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

TonB-ExbB-ExbD-like energy transduction systems are widespread among Gram-negative bacteria. While most species have only one copy of tonB-exbBD genes, the Pseudomonas 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 Pseudomonas aeruginosa Here, we show that the orthologous TonBm-PocA-PocB complex is important for polar localization of FlhF and flagella in Pseudomonas putida as well. Additionally, the system is necessary for maintaining membrane integrity, as the inactivation of the TonBm-PocAB complex results in increased membrane permeability, lowered stress tolerance, and conditional cell lysis. Interestingly, the functionality of TonBm-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 tonBm deletion strain, while only a few proteomic changes, mostly downregulation of outer membrane proteins, were determined in the stationary-phase ΔtonBm strain. We propose that this response in exponential phase, involving, inter alia, activation of AlgU and ColR regulons, can compensate for TonBm-PocAB's deficiency, while stationary-phase cells are unable to alleviate the lack of TonBm-PocAB. Our results suggest that mislocalization of flagella does not cause the membrane integrity problems; rather, the impaired membrane intactness of the TonBm-PocAB-deficient strain could be the reason for the random placement of flagella.IMPORTANCE The ubiquitous Pseudomonas 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 Pseudomonas species is of utmost importance. The stress tolerance of Pseudomonads is mainly achieved through the high barrier property of their membranes. Here, we present evidence that the TonB-ExbBD-like TonBm-PocAB system is involved in maintaining the membrane homeostasis of Pseudomonas putida, and its deficiency leads to lowered stress tolerance and conditional cell lysis.

Responses of Pseudomonas putida to Zinc Excess Determined at the Proteome Level: Pathways Dependent and Independent of ColRS.

Zinc is an important micronutrient for bacteria, but its excess is toxic. Recently, the ColRS two-component system was shown to detect and respond to zinc excess and to contribute to zinc tolerance of Pseudomonas putida. Here, we applied a label-free whole-cell proteome analysis to compare the zinc-induced responses of P. putida and colR knockout. We identified dozens of proteins that responded to zinc in a ColR-independent manner, among others, known metal efflux systems CzcCBA1, CzcCBA2, CadA2 and CzcD. Nine proteins were affected in a ColR-dependent manner, and besides known ColR targets, four new candidates for ColR regulon were identified. Despite the relatively modest ColR-dependent changes of wild-type, colR deficiency resulted in drastic proteome alterations, with 122 proteins up- and 62 down-regulated by zinc. This zinc-promoted response had remarkable overlap with the alternative sigma factor AlgU-controlled regulon in P. aeruginosa. The most prominent hallmark was a high induction of alginate biosynthesis proteins and regulators. This response likely alleviates the zinc stress, as the AlgU-regulated alginate regulator AmrZ was shown to contribute to zinc tolerance of colR knockout. Thus, the ColRS system is important for zinc homeostasis, and in its absence, alternative stress response pathways are activated to support the zinc tolerance.

The ColRS signal transduction system responds to the excess of external zinc, iron, manganese, and cadmium.

BACKGROUND: The ColRS two-component system has been shown to contribute to the membrane functionality and stress tolerance of Pseudomonas putida as well as to the virulence of Pseudomonas aeruginosa and plant pathogenic Xanthomonas species. However, the conditions activating the ColRS pathway and the signal(s) sensed by ColS have remained unknown. Here we aimed to analyze the role of the ColRS system in metal tolerance of P. putida and to test whether ColS can respond to metal excess. RESULTS: We show that the ColRS system is necessary for P. putida to tolerate the excess of iron and zinc, and that it also contributes to manganese and cadmium tolerance. Excess of iron, zinc, manganese or cadmium activates ColRS signaling and as a result modifies the expression of ColR-regulated genes. Our data suggest that the genes in the ColR regulon are functionally redundant, as several loci have to be deleted to observe a significant decrease in metal tolerance. Site-directed mutagenesis of ColS revealed that excess of iron and, surprisingly, also zinc are sensed by a conserved ExxE motif in ColS's periplasmic domain. While ColS is able to sense different metals, it still discriminates between the two oxidation states of iron, specifically responding to ferric and not ferrous iron. We propose a signal perception model involving a dimeric ColS, where each monomer donates one ExxE motif for metal binding. CONCLUSIONS: Several transition metals are essential for living organisms in certain amounts, but toxic in excess. We show that ColRS is a sensor system which detects and responds to the excess of physiologically important metals such as zinc, iron and manganese. Thus, the ColRS system is an important factor for metal homeostasis and tolerance in P. putida.

A moderate toxin, GraT, modulates growth rate and stress tolerance of Pseudomonas putida.

Chromosomal toxin-antitoxin (TA) systems are widespread among free-living bacteria and are supposedly involved in stress tolerance. Here, we report the first TA system identified in the soil bacterium Pseudomonas putida. The system, encoded by the loci PP1586-PP1585, is conserved in pseudomonads and belongs to the HigBA family. The new TA pair was named GraTA for the growth rate-affecting ability of GraT and the antidote activity of GraA. The GraTA system shares many features common to previously described type II TA systems. The overexpression of GraT is toxic to the antitoxin deletion mutants, since the toxin's neutralization is achieved by binding of the antitoxin. Also, the graTA operon structure and autoregulation by antitoxin resemble those of other TA loci. However, we were able to delete the antitoxin gene from the chromosome, which shows the unusually mild toxicity of innate GraT compared to previously described toxins. Furthermore, GraT is a temperature-dependent toxin, as its growth-regulating effect becomes more evident at lower temperatures. Besides affecting the growth rate, GraT also increases membrane permeability, resulting in higher sensitivity to some chemicals, e.g., NaCl and paraquat. Nevertheless, the active toxin helps the bacteria survive under different stressful conditions and increases their tolerance to several antibiotics, including streptomycin, kanamycin, and ciprofloxacin. Therefore, our data suggest that GraT may represent a new class of mild chromosomal regulatory toxins that have evolved to be less harmful to their host bacterium. Their moderate toxicity might allow finer growth and metabolism regulation than is possible with strong growth-arresting or bactericidal toxins.