Heterogeneity in flagella-mediated motility

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Many bacteria are motile by means of flagella, which provide an effective but metabolically expensive means of locomotion, adhesion, and environmental sensing. Numerous species, among which are several of the genus Shewanella, possess two distinct flagellar systems. Little is known about how such secondary systems are regulated. In previous studies we have demonstrated that S. putrefaciens CN-32 possesses a fully functional secondary system which, upon induction, results in formation of additional lateral filaments in addition to a primary polar one. In complex planktonic cultures, the population splits into several subpopulations with respect to flagellar expression: cells that do not express any flagella, cells that express one of the two flagellar system, and cells in which both flagellar systems are produced. This implicates that CN-32 employs a bet-hedging strategy with respect to motility. In the previous funding period, we have addressed the question of the benefit the expression of the secondary system entails. We could show that the subpopulation of cell with both one polar and one lateral filament was significantly more efficient in spreading than their counterparts with a single flagellar system only. We demonstrated that the presence of the secondary filament leads to a decrease in the average turning angle, thereby increasing the directional persistence of swimming. A complementary modeling approach using the trajectory parameters obtained experimentally confirmed more efficient spreading. We have therefore found a fundamentally novel means of improving effective swimming used by bacteria.In the follow-up studies proposed here we will further address the mechanism by which the secondary system exerts its function. To this end, we will use fluorescently labeled flagellar filaments to enable live imaging of swimming cells. To refine and validate the theoretical model, we will also study swimming in chemotactic gradients. To further characterize potential benficial role of the secondary system, we will determine the role of the system in cell-surface- and cell-cell-adhesion. In a complementary approach we will explore the regulation of the secondary flagellar system and how heterogeneity is established. To this end, we have already constructed fluorescence and luminescence reporter systems. By flow cytometry analysis we will determine shifts in the population heterogeneity upon environmental signals. In addition, we will specifically screen for potential regulating factors of the secondary system. For this, we will directly screen for potential DNA-binding proteins and employ two different transposon mutagenesis screens. Finally, we will transfer the secondary flagellar system into the closely related species S. oneidensis MR-1 to determine whether function and regulation are maintained among Shewanella sp. Both approaches are expected to reveal fundamentally novel aspects of flagella function und regulation.