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Understanding Gram-negative envelope biogenesis and/or antimicrobial resistance using genome-wide approaches

Primary Supervisor: Dr Manuel Banzhaf, School of Biosciences

Secondary supervisor: Patrick Moynihan

PhD project title: Understanding Gram-negative envelope biogenesis and/or antimicrobial resistance using genome-wide approaches

University of Registration: University of Birmingham

Project outline:

Gram-negative bacterial cell envelopes hold special interest because of their dual property as both structural elements, and permeability barriers. The permeability barrier is conferred by the asymmetric lipid bilayer referred to as the outer membrane, which restricts cell entry for toxic compounds including many antibiotics. Understanding which genes within the bacteria are playing a role in maintaining this envelope is fundamental to understanding its biology.

Despite this, genome-wide screens to assay envelope integrity or antimicrobial resistance in Gram-negative bacteria are largely missing. The projects outlined will fill this knowledge gap and by combining systems and molecular biology. The Ph.D. candidate can choose between a combination of the following research objectives (RO).

RO_1: Development of a robust high-throughput method to assay Gram-negative envelope integrity

Using state-of-the-art high-throughput screening facilities the goal is to develop a robust method that allows to screen single-deletion libraries of Gram-negative bacteria against perturbations (antimicrobials, food preservatives etc.) in order to identify key enzymes for Gram-negative envelope biosynthesis. To illustrate the nature of such a project, I advise to read publication 1.

Training: microbial systems biology - design, application data-analysis of high-throughput screen;

RO_2: Characterisation of key players involved in Gram-negative biogenesis

The goal is to decipher the molecular mechanism of pathways and complexes important for Gram-negative envelope integrity. Investigated hits can be derived of RO_1, or based on excising preliminary data the Banzhaf laboratory possess. The Ph.D candidate can get trained on a broad range of molecular biological techniques available at the Banzhaf laboratory and its collaborators. To illustrate the nature of such a project, I advise to read publication 2.

Training: microbial cell biology, biochemistry, structural biology (Dr. Andrew Lovering, UoB), microscopy (Dr. KC Huang, Stanford University).

RO_3: Chemical-genomics approaches to understand mode of action of antimicrobials and food preservatives.

Bacterial chemical-genomics screens can quantify the impact of each gene on the fitness of the organism subjected to a large number of chemical/environmental perturbations. Chemical-genomics enables the discovery of gene function and facilitates the mapping of pathways, often leading to the identification of drug primary/secondary targets. The goal of this project is to profile single-deletion libraries of Gram-negative bacteria against environmental stress, antimicrobials and food preservatives. This will likely identify insights in the mode of action of perturbation tested and may lead towards new insights in envelope biogenesis. To illustrate the nature of such a project, I advise to read publication 3.

Training: microbial systems biology - design, application data-analysis of high-throughput screen;

  1. Brochado, A.R., Telzerow, A., Bobonis, J., Banzhaf, M., Mateus, A., Selkrig, J., Huth, E., Bassler, S., Zamarreno Beas, J., Zietek, M., et al. (2018). Species-specific activity of antibacterial drug combinations. Nature
  1. Typas, A., Banzhaf, M., van den Berg van Saparoea, B., Verheul, J., Biboy, J., Nichols, R.J., Zietek, M., Beilharz, K., Kannenberg, K., von Rechenberg, M., et al. (2010). Regulation of peptidoglycan synthesis by outer-membrane proteins. Cell
  1. Kritikos, G., Banzhaf, M., Herrera-Dominguez, L., Koumoutsi, A., Wartel, M., Zietek, M., and Typas, A. (2017). A tool named Iris for versatile high-throughput phenotyping in microorganisms. Nature microbiology

BBSRC Strategic Research Priority: Sustainable Agriculture and Food: Microbial Food Safety & Understanding the Rules of Life: Microbiology & Systems Biology

Techniques that will be undertaken during the project:

  • Microbial systems biology (genome-wide screens)
  • Bacterial cell biology (advanced genetics, basic biochemistry, basic microscopy)
  • Structural biology (in collaboration with Patrick Moynihan and Andrew Lovering)
  • Advanced microscopy (in collaboration with KC Huang in Stanford)

Contact: Dr Manuel Banzhaf, University of Birmingham