Cell Cycle Dependent Orchestration of Surface Layer Biogenesis in Caulobacter crescentus

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.