Two independent spiral structures control cell shape in Caulobacter

Abstract

The actin homolog MreB contributes to bacterial cell shape. Here, we explore the role of the coexpressed MreC protein in Caulobacter and show that it forms a periplasmic spiral that is out of phase with the cytoplasmic MreB spiral. Both mreB and mreC are essential, and depletion of either protein results in a similar cell shape defect. MreB forms dynamic spirals in MreC-depleted cells, and MreC localizes helically in the presence of the MreB-inhibitor A22, indicating that each protein can form a spiral independently of the other. We show that the peptidoglycan transpeptidase Pbp2 also forms a helical pattern that partially colocalizes with MreC but not MreB. Perturbing either MreB (with A22) or MreC (with depletion) causes GFP-Pbp2 to mislocalize to the division plane, indicating that each is necessary but not sufficient to generate a helical Pbp2 pattern. We show that it is the division process that draws Pbp2 to midcell in the absence of MreB's regulation, because cells depleted of the tubulin homolog FtsZ maintain a helical Pbp2 localization in the presence of A22. By developing and employing a previously uncharacterized computational method for quantitating shape variance, we find that a FtsZ depletion can also partially rescue the A22-induced shape deformation. We conclude that MreB and MreC form spatially distinct and independently localized spirals and propose that MreB inhibits division plane localization of Pbp2, whereas MreC promotes lengthwise localization of Pbp2; together these two mechanism ensure a helical localization of Pbp2 and, thereby, the maintenance of proper cell morphology in Caulobacter.

Fig. 1.

MreC forms a helix that is nonoverlapping with MreB structures. (A) A schematic of the Caulobacter mre operon. (B) Cells of LS4272 expressing MreC-mRFP1, imaged with phase (Left) and fluorescence (Right). Arrows point to gaps at the division plane; arrowheads highlight puncta in the stalk. (C) MreC-mCherry imaged with deconvolution microscopy. From top to bottom, two optical sections (300 nm apart), a projection of seven Z sections, and a cartoon interpretation. Circle and line diagrams indicate the approximate position of the optical slice in a cross-section. (D) Cells of LS4279 expressing both MreC-mRFP1 and YFP-MreB imaged with (from left to right) phase, YFP, and mRFP1; images at Right are overlays of mRFP1 (magenta) and YFP (green) signals. (E) Deconvolution microscopy on GFP-MreB and MreC-mCherry expressed in LS4285. Shown from left to right are two optical sections (200 nm apart), a projection of 10 Z sections, and a cartoon interpretation. From top to bottom, images are GFP-MreB, MreC-mCherry, and an overlay (MreB in green and MreC in magenta). (Scale bars: 1 μm.)

Fig. 2.

mreC is essential, required for proper cell shape, and independent of MreB. (A) Viability of MreC-depletion strain (LS4275) grown in either permissive (red circles, +MreC) or nonpermissive (blue squares, -MreC) PYE media. (B) Phase (Left) and mRFP1 fluorescence (Right) of LS4275 grown with (Upper) or without (Lower) MreC-mRFP1 expression for 12 h in PYE. Fluorescence images were acquired by using identical exposure conditions and are scaled similarly. (C and D) Left is phase, Center is YFP-MreB, and Right is MreC-mRFP1 fluorescence. (C) LS4279 treated with A22 concurrent with MreC-mRFP1 and YFP-MreB expression for 6 h. (D) YFP-MreB localization in LS4278 after 24-h depletion of MreC-mRFP1 in M2GN. Arrows point to apparent helices and arrowheads to rings. (E and F) Top is phase, Middle is GFP (DivJ or PleC) and Bottom is MreC-mRFP1 fluorescence; arrows point to the stalked pole. Shown are representative cells expressing DivJ-GFP (LS4292) (E) or PleC-GFP (LS4293) (F) after 12 h of growth in PYE with (MreC⁺, Left) or without (MreC^-, Right) xylose. MreC-mRFP1 fluorescence in D-F has been enhanced to demonstrate the lack of protein in these cells. (Scale bars: 1 μm.)

Fig. 3.

Both MreC and MreB are required for helical GFP-Pbp2 localization. (A) Phase (Upper) and GFP fluorescence (Lower) images of LS4287 expressing GFP-Pbp2. (B and C) GFP-Pbp2 localized in the same cell with MreC-mCherry (LS4335) (B) or mCherry-MreB (LS4289) (C). Shown are projections of six to 10 deconvolved optical Z sections. From top to bottom are GFP (Pbp2), mCherry (MreB or MreC), and an overlay (Pbp2 in green, MreB or MreC in magenta, overlapping areas in white). Arrows in B highlight instances of colocalization; arrowheads in C point to the absence of Pbp2 at the division plane. (D and E) GFP-Pbp2 expressed while depleting for MreC for 24 h in M2GN (D) or treating with A22 (in M2GN + 0.03% xylose) for 6 h (E). Upper is phase; Lower is GFP fluorescence. Arrows denote division plane accumulations of GFP-Pbp2. (Scale bars: 1 μm.)

Fig. 4.

FtsZ depletion prevents A22-induced mislocalization of Pbp2 and change in cell shape. Swarmer cells of LS4290 were isolated and grown with or without FtsZ and with or without A22 for 6 h. Presented here is the primary mode of shape variation defined by PCA. Histograms display the distribution of parameter values (shapes) that were present in each population. Above the histograms are diagrams of cell shapes that correspond to the mean parameter value (μ) or various standard deviations (σ) away from the mean. To the right of each histogram are the median shape (a synthetic image generated from the PCA shape modes) and a representative real image (phase on left and GFP-Pbp2 on right) for that class.

Fig. 5.

Model for maintenance of proper cell morphology in Caulobacter.