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

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 control
biofilms 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