Multiple Flagellin Proteins Have Distinct and Synergistic Roles inAgrobacterium tumefaciensMotility

ABSTRACTRotary flagella propel bacteria through liquid and across semisolid environments. Flagella are composed of the basal body that constitutes the motor for rotation, the curved hook that connects to the basal body, and the flagellar filament that propels the cell. Flagellar filaments can be composed of a single flagellin protein, such as inEscherichia coli, or made up of multiple flagellins, such as inAgrobacterium tumefaciens. The four distinct flagellins FlaA, FlaB, FlaC, and FlaD produced by wild-typeA. tumefaciensare not redundant in function but have specific properties. FlaA and FlaB are much more abundant than FlaC and FlaD and are readily observable in mature flagellar filaments, when either FlaA or FlaB is fluorescently labeled. Cells producing FlaA with any one of the other three flagellins can generate functional filaments and thus are motile, but FlaA alone cannot constitute a functional filament. InflaAmutants that manifest swimming deficiencies, there are multiple ways by which these mutations can be phenotypically suppressed. These suppressor mutations primarily occur within or upstream of theflaBflagellin gene or in the transcription factorsciPregulating flagellin expression. The helical conformation of the flagellar filament appears to require a key asparagine residue present in FlaA and absent in other flagellins. However, FlaB can be spontaneously mutated to render helical flagella in the absence of FlaA, reflecting their overall similarity and perhaps the subtle differences in the specific functions they have evolved to fulfill.IMPORTANCEFlagellins are abundant bacterial proteins comprising the flagellar filaments that propel bacterial movement. Several members of the alphaproteobacterial group express multiple flagellins, in contrast to model systems, such as withEscherichia coli, which has one type of flagellin. The plant pathogenAgrobacterium tumefacienshas four flagellins, the abundant and readily detected FlaA and FlaB, and lower levels of FlaC and FlaD. Mutational analysis reveals that FlaA requires at least one of the other flagellins to function, asflaAmutants produce nonhelical flagella and cannot swim efficiently. Suppressor mutations can rescue this swimming defect through mutations in the remaining flagellins, including structural changes imparting helical shape to the flagella, and putative regulators. Our findings shed light on how multiple flagellins contribute to motility.