Characterisation of YtfM: a conserved protein implicated in outer membrane biogenesis in Gram-negative bacteria

Many bacteria, including some that cause disease in humans, animals and plants possess a protective surface membrane composed of protein, lipid and carbohydrate. This membrane is called the outer membrane and bacteria that posses an outer membrane are known as Gram-negative bacteria. The outer membrane provides a first line of defence against harmful substances such as antibiotics, and enables these bacteria to colonise a variety of different and often hostile environments. The outer membrane also provides protection to bacteria against our own defences during infection. Examples of Gram-negative bacteria that cause disease in humans include; Escherichia coli, which causes severe food poisoning and urinary tract infections and Pseudomonas aeruginosa, which causes a range of conditions in people with a weak immune system. A growing problem in the treatment of Gram-negative bacteria is an increasing incidence of antibiotic resistance. This situation is compounded since in recent years few new types of antibiotic active against Gram-negative bacteria have been discovered. One possible alternative to the discovery of new antibiotics is to make bacteria more sensitive to existing antibiotics. How bacteria synthesise their outer membrane is quite poorly understood, but it is known that if outer membrane formation is disrupted bacteria are much more easily killed by antibiotics, even becoming sensitive to antibiotics to which they were previously resistant. If we are able to identify the protein components involved in outer membrane formation we can either design or select molecules that interfere with it. In this way we can potentially both extend the usage of existing antibiotics and make bacteria sensitive to substances to which they were previously resistant. The overall goal of my research is to understand how the process of outer membrane biogenesis occurs and to identify the cellular components responsible for this process. We have recently identified an outer membrane protein (YtfM), which is required to render the outer membrane impermeable to large antibiotics. This protein is found in many Gram-negative bacteria indicating an important cellular function. It also shares important similarities with proteins that are known to be involved in the correct assembly of membrane proteins in Gram-negative bacteria, plants and animals. Our hypothesis is that YtfM is also involved in membrane protein biogenesis. We wish to identify the proteins that YtfM inserts into the outer membrane and also any other proteins that participate in this process by forming a complex with YtfM. We will then characterise the YtfM complex to gain a full understanding of how it functions.

The loss of proteins involved in biogenesis of the outer membrane of Gram-negative bacteria leads to a loss of membrane integrity that is characterised by a dramatic increase in sensitivity to certain antibiotics. We have shown that in E. coli the loss of YtfM, a member of the Omp85/YaeT superfamily of proteins gives rise to this phenotype. Omp85/YaeT family proteins are a conserved feature of the multi-protein complexes that responsible for the insertion of beta-barrel membrane proteins into the outer membrane of mitochondria, chloroplasts and Gram-negative bacteria. Our hypothesis is that YtfM forms part of a membrane bound protein complex responsible for the correct membrane insertion of a subset of the E. coli outer membrane proteome. The key aims of this research are to: (i) identify the substrate(s) of YtfM. (ii) Identify E. coli cell envelope proteins that interact with YtfM. (iii) Determine the oligomeric state of the native YtfM complex.