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Genetic analysis of the bacterial translocation machinery

Primary Supervisor: Dr Damon Huber, School of Biosciences

Secondary supervisor: Manuel Banzhaf

PhD project title: Genetic analysis of the bacterial translocation machinery

University of Registration: University of Birmingham

Project outline:

Every compartment of a cell contains proteins, and yet all of these proteins are initially synthesised in the cytoplasm. In most cells, the first step in the correct localisation of the vast majority of these proteins is translocation across or insertion into the cytoplasmic membrane by the Sec machinery. In bacteria, the core Sec machinery consists of an evolutionarily conserved protein-conducting channel in the cytoplasmic membrane, SecYEG, and a bacteria-specific ATPase, SecA. Numerous studies have probed the molecular mechanism of protein translocation by SecA and SecYEG in ever greater detail.

Nevertheless, there remain significant gaps in our knowledge about how the Sec machinery functions in living cells. For example, an array of auxiliary Sec components (e.g. SecD, SecF, YajC, YidC) associate with SecYEG in vivo and assist translocation. However, it is not clear what these proteins are doing. Furthermore, recent work by our group indicates that there is at least one previously undiscovered accessory Sec component (which we have named AscA) that assists translocation, and there could be more. Understanding the functions and molecular mechanisms of these components will be critical to understanding this fundamental biological process. In addition, insight into the mechanism of protein translocation in vivo would allow us to engineer cells that more efficiently produce secreted proteins and could lead to advances in protein production for biotechnology.

The goal of this project will be to determine the functions of these auxiliary and accessory Sec components in vivo. The student will use high-throughput genetic screening methods to discover these functions and potentially to discover new Sec accessory components. In addition, the student will cutting-edge methodologies in molecular genetics and biochemistry to investigate the molecular mechanism of these components in vivo.

Reference:

  1. Cranford Smith T, Jamshad M, Jeeves M, Alanazi M, Carter C, Lovett JE, Knowles T, Huber D. Iron Is a Physiological Ligand of SecA-like Metal Binding Domains. BioRxiv. 613315; doi: 10.1101/613315  

BBSRC Strategic Research Priority: Understanding the Rules of Life­­­: Microbiology

Techniques that will be undertaken during the project:

  • Molecular biology
  • Molecular genetics
  • High-throughput sequencing
  • High-throughput screening
  • Bioinformatics

Contact: Dr Damon Huber, University of Birmingham