Molecular basis of outer membrane stabilisation by the energised Tol-Pal system in Gram-negative bacteria

Bacteria are important for the health and well-being of most living organisms on earth. Examples in humans include their importance in our gut microbiomes for the digestion of food. Bacteria can also be pathogenic, meaning they can infect tissues and organs which, if left untreated, can be lethal. Much of modern medicine relies on the effective treatment of bacterial infections, such as pneumonia and sepsis, through the use of antibiotics. The rise of antibiotic resistance in bacteria is increasingly rendering many of our frontline antibiotics ineffective. The current proposal focuses on the outer membrane, one of the main factors contributing to antibiotic resistance in Gram-negative bacteria such as Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae. The outer membrane is a highly impermeable barrier that excludes many types of antibiotics that are otherwise effective against Gram-positive bacteria that lack an outer membrane. During the process of division in Gram-negative bacteria newly replicated daughter cells must have their individual outer membranes pinned to the underlying cell wall. This process requires the input of energy. Because the outer membrane is an 'energy-less' environment the requisite energy is provided by the electrochemical gradient across the inner membrane of the bacterium. A complex, multiprotein protein nanomachine known as Tol-Pal is tasked with this transfer of energy. Tol-Pal taps into this energy source to stabilise the outer membrane by an unknown mechanism. We discovered recently how Tol-Pal achieves this complex function, in the process identifying a biological mechanism we call 'mobilisation-and-capture'. The current proposal seeks to capitalise on these advances. We will investigate all the discrete interactions of the five Tol-Pal components and how they move in the membranes of the bacterium in order to understand how the cell's energy is exploited to pin the outer membrane to the cell wall. As part of this work, we will also exploit a new technology we have developed whereby energised complexes in Gram-negative bacteria can be trapped in their activated states for subsequent structural dissection using antibacterial proteins known as colicins. Through this work we will obtain the most detailed view yet of how the outer membrane is stabilised in Gram-negative bacteria and how nanomachines like Tol-Pal work to achieve this, knowledge that can be exploited in the future fight against bacterial infections.