Principles of Surface Layer Biogenesis in Caulobacter crescentus

原核生物是一组由细菌和古细菌组成的单细胞生物，形成由多层组成的细胞包膜，具有不同的生物功能。许多细菌和研究充分的古生菌的最外层由表面层（或S层）组成，这是一种包裹整个细胞的蛋白质壁。S层的细胞外蛋白质形成细胞结合的2D晶格，并且以如此高的数量产生，它们被认为是世界上最丰富的蛋白质家族。S层与细胞发育、应激耐受性、耐药性和感染有关。使用尖端的冷冻电子显微镜技术，我们打算建立一个显微图像，这些蛋白质致密层是如何在多个物种的微观水平上合成的，提高我们对原核生物膜如何形成和维持的理解。相关BBSRC优先领域对抗抗菌素耐药性：S层由许多病原菌形成，如艰难梭菌、炭疽杆菌和胎儿弯曲杆菌。S-层已经显示出增加细菌对抗微生物剂的抗性，其直接通过限制它们进入细胞，或间接通过促进耐受应力的生物膜的形成，其改变抗生素功效。了解S层的合成方法可以揭示旨在抑制其对生物膜形成和耐药性的贡献的治疗的潜在靶点。合成生物学：该项目将利用新月柄杆菌菌株（沿着其他潜在的细菌和古细菌物种），这些菌株被工程化以表达允许用一系列材料标记的S层。此外，C. Crescentus S-层长期以来作为蛋白质生产、蛋白质分析和2D生物材料形成的平台一直很有吸引力，这得益于S-层固有的稳定性、自繁殖和活细胞中的高拷贝数。建立我们关于S层如何分泌和形成的知识将在利用这些细胞进行合成生物学方法时提供有用的信息。

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.