Cell Diameter in Bacillus subtilis is Determined by the Opposing Actions of Two Distinct Cell Wall Synthetic Systems

Rod shaped bacteria grow by adding material into their cell wall via the action of two spatially distinct enzymatic systems: The Rod system moves around the cell circumference, while the class A penicillin-binding proteins (aPBPs) are unorganized. To understand how the combined action of these two systems defines bacterial dimensions, we examined how each system affects the growth and width of Bacillus subtilis, as well as the mechanical anisotropy and orientation of material within their sacculi. We find that rod diameter is not determined by MreB, rather it depends on the balance between the systems: The Rod system reduces diameter, while aPBPs increase it. RodA/PBP2A can both thin or widen cells, depending on its levels relative to MreBCD. Increased Rod system activity correlates with an increased density of directional MreB filaments, and a greater fraction of directionally moving PBP2A molecules. This increased circumferential synthesis increases the amount of oriented material within the sacculi, increasing their mechanical anisotropy and reinforcing rod shape. Together, these experiments explain how the combined action of the two main cell wall synthetic systems build rods of different widths, a model that appears generalizable: Escherichia coli containing Rod system mutants show the same relationship between the density of directionally moving MreB filaments and cell width.


151
We next examined how each PG synthetic system affected the rate of cell growth in our dual-induction strain using two

173
To gain a mechanistic link between the level of the PG synthetic systems to cell width, we sought to develop microscopic 174 measures of their activity. While the fraction of stationary aPBPs would be difficult to quantify, Rod complexes exhibit a 175 more quantifiable phenotype: As their motion is driven by PG synthesis, the cellular activity of the Rod system can be 176 measured by quantitating the number of directionally moving MreB filaments 37 . We developed an analysis method that, 177 using total internal reflection fluorescence microscopy (TIRFM) data, determines the density of MreB filaments moving 178 directionally around the cell. Filaments undergoing directed motion are detected by taking advantage of the temporal 179 correlation occurring between adjacent pixels across the cell width as objects move through them. These objects are then

200
To gain more insight into how an increased density of directional rod complexes can reduce rod width, we used polarization 201 microscopy to understand how increased circumferential synthesis affected the organization of material within the cell wall.

202
Polarization microscopy reports on both the angle and extent of orientation within optically anisotropic (or birefringent) 203 materials 39 , and has been used to assay the orientation of various materials, including plant cell walls [40][41][42]

214
Retardance is the differential optical path length for light polarized parallel and perpendicular to the axis of molecular 215 alignment; alternatively, it is defined as birefringence (∆n) times the physical path length through an anisotropic material.

216
Wide cells (high ponA, low mreBCD) had the lowest retardance, skinny cells (low ponA, high mreBCD) had the highest 217 retardance, and normal cells were in between ( Figure 4D). Because retardance depends on both the thickness of material 218 (path length) and the degree of its orientation (∆n), we normalized the retardance of each sample to the mean thickness of 219 the cell walls of each induction condition, obtained using transmission electron microscopy ( Figure S4A). This revealed that 220 the skinny cell walls had more highly ordered material (∆n) per path length (nm) of cell wall thickness, and that wide cell 221 walls had the least ( Figure 4E). Thus, in agreement with recent atomic force microscopy studies showing orientated glycans 222 in E. coli require MreB 16 , these experiments demonstrate that as Rod system activity increases, so does the amount of 223 oriented material in the wall.

225
The sacculi of E. coli and B. subtilis are mechanically anisotropic, stretching more along their length than across their width 226 44-46 . To test how the ratio of oriented to unoriented PG synthesis affected this property, we grew our dual-inducer strain at 227 the three mreBCD:ponA inductions above, labeled their walls with Alexa-488-D-amino carboxamides 4 , and assayed the 228 dimensions of their cell walls before and after hyperosmotic shocks (Figure 4F-G). This revealed that increased Rod system 229 activity correlated with an increased mechanical anisotropy of the sacculus: As we increased the expression of MreBCD 230 relative to PBP1A, rods shrank less across their width, and more along their length ( Figure 4H, S4B-C). Thus, the Rod 231 system acts to reinforce rod shape against internal turgor by promoting oriented PG synthesis around the rod. 232 233 E. coli Rod mutants also show a correlation between cell width and the density of directionally moving filaments.

234
Previous studies have examined how MreB and PBP2 mutations affect the shape of E. coli, hypothesizing that their 235 abnormal widths arise from changes in the curvature, twist, or angle of MreB filaments. Our observations in B. subtilis 236 suggested an alternative explanation: Abnormal width might arise from simply changing the amount of Rod system activity.

