Institute of Physics and Astronomy, University of Potsdamgrid.11348.3f, Potsdam, Germany.
mBio. 2022 Dec 20;13(6):e0218222. doi: 10.1128/mbio.02182-22. Epub 2022 Nov 21.
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 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. 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.
在土壤细菌恶臭假单胞菌中，鞭毛旋转的马达扭矩是由两个定子 MotAB 和 MotCD 产生的。在这里，我们构建了一个或两个定子敲除的突变株，并研究了它们在不同粘度的流体中和非均相结构化环境（半固体琼脂）中的游动能力。除了对单个细胞轨迹和扩展培养的相差成像外，双色荧光显微镜还使我们能够量化定子在使恶臭假单胞菌的三种不同游动模式中发挥的作用，其中鞭毛束推动、拉动或缠绕在细胞体周围。MotAB 定子对于液体中的游动能力是必需的，而在半固体琼脂中的扩展则不受影响。此外，如果敲除 MotAB 定子，在低粘度条件下形成缠绕模式的能力会受到严重损害，只有在增加粘度和在半固体琼脂中才能部分恢复。相比之下，如果缺失 MotCD 定子，在流体实验中细胞与野生型没有区别，但在半固体琼脂中扩展速度要慢得多。对微观轨迹的分析表明，MotCD 敲除株形成定殖簇，从而减少了游动细胞的数量，而游动速度不受影响。总之，两个定子确保了高效游动的野生型，使其能够在不同的环境条件下高效游动。由于其异质生境，土壤细菌恶臭假单胞菌需要在非常不同的环境条件下高效游动。在本文中，我们敲除了定子 MotAB 和 MotCD，以研究它们对恶臭假单胞菌游动能力的影响。虽然 MotAB 定子对于在流体中游动至关重要，但在半固体琼脂中，两个定子都足以维持快速游动的表型和缠绕模式的高频出现，已知缠绕模式有利于逃脱机械陷阱。然而，与 MotAB 敲除相比，MotCD 敲除细胞的培养物在琼脂中扩展得慢得多，因为它形成了非游动簇，从而减少了游动细胞的数量。
