Investigating the transcriptional regulation of the type VI secretion system and its post-translational assembly in Rhodobacter sphaeroides - IBB, ENW

VI型分泌系统（T6 SS）是许多革兰氏阴性细菌所具有的纳米机器。T6 SS在功能上和结构上类似于噬菌体收缩尾，作为反向噬菌体尾将效应蛋白从细胞质通过内膜和外膜转运到靶细胞中。通过T6 SS分泌蛋白到靶细胞中与病原体毒力和竞争性生态环境中的存活有关。自其最初在霍乱弧菌中发现以来，T6 SS已在许多非致病性生物体中鉴定，包括球形红细菌。由于其代谢多样性，类球红球菌能够占据过多的环境小生境，使其成为用于环境合成生物学应用的理想底盘，例如生物膜的控制和操纵，这在工业过程中是非常有问题的，在工业过程中，它们每年通过阻塞、降解和污染设备而花费数十亿美元。生物膜相关细菌对标准化学抗菌剂的敏感性低于它们的亲脂性对应物。它们与水合细胞外聚合物基质相关，据信这会阻碍抗生素的扩散。将毒性效应蛋白直接递送到靶细胞的细胞质中的分泌系统是用于生物膜控制的有希望的候选者。为了利用类球红球藻的T6 SS来设计用于此类应用的纳米机器，对T6 SS在转录和翻译后水平的调节的透彻理解对于利用这种分子机器用于此类合成生物学应用是必不可少的。本研究的目的是提供一个完整的描述T6 SS的调节在R.sphaeroides，阐明背后的控制机制T6 SS转录和翻译后组装成一个分子注射器的组件。这项研究还旨在确定球形红球藻T6 SS利用的效应蛋白，以期能够改变T6 SS的有效载荷。一个能够将具有特定活性的预定效应物递送到靶细胞的细胞质中的纳米机器将是合成生物学工具包中不可或缺的补充，提供了一种控制生物膜的新方法，或者如果该系统在更合适的底盘中表达，则将治疗剂直接递送到靶细胞中。BBSRC优先研究领域在此提案中提出：合成生物学-生物纳米科学：利用和开发基于细胞系统的合成分子（纳米）机器

The type VI secretion system (T6SS) is a nanomachine possessed by many gram negative bacteria. T6SS is functionally and structurally analogous to the bacteriophage contractile tail, operating as an inverted phage tail to translocate effector proteins from the cytoplasm, through both the inner and outer membranes, into a target cell. The secretion of proteins via T6SS into target cells has been implicated in pathogen virulence and survival in competitive ecological environments. Since its original discovery in Vibrio cholerae, T6SS has been identified in many non-pathogenic organisms including Rhodobacter sphaeroides. R.sphaeroides is able to occupy a plethora of environmental niches due to its metabolic diversity rendering it an ideal chassis for use in environmental synthetic biology applications, such as the control and manipulation of biofilms which are extremely problematic in industrial processes where they cost billions of dollars annually by blocking, degrading and contaminating equipment. Biofilm associated bacteria are less susceptible to standard chemical antibacterial agents than their planktonic counterparts. They are associated with a matrix of hydrated extracellular polymeric substances which is believed to hinder the diffusion of antibiotics. A secretion system which delivers toxic effector proteins directly into the cytoplasm of the target cell is a promising candidate for biofilm control. In order to harness the T6SS of R.sphaeroides to engineer a nano machine for such applications a thorough understanding of the regulation of T6SS at both the transcriptional and post-translational level is essential to harness this molecular machine for such synthetic biology applications. This research aims to provide a complete description of the regulation of T6SS in R.sphaeroides, elucidating the control mechanisms behind T6SS transcription and the post-translational assembly of its components into a molecular syringe. This research will also aim to determine the effector proteins utilised by the R.sphaeroides T6SS with a view to being able to alter the payload of T6SS. A nanomachine with the ability deliver predetermined effectors with specific activities into the cytoplasm of target cells would be an indispensable addition to the synthetic biology toolkit, presenting a novel way to controlbiofilms or, should the system be expressed in a more appropriate chassis, deliver therapeutics directly into target cells.BBSRC priority research areas addressed in this proposal: Synthetic biology - Bio nanoscience: Utilising and exploiting synthetic molecular (nano) machines based on cellular systems