237
We tested this by measuring the density of directional GFP-MreB filaments in these same mutant E. coli strains. As a 238 benchmark, we first assayed the width of E.coli as we titrated the expression of mreB-sw-msfGFP using CRISPRi against 239 msfGFP 47 28 . As in B. subtilis, this yielded an inverse relationship between directional MreB filament density and cell width 240 ( Figure 5A). Examining each group of mutants showed the same result: 1) An identical trend was observed for the 241 mutations hypothesized to change filament twist 19 , 2) as well as in the mutations designed to change filament curvature 24 .

242
And 3) notably, the same correlation between directional filament density and cell width was observed in strains where E.

243
coli mrdA (PBP2) was replaced with mrdA genes from other species 22 . TIRF-SIM imaging of these MreB mutants revealed 244 some insight into these effects (Movie SM4): Some of the wide mutants showed either A) longer but fewer filaments, or B) a 245 large fraction of immobile filaments. Conversely, some thinner mutants appeared to have more, but shorter filaments. Finally

253
The shape of bacteria is defined by their cell walls; these experiments demonstrate that the two systems that insert PG into 254 it have opposing roles on its shape. Due to the intrinsic orienting of MreB filaments around the rod width 11 , the Rod system 255 inserts circumferentially oriented material around the rod circumference, reducing its diameter. As the number of MreB 256 filaments increases, so does the fraction of directional enzymes and the amount of oriented material in the wall. In contrast, 257 the aPBPs do not move circumferentially, inserting material that isotropically enlarges the sacculus. Our data indicates the 258 macroscopic shape of the sacculus arises from the nature of the material inserted into it: The more it is oriented around the 259 rod circumference by the Rod complex, the less the rod stretches across its width, and the more it stretches along its length 260 ( Figure 5C).

262
If the balance between the two PG synthetic systems is perturbed, the shape of the sacculus becomes altered, though its 263 rate of expansion remains constant. As both systems utilize the same pool of lipid II 49 , the flux through each may depend on 264 their relative levels; if one is reduced, the flux through the other may increase. This would explain why disrupting the Rod 265 system causes cells to swell 27,29,50-52 : In the absence of Rod-mediated thinning, aPBPs add more material uniformly over the 266 cell surface. Likewise, in the absence of aPBP-mediated widening, increased flux through the Rod system would explain 267 why cells become extremely thin 17,53 . However, if both systems are equivalently reduced, cells grow with normal widths, but 268 at slower rates; as long as the activities are balanced -identical shape arises from the balanced levels of enzymes, but 269 growth is reduced due to their combined activity becoming limiting. This would explain why ponA mutations rescue mreB 270 deletions 54 ; equally crippling both systems may rebalance the activities such that the cell retains normal shape and viability.

272
Implications for the role of MreB in rod width determination.

273
Given that 1) mreBCD from the wide bacterium B. megaterium creates close to normal diameter B. subtilis rods, and 2) B.

274
subtilis diameter depends on mreBCD levels, we find it unlikely that any property of MreB filaments defines a given cell 275 diameter. Rather, MreB appears to be one component of a rod-thinning system, working in opposition to aPBP-mediated

295
While many different activities affect the shape of the sacculus, such as its cleavage by hydrolases or rigidification by wall 296 teichoic acids 11 , the first process defining its geometry is the spatial coordination controlling where glycans are inserted into 297 it. While these experiments give a coarse-grained description into how each synthetic system affects cell shape, a fine 298 scale, mechanistic understanding remains to be determined -How does aPBP activity make cells wider? How does 299 increased Rod activity make cells thinner? Understanding the physical mechanisms causing these changes will require not 300 only a better understanding of the molecular architecture of the sacculus, but also an investigation into how the enzymes 301 downstream of glycan insertion affect the shape and mechanics of sacculi as they subsequently modify, remodel, and break 302 down the nascently-polymerized glycan architecture.

733
(G) Schematic of the MreB data simulations. Simulations were run using custom MATLAB code, with the following parameters:

734
Maximum fluorescence intensity was 300 counts and noise was 10 (approximating the signal to noise conditions in our imaging).

735
The simulated cell had a width of 1 μm and a length of 4 μm. We tested the robustness of our tracking by varying one parameter 736 while the others were fixed at the following values: Filament density was 3.2/μm 2 (corresponding to 40 MreB filaments per cell), 737 filament velocity was 30 nm/sec, filament orientation was 0 degrees, and filament length was 250 nm. This data was tracked using

748
As above, standard TIRFM imaging followed by correlated motion analysis, and 2) TIRF-SIM imaging, followed by tracking 749 filaments with particle tracking (using the same settings as in (G) above).

849
Cell wall thickness measurements were performed using a custom-built MATLAB (Mathworks, MA) script. Image 850 intensity profiles extracted from lines were drawn perpendicular to a user-input line defining the middle of the cell wall.

