Understanding outer membrane protein biogenesis in Bacteroidota bacteria

Gram-negative bacteria are distinguished by the presence of an outer membrane (OM) at the cell periphery. This membrane is the site at which the bacterium interacts with its environment (or host if a pathogen), and provides the first line of defence against antibiotics, mechanical stresses, and immunological attacks. These functions depend on the presence of proteins that either span the outer membrane (Outer Membrane Proteins, or OMPs) or are anchored to the membrane by a lipid tail (lipoproteins). Sophisticated machinery is required to target, insert, and fold these proteins. Although this machinery is well-characterised in Escherichia coli and related organisms, it remains unclear how OM proteins are assembled in other major Gram-negative phyla. In this project we aim to elucidate novel features of OM protein biogenesis in bacteria of the phylum Bacteroidota (formerly Bacteroidetes). The Bacteriodota are key human commensals, but also include major anaerobic pathogens, and are environmentally abundant. The pathways for OM protein biogenesis in the Bacteroidota differ from the E. coli paradigm in at least two major ways that we will investigate. First, we have discovered that the Bam complex that is responsible for inserting OMPs into the OM has a very different composition from the E. coli paradigm and includes novel extracellular subunits. We now seek to identify the function(s) of these additional subunits and elucidate their molecular mechanism. Second, in contrast to E. coli, the cell surface of Bacteroidota bacteria is coated with abundant lipoproteins (surface lipoproteins, or SLPs) that must be exported across the OM by a currently unknown mechanism. We aim to identify and characterise this SLP transporter as well as the novel pathway that our work has shown is needed for delivering SLPs to the OM. To carry out these tasks we will employ in vivo and in vitro protein biochemistry, bacterial genetics, and structural analysis by cryoEM.