Project Summary Bacteria produce various macromolecular surface organelles that function in different processes. For many organelles, it is unclear how bacteria regulate their dimensions or even if a specific mechanism exists to govern the size or length of the organelle. The flagellum is a surface organelle that facilitates swimming motility and migration of pathogens to ideal niches in hosts to initiate infection and pathogenesis of disease. Two specific mechanisms have been discovered in flagellar biogenesis that regulate the length of the periplasmic rod and surface hook. However, a current hypothesis states that bacteria do not have a specific and active mechanism to regulate the length of the extracellular flagellar filament, which functions as the propeller of the flagellar motor and extends ~10 µm on the surface of model peritrichous bacteria such as Salmonella species and E. coli. Challenging this hypothesis are observations that many polarly-flagellated bacterial pathogens, such as Campylobacter jejuni, Vibrio cholerae, and Pseudomonas aeruginosa, produce flagellar filaments ~70% shorter than those of peritrichous bacteria. Furthermore, deletion of flaG that is conserved in these pathogens and many other polar flagellates but absent in peritrichous organisms, caused flagellar filaments to extend up to twice the length as those of WT cells. In addition, C. jejuni DflaG producing elongated flagellar filaments displayed a 28-fold defect for cecal colonization of the natural avian host, suggesting that properly regulating flagellar filament length is necessary for optimal colonization of a host. Together, these findings indicate that FlaG in polar flagellates is involved in a specific and active mechanism to control the extracellular length of the flagellar filament and is important for some pathogens to colonize hosts, but a molecular mechanism for how FlaG controls filament length is unknown. In this proposal, we will use C. jejuni as a model system for polar flagellates to understand how a bacterium employs FlaG to control the length of the flagellar filament by halting filament polymerization, an event that occurs at a large distance (~2-3 cell body lengths) from the bacterial surface. We will test two non-mutually exclusive hypotheses that FlaG controls flagellar filament length by the following mechanisms: 1) FlaG interfering with interactions between the filament-building flagellin subunits and the FliS chaperone to reduce flagellin secretion; and/or 2) FlaG functioning as a flagellin mimic to block flagellins from polymerizing into the filament at the tip of the flagellum. We will also investigate why production of elongated flagellar filaments by C. jejuni DflaG reduces the ability to colonize a host by analyzing the effects of elongated filaments on motility efficiency and velocity through different physiologically- relevant viscosities and secretion of proteins through the flagellum required for host interactions. Outcomes from t...