Biogenesis of Bordetella cell envelope components

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The envelope of Gram negative bacteria is complex. It comprises the inner membrane and the cell wall. The cell wall consists of a thin peptidoglycan structure surrounded by an asymmetric outer membrane bilayer consisting of phospholipids and lipopolysaccharides within which are attached or embedded various (lipo)proteins. The space between the inner and outer membranes is called the periplasm. In addition to functioning as a selective barrier and protecting bacteria from harsh conditions, the envelope plays a critical role in determining the outcome of interactions between the bacterium and its environment. Indeed, several components of the cell envelope are regulated in response to environmental conditions. Furthermore, the biogenesis of envelope components is also complex; targeting envelope components to specific locations requires dedicated molecular machinery and mechanisms that maintain the components in a state suited to being able to traverse one or more membranes. These mechanisms may also assist in promoting the function of the components once they have reached their destination. This proposal addresses components comprising the envelope of Bordetella species. Many Bordetella species are human or animal pathogens and there is much to learn about how the Bordetella envelope is made. We will focus on two aspects of the Bordetella envelope the biogenesis of surface proteins called autotransporters and a system that modifies the lipid A portion of lipopolysaccharide. We will characterize how two novel autotransporters with distinct and non-typical structures are secreted past the outer membrane. We will ascertain the requirement for accessory factors, how these autotransporter proteins interact with accessory factors, the role of the autotransporter C-terminus in effecting secretion, and how the proteins fold on the bacterial surface following secretion. With respect to lipid A modification, we will characterize the mechanism of how a group of proteins function together to modify lipid A with glucosamine at the inner membrane of the cell envelope. This process is highly regulated and is predicted to involve several regulatory systems, including a putative anti-sense RNA. We will investigate how this system is coordinated. Understanding how the envelope is built, especially in non model organisms can lead to uncovering fundamental principles of how membranes and membrane proteins and lipids are made and can also lead to improved methods for cell surface display or manipulation that can be used in bio and nanotechnology applications.