POLYAMINE HOMEOSTASIS CONTRIBUTES TO OXIDATIVE STRESS TOLERANCE IN Pseudomonas syringae

SOLMI LEANDRO; FR ROSSI; FERNANDO M. ROMERO; OSCAR A RUIZ; ANDRÉS GÁRRIZ

Buenos Aires

Congreso; Reunion conjunta SAIB-SAMIGE 2021; 2021

SAIB-SAMIGE

Putrescine (Put), and its aminopropylated derivative spermidine (Spd), are the main polyamines (PAs) found in bacteria and play crucial roles in the physiology of human pathogens. In this trend, it has been shown that PAs are essential in the responses of Escherichia coli and Pseudomonas aeruginosa to the oxidative stress. However, their participation in the responses to this stress in phytopathogenic bacteria has been poorly explored. With the purpose to unveil the potential functions played by PAsin these microorganisms, we first analyzed the variation in their concentrations in cultures of the bacterial model P. syringae pv. tomato DC3000 amended with H2O2. We observed a significant accumulation of Put in the culture supernatants, suggesting that the secretion of this molecule functions as an antioxidant. This is in agreement with previous reports demonstrating that extracellular PAs protect membrane phospholipids from oxidation. To corroborate this potential function played by extracellular Put, we grew cells in minimal medium supplemented with PAs and evaluated their survival in lethal concentrations of H2O2. Interestingly, the addition of Put increased stress tolerance, but its protective capacity is reduced in the presence of Spd. On these bases, we performed molecular dynamic simulations on a model bacterial outer membrane in order to obtain an understanding of the mechanism involved. The simulations suggested that even though both PAs engage in electrostatic interactions with the phosphate groups of the membrane, Spd makes stronger interactions. Thus, it has the potential to displace Put and minimize its protective role. On the other hand, it has been also shown that PA production is essential for stress tolerance. To examine the importance of this metabolic pathway, we study the phenotype of mutant strains perturbed in the synthesis of Put (ΔspeA/speC) and Sped (ΔspeE). This study showed that, whereas the ΔspeA/speC strain leads to susceptibility to oxidative stress, deletion of speE enhanced stress tolerance. Importantly, catalase activity was significantly increased in the later compared to the wild-type strain, suggesting that a higher rate of H2O2 decomposition is involved in this phenotype. To further examine the role of these enzymes, we constructed transcriptional fusions to monitor gene expression levels of the main catalases katG and katB using GFPuv as a reporter gene. Our results showed that only katG is upregulated in the ∆speE strain, indicating that this is the enzyme playing the major role in H2O2 degradation. Taking all the results together, we conceive that PA homeostasis contributes to the survival of P. syringae under an oxidative environment through multiple mechanisms. Thus, cells secrete Put in the presence of H2O2 to protect cell membranes, which also leads to a reduction in the intracellular concentration of Spd and the activation of catalase activity via katG upregulation.