OXIDATIVE STRESS RESPONSE OF MODEL BIOFILM SYSTEMS UNDER DIFFERENT ENVIRONMENTAL CUES

During my PhD, I focused on three important environmental bacteria, namely, Azotobacter vinelandii, Bacillus subtilis and Burkholderia thailandensis. In each model, I studied different mechanisms of oxidative stress, related to their role in the environment or, in the case of B. thailandensis, related to its condition of opportunistic pathogen of invertebrates and model for the human pathogen B. pseudomallei. In A. vinelandii, inactivation of the rhodanese‐like protein RhdA resulted in continuous generation of endogenous oxidative stress, promoting biofilm genesis, stimulating the activity of scavenging systems and triggering a switch between swarming and biofilmlike phenotypes. Furthermore, the oxidative stress affected the composition of the exopolymeric substances (EPS), resulting in the production of a polysaccharide‐rich extracellular polymeric matrix in mutant (part II, chapter 1). In B. subtilis, the antimicrobial mechanism of silver nanoparticles (Ag‐NPs) involve the production of reactive oxygen species (ROS), with possible consequences on soil bacteria. Sub‐lethal doses of Ag‐NPs increased the ROS formation in B. subtilis planktonic cells, but not in sessile cells, suggesting the presence of scavenging systems in biofilms. Consistently, proteomic analysis in Ag‐NPs‐treated biofilms showed increased production of proteins related to redox , quorum sensing and to stress response, thus suggesting a coordinated regulation of biofilm and stress response genes. Extracellular polysaccharide production and inorganic phosphate solubilization were also increased, possibly as part of a coordinated response to oxidative stress (part II, chapter 2). Finally we challenged B. thailandensis with phenazine methosulphate (PMS) to simulate the oxidative stress encountered in the soil and in the infected host. A new molecular approach to create mutants in Burkholderia spp. has been developed as part of this work (part II, chapter 3). In B. thailandensis biofilm, oxidative stress decreased as the biofilm reached the mature phase. The presence of PMS affected the biofilm morphology, triggering the production of more EPS. Interestingly , the deletion of the periplasmic superoxide dismutase, sodC, triggered polysaccharide production in biofilm cells (part III, chapter 1). My results demonstrate how the matrix production plays a pivotal role in protection from oxidative injuries in bacterial biofilm, both in Gram‐negative and Gram‐positive bacteria. The protection mechanisms activated by biofilm in response to oxidative stress can have important consequences on environmental biodiversity and in the balance between planktonic and biofilm cells.