Biogenesis and modification of the Bordetella cell envelope

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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 (LPS) within which are attached or embedded various proteins. The space between the inner and outer membranes is called the periplasm. In addition to functioning as a selective barrier to protect 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. 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 - a system that modifies the lipid A portion of lipopolysaccharide and the biogenesis of an unusual member of the autotransporter class of outer membrane proteins. We have discovered a 5 gene locus, called lgm, that modifies lipid A with glucosamine. These genes are arranged in what appears to be a non-contiguous operon, a recently described genetic architecture that has been shown to coordinate bacterial gene expression. Here, lgmA-lgmD are found in the sense strand, with lgmE found in the antisense strand partially overlapping lgmB. All genes are expressed which suggests that the lgm locus undergoes posttranscriptional control. Surprisingly, the anti-sense lgmE appears to encode a membrane protein whose expression may be involved in posttranslational control of this locus. We will examine how the lgm locus is regulated temporally and in response to various stimuli. We also propose that the Lgm proteins function together as a complex and in a coordinated manner. We have developed an expression system that is able to make 10-20mg/L quantities of components of this system. This will allow us to characterize these proteins structurally and develop hypotheses of how this locus is regulated transcriptionally, post-transcriptionally, translationally, and post-translationally. Autotransporter protein biogenesis requires that the protein be suitably targeted to the outer membrane while being maintained in a translocation-competent state. TcfA is an unusual autotransporter protein that is only found in Bordetella. It is mis-targeted when expressed in E. coli, suggesting that Bordetella possess a unique system to localize TcfA to the surface. We will determine the requirements of TcfA localization. Uncovering fundamental principles of how the envelope is built in both model and non-model organisms can lead to improved methods for cell surface manipulation that can be used in bio and nanotechnology applications.