Psl exopolysaccharide regulates biofilm formation and environmental adaptation in Pseudomonas fluorescens PF07

Pseudomonas fluorescens PF07, a prevalent psychrotrophic spoilage bacterium, relies heavily on robust biofilm formation to persist in food environments and exert spoilage effects. Nevertheless, the distinct contributions of exopolysaccharides to its biofilm maturation and stress adaptation remain unclear. Herein, we systematically characterized the genetic determinants and biological functions of three exopolysaccharides (Psl, alginate and PNAG) in modulating PF07 biofilm development and resistance to food-processing-associated stressors. Single, double, and triple deletion mutants targeting the pslB, alg8, and pgaC genes were constructed to evaluate the contributions of these biosynthetic systems, and transcriptomic analysis was performed to explore the underlying responses. Deletion of pslB markedly impaired biofilm formation, pellicle development, and extracellular matrix production, whereas deficiency of alg8 or pgaC caused only minor phenotypic changes compared with the wild-type strain, indicating that Psl is the primary structural determinant of the PF07 biofilm matrix. Furthermore, double- and triple-mutants lacking pslB exhibited more severe reductions in biofilm biomass and thickness, suggesting that alginate and PNAG make supplementary but secondary contributions to the Psl-dominated biofilm matrix. In addition, ΔpslB exhibited substantially reduced biofilm formation under low temperature and ionic stress and decreased tolerance to sodium hypochlorite and benzalkonium chloride, in contrast with limited alterations for the Δalg8 and ΔpgaC mutants. Consistently, catalase and superoxide dismutase activities were significantly weakened in the ΔpslB mutant but largely unaltered in the Δalg8 mutant, indicating that Psl deficiency was associated with reduced antioxidant enzyme activities and suggesting a potential role of Psl in the antioxidant response of PF07. Transcriptomic analysis further revealed significant changes in genes associated with stress adaptation and metabolism, including the upregulation of aqpZ, the downregulation of osmY, and alterations in multiple metabolism-related genes. Thus, these findings demonstrate that Psl is a pivotal structural polysaccharide in the biofilm matrix of P. fluorescens PF07 and plays critical roles in biofilm structure and tolerance to cold, ionic, and oxidative stresses, providing new insights into potential strategies for controlling P. fluorescens biofilms in food-processing environments.