Principles of Surface Layer Biogenesis in Caulobacter crescentus

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Non-specialist Summary Prokaryotes, a group of single-celled organisms comprised of bacteria and archaea, form a cell envelope composed of multiple layers that serve variable biological functions. The outermost layer of many bacteria and well-studied archaea consists of a surface layer (or S-layer), a protein-based wall that encapsulates the entire cell. The extracellular proteins of the S-layer form a cell-bound 2D lattice, and are produced at such high numbers they are thought to be the most abundant family of protein in the world. The S-layer has been implicated in cell development, stress tolerance, drug resistance, and infection. Using cutting-edge cryo-electron microscopy techniques, we intend to build a microscopic image of how these protein-dense layers are synthesised on a microscopic level across multiple species, improving our understanding of how prokaryotic membranes are formed and maintained. Relevant BBSRC Priority Areas Combatting antimicrobial resistance: S-layers are formed by many pathogenic bacteria, such as Clostridium difficile, Bacillus anthracis, and Campylobacter fetus. S-layers have been shown to increase bacterial resistance to antimicrobials either directly by limiting their passage into the cell, or indirectly by promoting the formation of stress-tolerant biofilms which aberrate antibiotic efficacy. Understanding the methods by which S-layers are synthesised could reveal potential targets for therapies aimed at inhibiting their contribution to biofilm formation and drug resistance. Synthetic biology: This project will utilise strains of Caulobacter crescentus (along with other potential bacterial and archaeal species) engineered to express S-layers permissive to labelling with a range of materials. Additionally, the C. crescentus S-layer has long been attractive as a platform for protein production, protein analysis, and formation of 2D biomaterials, aided by the S-layers inherent stability, self-propagation, and high copy number in living cells. Building our knowledge of how the S-layer is secreted and formed will provide useful information when utilising these cells for synthetic biology approaches.