Understanding outer membrane protein complex assembly in the Bacteroidetes

Bacteria can be divided into two types. Gram-positive bacteria have a thick cell wall on the cell surface and a single, cytoplasmic membrane. By contrast, Gram-negative bacteria such as E. coli have two membranes: a cytoplasmic (inner) membrane (IM) and an outer membrane (OM). Both membranes are separated by the periplasmic space that contains a thin cell wall. The OM is a unique lipid bilayer that is essential for most Gram-negative bacteria for two reasons. First, the OM is important for the mechanical stability of the cell. Second, the OM creates a physical barrier that prevents entry of harmful small molecules such as antibiotics, and therefore forms a protective layer on the outside of the cell. However, for the bacterium to grow, OM channels and other OM proteins ("OMPs") are needed, for example for the uptake of nutrients.

Like all proteins, OMPs are made in the cytoplasmic space in an unfolded form. Therefore, in Gram-negative bacteria, the newly-made, unfolded OMPs have to cross the IM and the periplasmic space before they arrive at their OM destination. There, they are folded into their active form and inserted into the OM. Both processes are carried out by an OMP complex named BAM, which stands for barrel assembly machine due to the fact that many OMPs form barrel-like structures. Due to its important functions, BAM is essential for the viability of all Gram-negative bacteria, and much research has been devoted in the past 20 years to determine how BAM works. However, most of this research has been done in model bacteria such as E. coli, and it is not clear whether BAM from very different bacteria, such as those from the human gut, has the same structure and functions in the same way as BAM from, for example, E. coli.

From preliminary experiments that form the basis of this proposal, we have good evidence that the BAM complex from an abundant group of human gut bacteria is in fact very different from E. coli BAM, both in structure but likely also regarding its functions. This proposal will characterise the functions of this novel BAM complex (named BtBAM) via three research aims.

In the first aim, we will determine the structure of BtBAM to a level that will allow building of complete atomic models for all the seven proteins that form the complex. This will be done via cryogenic electron microscopy (cryo-EM), in which individual, frozen protein molecules can be visualised and imaged at high magnifications. Knowing the detailed structure of BtBAM is important because it will allow us to hypothesise how all the individual components of this protein machine work together, much like it is only possible to understand how a car engine works if one can see what it looks like.

In the second aim, we will do experiments to get more information on the function of the different protein components of BtBAM. We will remove one or more components, followed by experiments to understand how that affects BAM function. For example, we can culture "mutant" bacteria with the modified BAM complex and compare growth with bacteria that still have the original complex. Due to its importance, bacteria with a defective BAM will likely grow slower, or not at all. For making more subtle changes in BtBAM, the structural information from aim 1 is also important, because it suggests where changes should be made. Besides growth experiments, we will also analyse which OMPs are present in the bacteria and their quantities in the OM, something that is likely to be affected if BAM doesn't function properly.

The final, third aim will be to study the function(s) of BtBAM in the test tube, rather than in live bacteria, to visualise more directly what BAM does.

Together, these experiments will clarify the structure and function of a BAM complex that is likely to be very different from that of model bacteria like E. coli. The project will show that even fundamental processes like OMP biogenesis can differ substantially in bacteria.

Gram-negative bacteria like E. coli have a cell envelope consisting of the cytoplasmic (inner) membrane (IM) and the outer membrane (OM). The OM is a unique bilayer and essential for viability of most Gram-negative bacteria. Due to the need to acquire nutrients and to interact with the extracellular environment, the OM contains peripheral lipoproteins and integral beta-barrel proteins. In model bacteria like E. coli, very few lipoproteins are exposed to the extracellular environment, and how they achieve this topology is mostly unclear. On the other hand, beta-barrel OM proteins (OMPs) are folded and integrated into the OM by a conserved, essential protein complex named BAM (beta-barrel assembly machine). While much is known about BAM structure and function, virtually all this knowledge is based on Proteobacterial research, and it is not clear whether BAM is different in other phyla.

Unlike Proteobacteria, the Bacteroidetes are characterised by the presence of many, functionally important, surface lipoproteins (SLPs), most of which form stable OMP complexes with beta-barrels. Reasoning that a putative SLP flippase might be associated with the BAM complex, we His-tagged BamA from Bacteroides thetaiotaomicron and, surprisingly, discovered that it forms a hitherto-unknown, seven-protein complex with a large extracellular domain, named BtBAM. We hypothesise that BtBAM forms a folding cage for the assembly of newly-flipped SLP and beta-barrels. This proposal will test this hypothesis via an interdisciplinary approach involving cryo-EM, quantitative proteomics, and in vivo and in vitro functional assays. The results will transform our knowledge on the fundamental process of OMP biogenesis within a widespread and important group of Gram-negative bacteria. In addition, given that the BAM complex is an attractive antibiotics target, large extracellular domains like that of BtBAM may provide opportunities to target OMP biogenesis in pathogenic Bacteroidetes.