Abstract
The peptidoglycan cell wall is an integral organelle critical for bacterial cell shape and stability. Proper cell wall construction requires the interaction of synthesis enzymes and the cytoskeleton, but it is unclear how the activities of individual proteins are coordinated to preserve the morphology and integrity of the cell wall during growth. To elucidate this coordination, we used single-molecule imaging to follow the behaviours of the two major peptidoglycan synthases in live, elongating Escherichia coli cells and after perturbation. We observed heterogeneous localization dynamics of penicillin-binding protein (PBP) 1A, the synthase predominantly associated with cell wall elongation, with individual PBP1A molecules distributed between mobile and immobile populations. Perturbations to PBP1A activity, either directly through antibiotics or indirectly through PBP1A's interaction with its lipoprotein activator or other synthases, shifted the fraction of mobile molecules. Our results suggest that multiple levels of regulation control the activity of enzymes to coordinate peptidoglycan synthesis.
Highlights
The peptidoglycan cell wall is an integral organelle critical for bacterial cell shape and stability
PAmCherry-PBP1A/B molecules in live E. coli cells growing at 30 °C were imaged with total internal reflection fluorescence (TIRF) microscopy every 30 ms (Fig. 1a)
PBP1A and PBP1B inactivation with cefsulodin had no effect on growth rate during elongation (Supplementary Fig. 9), in agreement with the previous observation that MreB speed is not affected by cefsulodin treatment[22], suggesting that either PBP1A is not essential during elongation or that another peptidoglycan synthase is active in its place
Summary
The peptidoglycan cell wall is an integral organelle critical for bacterial cell shape and stability. We observed a significant decrease in diffusive motion in both the presence (Fig. 2a, Po0.0001, permutation test) and absence (Supplementary Fig. 5, Po0.0001, permutation test) of PBP1B, suggesting that the motion of PBP1A reflects its enzymatic state inside living cells and that motion slows when PBP1A’s transpeptidase activity is inhibited.
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