The TonB_m-PocAB System Is Required for Maintenance of Membrane Integrity and Polar Position of Flagella in Pseudomonas putida

Abstract

TonB-ExbB-ExbD-like energy transduction systems are widespread among Gram-negative bacteria. While most species have only one copy of <i>tonB</i>-e<i>xbBD</i> genes, the <i>Pseudomonas</i> species possess more TonB-ExbBD homologues. One of them, the TonB3-PocA-PocB complex, was recently shown to be required for polar localization of FlhF and, thus, the flagella in <i>Pseudomonas aeruginosa</i> Here, we show that the orthologous TonB_m-PocA-PocB complex is important for polar localization of FlhF and flagella in <i>Pseudomonas putida</i> as well. Additionally, the system is necessary for maintaining membrane integrity, as the inactivation of the TonB_m-PocAB complex results in increased membrane permeability, lowered stress tolerance, and conditional cell lysis. Interestingly, the functionality of TonB_m-PocAB complex is more important for stationary than for exponentially growing bacteria. The whole-cell proteome analysis provided a likely explanation for this growth phase dependence, as extensive reprogramming was disclosed in an exponentially growing <i>tonB_m</i> deletion strain, while only a few proteomic changes, mostly downregulation of outer membrane proteins, were determined in the stationary-phase <i>ΔtonB_m</i> strain. We propose that this response in exponential phase, involving, <i>inter alia</i>, activation of AlgU and ColR regulons, can compensate for TonB_m-PocAB's deficiency, while stationary-phase cells are unable to alleviate the lack of TonB_m-PocAB. Our results suggest that mislocalization of flagella does not cause the membrane integrity problems; rather, the impaired membrane intactness of the TonB_m-PocAB-deficient strain could be the reason for the random placement of flagella.<b>IMPORTANCE</b> The ubiquitous <i>Pseudomonas</i> species are well adapted to survive in a wide variety of environments. Their success relies on their versatile metabolic, signaling, and transport ability but also on their high intrinsic tolerance to various stress factors. This is why the study of the stress-surviving mechanisms of <i>Pseudomonas</i> species is of utmost importance. The stress tolerance of <i>Pseudomonads</i> is mainly achieved through the high barrier property of their membranes. Here, we present evidence that the TonB-ExbBD-like TonB_m-PocAB system is involved in maintaining the membrane homeostasis of <i>Pseudomonas putida</i>, and its deficiency leads to lowered stress tolerance and conditional cell lysis.