Veronika Pfeifer, Sönke Beier, Zahra Alirezaeizanjani, Carsten Beta

• In the soil bacterium Pseudomonas putida, the motor torque for flagellar rotation is generated by the two stators MotAB and MotCD. Here, we construct mutant strains in which one or both stators are knocked out and investigate their swimming motility in fluids of different viscosity and in heterogeneous structured environments (semisolid agar). Besides phase-contrast imaging of single-cell trajectories and spreading cultures, dual-color fluorescence microscopy allows us to quantify the role of the stators in enabling P. putida's three different swimming modes, where the flagellar bundle pushes, pulls, or wraps around the cell body. The MotAB stator is essential for swimming motility in liquids, while spreading in semisolid agar is not affected. Moreover, if the MotAB stator is knocked out, wrapped mode formation under low-viscosity conditions is strongly impaired and only partly restored for increased viscosity and in semisolid agar. In contrast, when the MotCD stator is missing, cells are indistinguishable from the wild typeIn the soil bacterium Pseudomonas putida, the motor torque for flagellar rotation is generated by the two stators MotAB and MotCD. Here, we construct mutant strains in which one or both stators are knocked out and investigate their swimming motility in fluids of different viscosity and in heterogeneous structured environments (semisolid agar). Besides phase-contrast imaging of single-cell trajectories and spreading cultures, dual-color fluorescence microscopy allows us to quantify the role of the stators in enabling P. putida's three different swimming modes, where the flagellar bundle pushes, pulls, or wraps around the cell body. The MotAB stator is essential for swimming motility in liquids, while spreading in semisolid agar is not affected. Moreover, if the MotAB stator is knocked out, wrapped mode formation under low-viscosity conditions is strongly impaired and only partly restored for increased viscosity and in semisolid agar. In contrast, when the MotCD stator is missing, cells are indistinguishable from the wild type in fluid experiments but spread much more slowly in semisolid agar. Analysis of the microscopic trajectories reveals that the MotCD knockout strain forms sessile clusters, thereby reducing the number of motile cells, while the swimming speed is unaffected. Together, both stators ensure a robust wild type that swims efficiently under different environmental conditions. IMPORTANCE Because of its heterogeneous habitat, the soil bacterium Pseudomonas putida needs to swim efficiently under very different environmental conditions. In this paper, we knocked out the stators MotAB and MotCD to investigate their impact on the swimming motility of P. putida. While the MotAB stator is crucial for swimming in fluids, in semisolid agar, both stators are sufficient to sustain a fast-swimming phenotype and increased frequencies of the wrapped mode, which is known to be beneficial for escaping mechanical traps. However, in contrast to the MotAB knockout, a culture of MotCD knockout cells spreads much more slowly in the agar, as it forms nonmotile clusters that reduce the number of motile cells. Because of its heterogeneous habitat, the soil bacterium Pseudomonas putida needs to swim efficiently under very different environmental conditions. In this paper, we knocked out the stators MotAB and MotCD to investigate their impact on the swimming motility of P. putida.…