Cell shape and cell-envelope integrity of bacteria are determined by the peptidoglycan cell wall. In rod-shaped Escherichia coli, two conserved sets of machinery are essential for cell-wall insertion in the cylindrical part of the cell: the Rod complex and the class-A penicillin-binding proteins (aPBPs). While the Rod complex governs rod-like cell shape, aPBP function is less well understood. aPBPs were previously hypothesized to either work in concert with the Rod complex or to independently repair cell-wall defects. First, we demonstrate through modulation of enzyme levels that aPBPs do not contribute to rod-like cell shape but are required for mechanical stability, supporting their independent activity. By combining measurements of cell-wall stiffness, cell-wall insertion, and PBP1b motion at the single-molecule level, we then present evidence that PBP1b, the major aPBP, contributes to cell-wall integrity by repairing cell wall defects.
The peptidoglycan (PG) sacculus provides bacteria with the mechanical strength to maintain cell shape and resist osmotic stress. Enlargement of the mesh‐like sacculus requires the combined activity of peptidoglycan synthases and hydrolases. In Escherichia coli, the activity of two PG synthases is driven by lipoproteins anchored in the outer membrane (OM). However, the regulation of PG hydrolases is less well understood, with only regulators for PG amidases having been described. Here, we identify the OM lipoprotein NlpI as a general adaptor protein for PG hydrolases. NlpI binds to different classes of hydrolases and can specifically form complexes with various PG endopeptidases. In addition, NlpI seems to contribute both to PG elongation and division biosynthetic complexes based on its localization and genetic interactions. Consistent with such a role, we reconstitute PG multi‐enzyme complexes containing NlpI, the PG synthesis regulator LpoA, its cognate bifunctional synthase, PBP1A, and different endopeptidases. Our results indicate that peptidoglycan regulators and adaptors are part of PG biosynthetic multi‐enzyme complexes, regulating and potentially coordinating the spatiotemporal action of PG synthases and hydrolases.
Background:The YvcK protein is essential for Bacillus subtilis growth on gluconeogenic conditions; its overproduction rescues an mreB mutant. Results: PrkC phosphorylates YvcK; this phosphorylation is not required for growth on gluconeogenic conditions but is necessary for mreB rescue. Conclusion: YvcK phosphorylation is specifically involved in B. subtilis morphogenesis. Significance: This phosphorylation-based regulatory mechanism could be widespread in bacteria.
8Cells exhibit a high degree of intracellular crowding. To control the level of crowding during growth cells 9 must increase their volumes in response to the accumulation of biomass. Using Escherichia coli as a model 10 organism, we found that cells control cell volume indirectly, by increasing cell-surface area in proportion to 11 biomass growth. Thus, dry-mass density, a readout of intracellular crowding, varies in proportion to the 12 surface-to-volume ratio, both during the cell cycle and during perturbations such as nutrient shifts. On long 13 time scales after shifts, initial dry-mass density is nearly restored by slow variations of the surface-to-mass 14 ratio. Contrary to a long-standing paradigm, cell-envelope expansion is controlled independently of cell-15 wall synthesis but responds to the activity of cell-wall cleaving hydrolases. Finally, we observed rapid 16 changes of Turgor pressure after nutrient shifts, which were likely responsible for initial changes of cell 17 diameter and dry-mass-density. Together, our experiments reveal important regulatory relationships for 18 cell volume and shape.
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