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(T6SS)是许多革兰氏阴性菌所拥有的纳米机器。T6SS在功能和结构上类似于噬菌体收缩尾巴，作为一个倒置的噬菌体尾巴，将效应蛋白从细胞质通过内膜和外膜转移到靶细胞。通过T6SS分泌到靶细胞的蛋白质与病原菌的毒力和在竞争生态环境中的生存有关。自从最初在霍乱弧菌中发现T6SS以来，已经在包括球形红杆菌在内的许多非致病生物中发现了T6SS。由于其代谢多样性，球形罗氏杆菌能够占据过多的环境利基，使其成为环境合成生物学应用的理想底盘，例如在工业过程中生物膜的控制和操作是极其困难的，在工业过程中，由于阻挡、降解和污染设备，这些生物膜每年要花费数十亿美元。与浮游细菌相比，生物膜相关细菌对标准化学抗菌剂的敏感性较低。它们与一种水合的胞外聚合物基质有关，据信这会阻碍抗生素的扩散。将有毒效应蛋白直接输送到靶细胞细胞质的分泌系统是生物被膜控制的一个很有前途的候选系统。为了利用球藻的T6SS来设计用于此类应用的纳米机器，彻底了解T6SS在转录和翻译后水平的调节对于利用这一分子机器用于此类合成生物学应用是至关重要的。本研究旨在对T6SS在球藻中的调控提供完整的描述，阐明T6SS转录及其组分翻译后组装成分子注射器的调控机制。这项研究还旨在确定球孢杆菌T6SS所利用的效应蛋白，以期能够改变T6SS的有效载荷。一种能够将具有特定活性的预定效应器输送到靶细胞细胞质的纳米机器将是合成生物学工具包不可或缺的补充，它将提供一种控制生物膜的新方法，或者，如果系统在更合适的底盘中表达，则直接将治疗药物输送到靶细胞。BBSRC优先研究领域：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