Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA.
School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts, USA.
Appl Environ Microbiol. 2019 Jun 17;85(13). doi: 10.1128/AEM.00434-19. Print 2019 Jul 1.
Curli are amyloid proteins that are assembled into extracellular polymeric fibers by bacteria during biofilm formation. The beta-sheet-rich protein CsgA, the primary structural component of the fibers, is secreted through dedicated machinery and self-assembles into cell-anchored fibers many times longer than the cell. Here, we have developed an fluorescence assay for curli production that exploits the fluorescent properties of Congo red (CR) dye when bound to amyloid, allowing for rapid and robust curli quantification. We initially evaluated three amyloid-binding dyes for the fluorescent detection of curli in bacterial culture and found only Congo red compatible with quantification. We further characterized the fluorescent properties of the dye directly in bacterial culture and calibrated the fluorescence using purified CsgA protein. We then used the Congo red assay to rapidly develop and characterize inducible curli-producing constructs in both an MC4100-derived lab strain of and a derivative of the probiotic strain Nissle. This technique can be used to evaluate curli production in a minimally invasive manner using a range of equipment, simplifying curli quantification and the development of novel engineered curli systems. Curli are proteins produced by many bacteria as a structural component of biofilms, and they have recently emerged as a platform for fabrication of biological materials. Curli fibers are very robust and resistant to degradation, and the curli subunits can tolerate many protein fusions, facilitating the biosynthesis of novel functional materials. A serious bottleneck in the development of more sophisticated engineered curli systems is the rapid quantification of curli production by the bacteria. In this work we address this issue by developing a technique to monitor curli production directly in bacterial cultures, allowing for rapid curli quantification in a manner compatible with many powerful high-throughput techniques that can be used to engineer complex biological material systems.
卷曲菌是一种在生物膜形成过程中由细菌组装成细胞外聚合纤维的淀粉样蛋白。纤维的主要结构成分富含β-折叠的 CsgA 蛋白，通过专用机制分泌，并自我组装成比细胞长许多倍的细胞锚定纤维。在这里，我们开发了一种用于卷曲菌产生的荧光测定法，该方法利用刚果红（CR）染料与淀粉样蛋白结合时的荧光特性，可快速、稳健地定量卷曲菌。我们最初评估了三种用于细菌培养物中卷曲菌荧光检测的淀粉样蛋白结合染料，发现只有刚果红与定量兼容。我们进一步在细菌培养物中直接表征了染料的荧光特性，并使用纯化的 CsgA 蛋白对荧光进行了校准。然后，我们使用刚果红测定法在 MC4100 衍生的实验室菌株和益生菌菌株 Nissle 的衍生物中快速开发和表征诱导产生卷曲菌的构建体。该技术可以使用各种设备以微创的方式评估卷曲菌的产生，简化卷曲菌的定量和新型工程卷曲菌系统的开发。卷曲菌是许多细菌作为生物膜结构成分产生的蛋白质，它们最近已成为制造生物材料的平台。卷曲菌纤维非常坚固且不易降解，并且卷曲菌亚基可以耐受许多蛋白质融合，从而促进新型功能材料的生物合成。更复杂的工程卷曲菌系统的开发中的一个严重瓶颈是快速定量细菌产生的卷曲菌。在这项工作中，我们通过开发一种直接在细菌培养物中监测卷曲菌产生的技术来解决这个问题，该技术可以快速定量卷曲菌，并且与许多强大的高通量技术兼容，这些技术可用于设计复杂的生物材料系统。
