Understanding the role of the Bam complex in the biogenesis of outer membrane proteins

Bacterial species can be divided into two basics groups: Gram-positive and Gram-negative. The major difference between these two groups is the number of cell membranes that each possesses. Thus, Gram-positive bacteria possess one membrane called the cytoplasmic membrane and Gram-negative bacteria possess two called the inner membrane and the outer membrane. The cytoplasmic membrane of Gram-positive bacteria and the inner membrane of Gram-negative bacteria share many of the same features and function in much the same way. In the case of Gram-negative bacteria the outer membrane is essential for survival by providing protection against a myriad of antibacterial agents. However, the presence of the outer membrane also poses problems for the bacterium; toxic substances produced by the bacterium have to be transported across the outer membrane out of the cell and nutrients have to be harvested from outside the cell and transported across the outer membrane into the cell. Additionally, survival of bacteria within a particular environment often requires the production of proteins which are inserted into the membrane and secreted outside the cell. These factors have necessitated the development of sophisticated biological machines to assemble components of the outer membrane and to secrete proteins outside the cell.

One component of this assembly machinery that was recently discovered is a protein termed Omp85. This protein is found in all Gram-negative bacteria and is essential for bacterial survival. Interestingly, this protein is also found in mitochondria. These are minute organs that supply energy to human, animal and plant cells; without the Omp85 protein the mitochondria cannot function and the cells cannot survive. In addition, this protein is also found in chloroplasts; the minute organs of plants cells than allow plants to convert carbon dioxide to oxygen; Omp85 is also essential for survival of chloroplasts.

The Omp85 homologues represent a fundamentally and important target for drugs. Since Omp85 is conserved in all Gram-negative bacterial species it may be possible to design strategies to inhibit the function of Omp85 and thus block pathogenesis of many diseases. However, with little understanding of how Omp85 functions this therapeutic potential cannot be effectively unlocked or exploited. This project aims to investigate how this protein functions by using a variety of techniques including genetic mutations and biochemistry.

The Escherichia coli protein YaeT (BamA) belongs to the Omp85 family of essential outer membrane proteins in Gram-negative bacteria. However, there is little biochemical understanding of how this protein functions. YaeT is an excellent choice for investigation due to the comprehensive understanding of the physiology and membrane structure of the model organism E. coli. We aim to

1. Perform structure-function analysis of the protein by defining regions critical for function. By monitoring the location of our tagged YaeT molecule in a YaeT competent background and subsequently expressing the same molecule in a depletion strain we will demonstrate which domains are critical for function and those that are critical for the structural integrity of the molecule.

2. Define which domains have affinity for the C-terminal tri-peptide motif of outer membrane proteins. Previous investigations have suggested that YaeT binds to peptides derived from PhoE. Using a variety of biophysical measurements we will demonstrate whether the POTRA domains are specifically capable of binding these peptides as suggested by the current dogma, and whether this is dependent on the C-terminal motif.

3. Determine whether the proteins function in a species specific manner. Current theories suggest Omp85 proteins are species specific. We have demonstrated that the Omp85-like protein from Salmonella enterica Typhimurium is capable of complementing an E. coli YaeT depletion strain, thus it is not strictly species specific. Using chimeric proteins and a variety of homologues we will demonstrate whether Omp85-like proteins from other species are capable of complementing the E. coli YaeT synthetic lethal effect, determining the level of species specificity and domains specificity.