Summary Surface layers (S-layers) are proteinaceous crystalline coats that constitute the outermost component of most prokaryotic cell envelopes. In this study, we have investigated the role of metal ions in the formation of the Caulobacter crescentus S-layer using high-resolution structural and cell biology techniques, as well as molecular simulations. Utilizing optical microscopy of fluorescently tagged S-layers, we show that calcium ions facilitate S-layer lattice formation and cell-surface binding. We report all-atom molecular dynamics simulations of the S-layer lattice, revealing the importance of bound metal ions. Finally, using electron cryomicroscopy and long-wavelength X-ray diffraction experiments, we mapped the positions of metal ions in the S-layer at near-atomic resolution, supporting our insights from the cellular and simulations data. Our findings contribute to the understanding of how C. crescentus cells form a regularly arranged S-layer on their surface, with implications on fundamental S-layer biology and the synthetic biology of self-assembling biomaterials.

Surface layers (S-layers) are proteinaceous, two-dimensional crystals that constitute the outermost components of many prokaryotic cell envelopes. In this study, we investigated principles of S-layer biogenesis on the outer membrane in the bacterial model organism Caulobacter crescentus. Fluorescent microscopy revealed localised incorporation of new S-layer at the poles and mid-cell, consistent with elongation and division phases of the cell cycle. Next, light microscopy and electron cryotomography investigations of drug-treated bacteria revealed that bacterial actin homologue MreB is crucial for localised S-layer insertion. We further uncovered that S-layer biogenesis follows new peptidoglycan synthesis and localises to regions of high cell wall turnover. Finally, correlated cryo-light microscopy and electron cryotomographic analysis of regions of S-layer insertion showed the presence of gaps in the hexagonal S-layer lattice, contrasting with other S-layers completed by defined symmetric defects. Our findings provide insight into how C. crescentus cells form an ordered S-layer on their surface, providing evidence for coordination between the biogenesis of the cell envelope at multiple levels.