markiewicz et al, 1983): (1) disaccharide tetrapeptide, (2) disaccharide pentapeptide(Gly5), (3) disaccharide pentapeptide, (4) bis-disaccharide tetrapentapeptide(Gly5), (5) bis-disaccharide tetratetrapeptide and (6) bis-disaccharide tetrapentapeptide. (E) Schematic of how a cell length gradient along the long axis of the sidewall constitutes cell curvature. The sidewall displays a smooth gradient of lengths from the longest line, at the middle of the outer curvature (line a), through line b, at the middle of the gradient, to the shortest line, at the middle of the inner curvature (line c). (F) Transmission EM of peptidoglycan sacculi from a creS-overexpressing strain (CB15N/pJS14creS; CJW1430) and a ΔcreS strain carrying an empty vector (CB15N ΔcreS/pJS14; CJW2855) chased for 90 min after growth in D-Cys to reveal regions of new peptidoglycan insertion. D-Cys residues were biotinylated, then the sacculi were labelled with anti-biotin and gold-coupled anti-rabbit antibodies. Areas devoid of gold label show regions where new peptidoglycan material was incorporated during the chase. The antibodies used to detect the biotinylated D-Cys residues appeared to clump together. Measured lengths of the cleared region on each side of the sacculus are shown with broken lines. PHB, polyhydroxybutyrate granules. Bar, 0.5 μm.

Figure 4

Physical force can alter growth to produce cell curvature. (A) Time-lapse series of a ΔcreS cell (CJW1819; CB15N ΔcreS ftsZ∷pBJM1) grown in a circular agarose microchamber. Cell division was blocked by FtsZ depletion to promote cell elongation and contact with the chamber wall. Bar, 2 μm. (B) A CJW1819 cell released from a circular chamber, placed on an agarose pad (containing medium), and imaged just after release and then after 2 or 4 h of growth. (C) A cell released from a circular chamber and placed on an agarose pad containing 40 μg/ml chloramphenicol to block cell growth. Images show cell just after release and 1 and 9.5 h later.

Figure 5

Crescentin can produce cell curvature in E. coli. (A) Composite phase-contrast image (assembled from overlapping frames) of CJW2193 (E. coli MC1000/pBAD18[Cm]creS) cells grown in M9-glycerol supplemented with 0.2% arabinose (to induce crescentin production) for 4 h. The arrows indicate elongated, helical cells. The inset (bar 5 μm) shows the morphology of CJW2193 cells when grown in the presence of 0.2% glucose, which represses crescentin production. Bar, 10 μm. (B) CJW2194 cells (MC1000/pBAD18creS-tc) grown for 4 h in M9-glycerol with 0.2% arabinose and stained with FlAsH to visualize the crescentin-TC structure. Arrows show cell chaining, presumably caused by interference at cell division sites by the crescentin structure. Bar, 2 μm. (C) CJW2193 cells (E. coli MC1000/pBAD18[Cm]creS) grown in M9-glycerol with 0.2% glucose for 3.5 h (to repress crescentin production) or 0.2% arabinose for 4 h (to induce crescentin synthesis) in the presence of cephalexin (to block cell division). Bar, 5 μm. (D) SEM of a cephalexin-treated CJW2193 E. coli cell producing crescentin for 4.5 h. (E) Cephalexin-treated CJW2194 (MC1000/pBAD18[Cm]creS-tc) grown in M9-glycerol with arabinose for 4.5 h and stained with FlAsH to visualize the crescentin-TC structure. Bars, 2 μm.

Figure 6

Crescentin requires its basic N-terminus for cell envelope attachment. (A) FlAsH-stained crescentin_ΔN27-TC (red, laid over phase contrast, cyan) produced in a ΔcreS background (CJW2927; CB15N ΔcreS/pMR20creSΔN27-tc). A FlAsH-only micrograph is also provided for better visualization of the fluorescent signal. Bar, 2 μm. (B) FlAsH-stained crescentin_K12Q-TC (red, laid over phase contrast, cyan) produced in a ΔcreS background (CJW2818; CB15N ΔcreS/pMR10creS_K12Q-tc). Arrow indicates a cell in which the crescentin_K12Q-TC structure has detached from the cell membrane. Bar, 1 μm. (C) Cephalexin-treated CJW2199 cells (MC1000/pBAD18creSΔN27-tc) producing crescentin_ΔN27-TC induced for 4 h. Bar, 5 μm.

Figure 7

Model for crescentin action. (A) A rod-shaped cell lacking crescentin. Turgor pressure strains peptide bridges (red), which favours hydrolysis and new wall insertion. The glycan strands are represented as perpendicular to the long axis of the cell according to the prevailing view, but the model holds irrespective of the orientation of the glycan strands, as the peptide bridges are the extensible elements of the peptidoglycan. In such a cell, turgor pressure produces equal stress all around the circumference of the sidewall (3D view, a, b, c), and thus wall insertion is equally favoured all around the sidewall circumference. Cell elongation then causes straight elongation into a longer rod. (B) A cell with a crescentin structure (blue), which is affixed to the cell membrane in a stretched configuration. This in turn produces a compressive force on the cell wall (blue arrows), which slightly reduces local peptide bridge strain in a line corresponding to c in the 3D view. This small reduction in peptide bridge strain at line c is distributed in a smooth gradient of strain around the circumference of the sidewall because of the elasticity of the peptidoglycan fabric. Although it is too small a force to produce discernable morphological effects, the gradient of strain sets up a gradient of sidewall growth rates. Cell elongation under these conditions produces a gradient of cell lengths (from line a to line c), and thus curved morphology.