Delivery and clearance of outer membrane proteins to the bacterial outer membrane

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Gram-negative bacteria are a type of micro-organism that are absolutely pervasive in Nature, and we lead lives that are intimately entangled with their biology. such bacteria and have enormous implications for our everyday lives, because they live in our guts, infect our bodies, and are in our environment. They therefore impact human health and wellbeing, are important for biotechnology, sustainable agricultural economies and much more. Gram-negative bacteria differ from other bacteria in that they possess an outer membrane (or OM) that is rich in proteins and complex lipopolysaccharides (LPS). This OM plays a vital role in protecting the bacteria from their environment, for example it is a formidable barrier to toxins, including those such antibiotics that we intentionally use to try to poison them. The OM is critical for their normal growth, and for their pathogenesis in plant, animal and human disease. However, the creation of the OM is uniquely challenging because each component from which it is made, whether that be a protein or lipid molecule, is made inside the bacterial cell, and exported across the inner membrane to a space between the inner and outer membranes called the periplasm. Those components then have to before be incorporated into the OM. The periplasm is devoid of ATP, the molecule that is typically used to power such processes across Nature and so the choreography of the different steps in OM biogenesis must be powered and controlled in a different way to typical protein machineries inside the cell. Bacteria have thus evolved elaborate machineries to build, maintain and adapt their OMs, dependent on growth conditions. A single, essential protein complex, the beta-barrel assembly machinery (or BAM complex), is required to fold and insert outer membrane proteins (OMPs) into the OM. Although substantial recent progress has revealed the structure of BAM and given initial insights into the mechanisms by which OMPs may be inserted into the OM, two critical areas of BAM function remain poorly characterised, namely how are OMPs delivered to BAM and what happens if OMP folding stalls? The rates of OMP synthesis, folding and degradation are finely balanced, and their dysregulation can be bactericidal1. Understanding how these processes operate is thus crucial to understanding OM biogenesis and may reveal an Achilles heel by which bacterial growth can be halted. The focus of this application is to provide new insight into these important questions. We will use an integrated structural molecular biology approach to determine the 3D structure of complexes between BAM and the periplasmic molecular chaperone SurA, which delivers OMPs to BAM, and complexes that include substrate OMPs. We will then use mass spectrometry methods to look at the dynamics and interactions made in such complexes, and functional assays to understand how OMP delivery works. We will also look at what happens when OMP folding goes wrong, and how BAM cooperates with proteins that degrade misfolded OMPs, using the same toolkit of techniques.