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 resem-ble 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 GraTmay represent a new class of mild chromo-somal regulatory toxins that have evolved to be less harmful to their host bacterium. Their moderate toxicity might allow finer growth andmetabolism regulation than is possible with strong growth-arresting or bactericidal toxins. Toxin-antitoxin (TA) systems are widespread among bacteria.These gene cassettes consist of two small genes which code fo