Determinants Influencing Polar Flagellar Biosynthesis and Cell Division in Campylobacter jejuni

Campylobacter jejuni is a worldwide leading cause of bacterial gastrointestinal disease. The natural habitat of this organism is the gastrointestinal tracts of warm-blooded animals, especially poultry, where the bacterium promotoes a harmless commensal colonization. The abundance of C. jejuni in poultry creates a risk for food-borne infections to human populations. Flagellar motility by C. jejuni is required to colonize both human and animal hosts. For motility, C. jejuni produces amphitrichous flagella, resulting in the formation of a single flagellum at both poles. This work explored factors that regulate numerical and spatial parameters for amphitrichious flagellation. Two factors that have been identified to control flagellar placement and numbers in polarly-flagellated bacteria are the FlhF GTPase and the FlhG ATPase. FlhF has been shown to be required for regulation of flagellar gene expression and flagellar placement in some Pseudomonas and Vibrio species. Characterization of FlhF in C. jejuni was accomplished by creating point mutants in C-terminal GTPase domain of FlhF to decrease its GTPase activity. GTPase mutants, unlike mutants that lack FlhF, did not have a significant reduction in sigma54-dependent flagellar gene expression. Instead, a significant proportion of the population produced flagella at lateral sites or produced multiple flagella at a pole, whereas wild-type bacteria produced single polar flagella. Further experiments suggested that FlhF functions downstream of the FlgSR-flagellar export apparatus (FEA) pathway to activate sigma54-dependent flagellar gene expression. Thus, our data suggested that FlhF and its GTPase activity are required for distinct processes in flagellar gene regulation. FlhG has been shown to control flagellar numbers in Pseudomonas and Vibrio species. We examined flhG mutants and confirmed that FlhG regulates flagellar numbers. C. jejuni flhG mutants also demonstrated a minicell phenotype, which is the result of division erroneously occurring at polar regions. Further examination revealed that FlhG and the flagellar base components compose a novel division inhibition system to spatially prevent polar division and encourage septation at the cellular midpoint for symmetrical division. This work greatly extends our understanding of factors that govern spatial and numerical patterns of polar flagellation and has identified an unprecedented system to spatially regulate division in bacteria.