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Elucidating the mechanisms of outer membrane protein biogenesis

Principal Supervisor: Dr Tim Knowles - School of Biosciences

Co-supervisor: Ian Henderson

PhD project title: Elucidating the mechanisms of outer membrane protein biogenesis

University of Registration: University of Birmingham

Project outline:

This project focuses on the biophysical characterisation of the mechanisms involved in outer membrane protein biogenesis in Gram-negative bacteria with the overall aim of identifying novel targets for the development of the next generation of antimicrobials. The emergence of bacteria that are resistant to available antibiotics represents an enormous and growing global threat requiring new targets and strategies to combat infection. Multidrug resistance is most serious for Gram-negative bacteria, with essentially few antibiotics under development or likely to be available for clinical use in the near future. The understanding of the Gram-negative bacterial outer membrane (OM) is critical to developing new antimicrobial agents.

Proteins present within this membrane are the prime instruments of microbial warfare and play key roles in microbial pathogenesis, virulence and multidrug resistance, mediating many of the lethal processes responsible for infection and disease progression. Outer membrane proteins (OMPs) are also essential for cellular homeostasis allowing excretion of toxic substances, such as antibiotics, and uptake of nutrients.

The research in the laboratory of Dr Tim Knowles is focused on elucidating the mechanisms involved in the fundamental processes of OM biogenesis in Gram-negative bacteria and has several important objectives: (1) to provide fundamental information about how Gram-negative bacteria form and therefore by proxy mitochondria and chloroplasts. (2) To provide new opportunities to attenuate bacteria in the pursuit of anti-infective strategies. Current antibiotics predominantly target peptidoglycan synthesis and have been very effective in the past. Targeting OM biogenesis offers the potential for a whole new class of antimicrobials urgently required to stay ahead of bacterial resistance.

Focus - The Bam complex

A single OM complex, the β-Barrel assembly machine (Bam) complex, has been recognized as essential for the efficient insertion of almost all OMPs into the OM. It is ubiquitous throughout Gram-negative bacteria, however little is known about how it functions. The structures of the individual components have recently been identified but little information about how the components function as part of the complex nor how this complex can insert the myriad OMPs targeted to the OM has been determined. We are using novel techniques including neutron scattering, NMR and neutron reflectometry to probe the structure of this complex and how it functions. This understanding is critical as the design of compounds that inhibit this process would impede OMP biogenesis and therefore essential physiological, pathogenic and drug resistance functions.

To probe the mechanisms by which this fundamental process occurs will require a multidisciplinary approach, working in the fields of both biophysics and molecular biology and using the latest structural biology techniques including nuclear magnetic resonance, neutron and X-ray scattering, neutron reflectometry, electron microscopy and X-ray crystallography.

References:

  1. Browning, D.F., et al., Cross-species chimeras reveal BamA POTRA and beta-barrel domains must be fine-tuned for efficient OMP insertion. Mol Microbiol, 2015. 97(4): p. 646-59.
  2. Knowles, T.J., et al., Structure and function of BamE within the outer membrane and the beta-barrel assembly machine. EMBO Rep, 2011. 12(2): p. 123-8.

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

  • Electron microscopy
  • Nuclear Magnetic Resonance spectroscopy
  • Neutron Scattering
  • X-ray Scattering
  • X-ray crystallography
  • Novel polymer solubilisation techniques,
  • Mass spectrometry
  • Linear dichroism
  • Circular dichroism
  • Analytical ultracentrifugation
  • Isothermal titration calorimetry
  • Surface plasmon resonance
  • Protein expression and purification
  • Mutagenesis
  • SMALPs
  • Enzymatic activity
  • Ligand screening assays
  • Protein structure calculation and modelling
  • Cell-based assays
  • Western blots
  • SDS_PAGE
  • Genome sequencing

Contact: Dr Tim Knowles, University of Birmingham