Characterization of LuxI/LuxR and their regulation involved in biofilm formation and stress resistance in fish spoilers Pseudomonas fluorescens

Quorum sensing (QS) is crucial for adaption and development of foodborne bacteria in diverse environments. Pseudomonas fluorescens PF07 with QS mediated acylated homoserine lactones (AHLs) activity was isolated from spoiled large yellow croaker (Pseudosciaena crocea). In this study AHL-mediated QS system was characterized and their roles in biofilm formation, motility, stress response and spoilage of P. fluorescens were evaluated. A LuxI/LuxR homolog consisting of a conserved AHL synthase gene (luxI) and a transcriptional regulator gene (luxR) was identified in the strain. Two in-frame deletion mutants of luxI and luxR, ∆luxI and ∆luxR, were constructed to explore their QS signaling function in P. fluorescens. Three types of AHLs were detected in PF07 culture by LC-MS/MS, and N-butanoyl-l-homoserine lactone (C4-HSL) was a major signal molecule. The C4-HSL activities was almost abolished in ∆luxI, and decreased greatly in ∆luxR. Compared with wild type (WT) strain, both ∆luxI and ∆luxR showed the significant decrease of biofilm biomass and expolysaccharide production, resulting in thinner and incompact biofilm structure, but promoted swimming motility. The resistance of P. fluorescens to H2O2, heat, NaCl and crystal violet apparently declined in two mutants compared to WT. Spoilage factors, siderophore and protease, apparently attenuated due to deletion of luxI or luxR gene, while the growth and TVB-N production did not differ. Furthermore, the changes of the biofilm formation, motility and protease in ∆luxI strain were partially restored by the exogenous C4-HSL. In agreement with the effect of two mutants on various phenotypes, the transcriptions of alg, lapA, flgA, rpoS, and aprX were significantly down-regulated, and flgA was up-regulated in ∆luxI and ∆luxR. Therefore, the present study highlighted that the co-operation of LuxI/LuxR homolog was an important QS regulator in biofilm formation, motility and spoilage potential, and hinted its positive regulation of stress resistance with RpoS in P. fluorescens.