Principles of Bacterial Cell-Size Determination Revealed by Cell-Wall Synthesis Perturbations

• Heterologous expression of cell-wall enzymes complements growth and alters cell width
• Cell-wall enzyme perturbation does not affect chemical composition of peptidoglycan
• Orientation of MreB motion and cell twisting are correlated with cell width
• Quantification of growth behaviors reveals different roles for MreB and PBP2

SummaryAlthough bacterial cell morphology is tightly controlled, the principles of size regulation remain elusive. In Escherichia coli, perturbation of cell-wall synthesis often results in similar morphologies, making it difficult to deconvolve the complex genotype-phenotype relationships underlying morphogenesis. Here we modulated cell width through heterologous expression of sequences encoding the essential enzyme PBP2 and through sublethal treatments with drugs that inhibit PBP2 and the MreB cytoskeleton. We quantified the biochemical and biophysical properties of the cell wall across a wide range of cell sizes. We find that, although cell-wall chemical composition is unaltered, MreB dynamics, cell twisting, and cellular mechanics exhibit systematic large-scale changes consistent with altered chirality and a more isotropic cell wall. This multiscale analysis enabled identification of distinct roles for MreB and PBP2, despite having similar morphological effects when depleted. Altogether, our results highlight the robustness of cell-wall synthesis and physical principles dictating cell-size control.

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