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 .

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 bright-field imaging of single cell trajectories and spreading cultures, dual color fluorescence microscopy allows us to quantify the role of the stators in forming 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 slower 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.IMPORTANCEBecause 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 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 know to be beneficial for escaping mechanical traps. However, in contrast to the MotAB knock-out, a culture of MotCD knock-out cells spreads much slower in the agar as it forms non-motile clusters that reduce the amount of motile cells.