Akbary, Z. | bioRxiv Science

bioRxiv2025

Peptidoglycan turnover promotes active transport of protein through the bacterial cell wall

The bacterial cell wall is a critical load-bearing structure, but is not thought to be an important permeability barrier since proteins freely diffuse through isolated cell wall sacculi and bacteria secrete proteins without the aid of any known channels or transporters in the wall. Using new genetically encoded probes to measure the permeability of the cell in situ at the single-cell level, we discovered that the size threshold determining whether proteins can pass through the Bacillus subtilis sacculus is smaller than was previously thought. We found that transport of small proteins (<10 kDa) through the sacculus was consistent with passive diffusion through discrete pores, while larger proteins (>15 kDa) required the generation of larger pores by inducing peptidoglycan hydrolysis unbalanced by synthesis. These data are consistent with physics-based models of diffusion through a random percolation network of finite thickness. Conversely, the ability of the innate immune factor phospholipase (15.2 kDa) to kill B. subtilis was inhibited by membrane de-polarization. The protective effect of de-polarization was dependent on latent peptidoglycan synthesis (decoupled from cell growth) by PBP1 - highlighting a new role for this enzyme - and on reduced peptidoglycan hydrolysis. These results demonstrate that the rapid peptidoglycan turnover that drives cell growth also promotes movement of phospholipase across the cell wall, identifying a quintessentially bacterial mechanism of active transport. Significance StatementGram-positive bacteria, which include many serious pathogens like Staphylococcus aureus, Listeria monocytogenes, and Clostridium difficile, are defined by their thick peptidoglycan cell wall. Here, we demonstrate that this structure is a critical permeability barrier that blocks antibacterial proteins like those used by the human innate immune system. Furthermore, this barrier function depends on the physiological state of the cell: the wall of non-growing cells is less permeable because peptidoglycan turnover during growth actively promotes transport of specific proteins through the cell wall. This prokaryotic paradigm for molecular transport has important implications for host-pathogen interactions since pathogenic bacteria often assume both non-growing and growing states during infection.

microbiology↗

bioRxiv2024

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

microbiology↗

bioRxiv2022

The cell wall polymer initiates plasma membrane partitioning in mycobacteria

Lateral partitioning of proteins and lipids shapes membrane function. In model membranes, partitioning can be influenced by interactions with other membranes and solid supports. While cellular membranes can departition in response to various perturbations, including disruption of bilayer-extrinsic structures, the mechanisms by which they partition de novo are largely unknown. The plasma membrane of Mycobacterium smegmatis can be spatially and biochemically departitioned by the fluidizing agent benzyl alcohol. By screening for mutants that are sensitive to benzyl alcohol, we show that the bifunctional cell wall synthase PonA2 promotes membrane partitioning and cell growth upon fluidizer washout. PonA2s role in membrane repartitioning and regrowth depends solely on its conserved transglycosylase domain. We find that the cell wall polymer, but not active cell wall polymerization, is critical for membrane partitioning. Our work highlights a key initiating role for bilayer-extrinsic structures in patterning cellular membranes.

molecular biology↗