851
The distance between the two lowest points below a threshold within 40 nm of the middle of the cell wall was  Morphometrics software package 2 . Each "Width" data point (Figures 1-4) is calculated from at least 79 cells, but 863 most typically hundreds (see Table S1) from multiple fields of view across different areas of the agarose pad. Key 864 points in these experiments were repeated multiple times on independent days; including induction conditions for 865 bMD545, bMD598, and bMD620 that resulted in WT width and the extremes of thinning and widening; and PY79 866 measured in parallel as a width control. All repeat measurements gave similar mean values.

868 869
Measurements of single-cell growth rate

870
Cultures grown to an OD600 below 0.3 were concentrated by centrifugation at 6,000 x g for 30 sec. The cell pellet was 871 resuspended in growth medium, and applied to No. 1.5 glass-bottomed dishes (MatTek Corp., MA). All cells were 872 imaged under a 2% agarose pad containing growth medium, with the top surface exposed to air, in a chamber heated 873 to 37°C. Phase-contrast microscopy was performed using a Nikon Eclipse Ti equipped with a Nikon Plan Apo λ 874 100×/1.4NA objective and an Andor camera. We used a custom-built package in MATLAB to perform segmentation 875 on phase-contrast time-lapse movies, then calculated the growth rate of the surface area of single B. subtilis chains.

876
Each data point for the single-cell growth rates (Figure 2D)

889
Cultures grown to an OD600 below 0.3 were labelled with 10 nM JF549 3 , and were concentrated by centrifugation at 890 6,000 x g for 30 sec. The cell pellet was resuspended in growth medium and applied to ethanol-cleaned No. 1.5 891 coverslips. All cells were imaged under a 2% agarose pad containing growth medium with the top surface exposed to 892 air, in a chamber heated to 37°C. TIRFM and phase-contrast microscopy were performed using a Nikon Eclipse Ti 893 equipped with a Nikon Plan Apo λ 100×/1.45NA objective and a Hamamatsu ORCA-Flash4.0 V2 sCMOS camera.

894
Fluorescence time-lapse images were collected by continuous acquisition with 300 msec exposures. All data are from 895 a single experiment, where cells were induced at different levels, and tracks from >20 cells were used for analysis of 896 each data point.

898
Analysis of the density of directionally moving MreB Filaments.

899
Phase images of bacteria were segmented using Morphometrics 4 , and the width and length of each cell was 900 calculated. Next, the fluorescence time-lapses were analyzed based on the segmentation mask of the phase image 901 ( Figure S3C). Filament counting was performed in several steps ( Figure S3). First, kymographs were generated for 902 each row of pixels along the midline of the cell. Next, the kymographs for each row were placed side by side, 903 converting the TIRF time lapse data into a single 2D image (Figure S3D). To identify filaments in the kymograph, 904 closed contours were generated in the 2D image ( Figure S3E). We only selected contours within a given size range 905 (0.04 μm 2 to 0.17 μm 2 ). For these contours, we calculated the total intensity (the sum of the intensities of the pixels in 906 the contour), the centroid, the velocity (calculated from the slope of the major axis line of the contour) (Figure S3F),

907
and time (from the centroid). Next, to identify cases where the same MreB filament appears in multiple sequential 908 kymographs, each object in a given kymograph is linked to a corresponding object in the next and previous 909 kymographs based on the above properties of the object (see Figure S3F for details). Finally, the counting is verified 910 manually by numbering each filament on the 2D image ( Figure 3A). To test the performance of the filament counting,

911
we analyzed simulated data with different filament density, velocity, and orientation settings ( Figure S3).

919
The Image Correlation Spectroscopy 5 MATLAB package was used for the simulation of MreB moving around the cell.

920
The following parameters were set for the MreB simulations: velocity, orientation, filament number and filament length.

921
The default velocity setting is 30 nm/sec and the default orientation is 0, which means MreB moves perpendicular to

949
Particle tracking was performed using the software package FIJI 6 and the TrackMate v3.8 7 plugin. For calculation of 950 particle velocity, the scaling exponent α, and track orientations relative to the midline of the cell, only tracks persisting 951 for 7 frames or longer were used. Particle velocity for each track was calculated from nonlinear least squares fitting 952 using the equation MSD(t) = 4Dt + (vt) 2 , where MSD is mean squared displacement, t is time interval, D is the 953 diffusion coefficient, and v is speed. The maximum time interval used was 80% of the track length. To filter for 954 directionally moving tracks, we discarded those with a velocity lower than 0.01 nm/sec. Tracks were also excluded if 955 the R 2 for log MSD versus log t was less than 0.95, indicating a poor ability to fit the MSD curve.

958
Overnight, exponentially growing cultures (as described in "Media and culture conditions") were diluted into fresh CH 959 medium, grown at 37°C to an OD600 of 0.1 to 0.

978
We used complementarity-based CRISPR knockdown to titrate the MreB expression level in E. coli. The degree of 979 MreB-SW-msfGFP repression is controlled by introducing mismatches between the guide RNA and the target DNA 9 .