Wall teichoic acids regulate peptidoglycan synthesis by paving cell wall microstructure

The cell wall is a polymeric exoskeleton that defines the size and shape of bacteria; it is composed of peptidoglycan and, in Gram-positive bacteria, wall teichoic acid polymers. Two systems synthesize peptidoglycan in rod-shaped bacteria, which are found pervasively across bacterial clades: the multi-protein Rod complexes synthesize anisotropic peptidoglycan, which is required for rod shape because it reinforces the cell wall along its circumference1-3, whereas the non-essential enzyme PBP1 synthesizes isotropic peptidoglycan1,4 (Fig. 1A). In Gram-positive bacteria, rod shape also requires wall teichoic acids5 for unknown reasons. Here, we show that wall teichoic acids promote rod shape by preventing the formation of nanoscopic pores in the Bacillus subtilis cell wall, which lead to amorphous growth by activating PBP1 and inhibiting Rod complexes. Depleting wall teichoic acids resulted in pores within minutes, coinciding with a rapid increase in PBP1-mediated synthesis. PBP1s ability to sustain teichoic acid-less growth depended on its intrinsically disordered domain. In contrast to previous steady-state measurements6,7, we found that wall teichoic acid depletion caused the transient arrest of Rod complexes prior to the onset of amorphous growth. Finally, one of the two synthetically lethal cell wall hydrolases in B. subtilis8, LytE, became essential during wall teichoic acid depletion, meaning that PBP1 and LytE execute a novel, amorphous mode of growth. Collectively, our results identify the cell wall, via its molecular-scale structure, as a non-canonical auto-regulator of its own synthesis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/610702v3_fig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@154996forg.highwire.dtl.DTLVardef@12561c3org.highwire.dtl.DTLVardef@1356cacorg.highwire.dtl.DTLVardef@719d22_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1:C_FLOATNO Inhibiting wall teichoic acid synthesis decreases Rod complex activity prior to cell shape loss.(A) Schematic of Gram-positive cell wall synthesis. (B) Cell length growth rate [Figure 1] for wild-type cells during 0.5 g/mL tunicamycin treatment. Blue line shows smoothed population median, error bars show standard deviation. Data shown for 2,048 discrete cell tracks from 3 biological replicates. Inset: micrographs taken (i) before and (ii) after 45 min tunicamycin treatment. Scale bars 5 m. (C) Time course for the percentage of processive Mbl filaments during 0.5 g/mL tunicamycin treatment. Error bars show 95% confidence intervals across biological replicates (bootstrap analysis). Analysis performed on 62,901 discrete filament tracks from 4 biological replicates. Inset shows representative fluorescent kymographs of Mbl motion across the cell waist in (i) LB and (ii) after 30 min tunicamycin treatment. Red dotted line follows a processive Mbl filament. Scale bars 1 m. (D) Mean fluorescent puncta per unit cell length for wild-type, {Delta}ponA and ponATP-cells during tunicamycin treatment, labeled with fluorescent D-amino acids. Error bars show 95% confidence intervals. Analysis performed over 2,017 segmentations from 4 biological replicates (wild-type), 857 segmentations from 3 biological replicates ({Delta}ponA) and 257 cells from 2 biological replicates (ponATP-). All timepoints show statistical significance for difference in mean between cell types (Students T-test, P<0.01). Dots show individual replicates. Measurements at 50 min tunicamycin treatment used a lower exposure time so are plotted separately. Inset shows micrographs of (i,iii,v) wild-type and (ii,iv,vi) {Delta}ponA cells at (i,ii) 0 min, (iii,iv) 30 min, and (v,vi) 50 min of treatment. Micrographs are identically saturated across cell types. Scale bars 5 m. (E) Percentage of peptidoglycan subunits in monomers, measured by HPLC (Methods). Two biological replicates per strain. Mean values calculated across biological replicates. Error bars show 95% confidence intervals. Individual datapoints shown in red. Statistical significance calculated using Students T-test, P<0.05. C_FIG