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How key protein interactions drive bacterial flagella assembly

Primary Supervisor: Dr Chrystala Constantinidou, WMS

Secondary supervisor: Dr Nick Waterfield

PhD project title: How key protein interactions drive bacterial flagella assembly

University of Registration: University of Warwick.

Project outline:

Overview

Studies will focus on investigating how specific amino acid mutations in key flagella structural proteins affect protein-protein interactions, the rate of assembly, placement, structure and function of the C. jejuni flagella.

Background

Flagella are complex structures formed by up to 100 different proteins with different structural and regulatory functions. They are one of the main organelles that impart locomotion on bacteria, allowing them to move towards favourable environments. The roles of flagella in motility and chemotaxis have always been considered important for the virulence and survival of bacterial pathogens. More recently, accumulating evidence has been pinpointing to more diverse roles such as adhesion, biofilm formation, virulence factor secretion and modulation of the immune system of eukaryotic cells.

It is not therefore surprising that the structure and regulation of flagella has been extensively studied especially in model organisms such as E. coli and Salmonella. Fundamentally these macromolecular structures look similar across different species. The assembly of the structural proteins follows a specific order, beginning from the inner end of the construction (cytoplasm and inner membrane) and finishing at the outer end of the organelle outside the bacterium. Flagellar gene expression is also mediated in a hierarchical manner ensuring the timely expression of the flagella structural components. Adding to the complexity is the diverse patterns and numbers of flagella different bacteria species possess. It is therefore unsurprising that the regulatory networks that govern the expression of the flagella- associated genes also vary; for example bipolar bacteria like C. jejuni and unipolar bacteria such as H. pylori, lack the flagella master regulon present in E. coli and Salmonella.

flhF is a gene thought to play a key role in the temporal and spatial activation of the genes required for the completion of the flagella structure in polar bacteria. Evidence of its exact role from studies in a number of different bacterial species, is so far equivocal. C. jejuni cells lacking flhF do not make flagella; none the less in our lab we have isolated pseudorevertants that are motile. Studies based on these pseudoreverants have helped us gather evidence that suggests that FlhF plays a role (directly or indirectly) in ensuring the initial flagella structure embedded in the inner membrane has the correct conformation so as to enable the efficient secretion of the rest of the flagella components. A recent study in the Vibrio species suggested that FlhF plays a role in the recruitment of these early flagellar components to the bacterial poles and excitedly the structure of the main component of this early structure, FliF, has finally been elucidated for Salmonella earlier this year.

We will use our novel and unique set of pseudorevertants to determine which protein-protein interactions and structural changes are crucial both in generating the signals required for the initiation of the flagella assembly and targeting and ensuring efficient energy transduction required for both flagella assembly and motility.

The project will complement ongoing work focused on computational modeling and cryoEM analysis of the C. jejuni flagella structures in collaboration with Dr Peter Bond at the A*STAR Bioinformatics Institute, Singapore and Dr Saskia Bakker at the Advanced Bioimaging RTP at University of Warwick.

References:

  1. Johnson, S., Fong, Y.H., Deme, J.C. et al. Symmetry mismatch in the MS-ring of the bacterial flagellar rotor explains the structural coordination of secretion and rotation. Nat Microbiol 5, 966–975 (2020). https://doi.org/10.1038/s41564-020-0703-3
  2. Terashima, H., Hirano, K., et al. Assembly Mechanism of a Supramolecular MS-Ring Complex To Initiate Bacterial Flagellar Biogenesis in Vibrio Species. Journal of Bacteriology, 202 (16), e00236-20 (2020). https://doi.org/10.1128/JB.00236-20
  3. Lertsethtakarn, P., Ottemann, K.M., and Hendrixson, D.R. Motility and chemotaxis in Campylobacter and Helicobacter .” Annual review of microbiology, 65, 389-410, (2011). https://doi:10.1146/annurev-micro-090110-102908

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

      Techniques that will be undertaken during the project:

      • Fundamental microbiology skills including working with Cat 2 pathogenic bacteria and carrying out phenotypic assays such as motility
      • Basic molecular biology techniques, including cloning, protein expression and mutagenesis
      • Protein-protein interactions using a variety of biochemical techniques including co-immunoprecipiation, cross-linking followed by mass spectroscopy, subcellular fractionation
      • Proteomics experiments and data analysis
      • Microscopy (CryoEM/Immunofluorescence)

      Contact: Dr Chrystala Constantinidou, University of Warwick