Abstract
We recently reported that Pseudomonas fluorescens PF07, an isolate from refrigerated marine fish, produces the functional amyloid Fap as the major component of its biofilm matrix and that transcription of the fap gene cluster is directly regulated by BrfA, a novel c-di-GMP-responsive transcription regulator. As a psychrotrophic food spoiler, P. fluorescens encounters temperature fluctuations during food processing and distribution; however, the effects of temperature on its biofilm formation remain poorly characterized. Here, we show that reduced temperatures (4°C and 15°C) significantly attenuate macrocolony, pellicle, and solid-surface-associated biofilm formation in PF07 compared to 28°C. Mechanistically, low temperatures suppress Fap-dependent biofilm formation by downregulating intracellular c-di-GMP levels via the coordinated control of two key enzymes: a novel diguanylate cyclase, DebA, and a cold-adapted phosphodiesterase BifA. At 28°C, DebA is highly expressed and maintains robust catalytic activity via its PAS domain, while BifA exhibits low activity due to poor thermostability; these effects together drive c-di-GMP accumulation, fap expression, and biofilm formation. Conversely, low temperatures reduce DebA expression and activity, while BifA retains exceptional cold tolerance to accelerate c-di-GMP degradation, thereby suppressing fap expression and biofilm formation. This study delineates a novel temperature-responsive c-di-GMP signaling pathway in the psychrotrophic food spoiler, P. fluorescens PF07. IMPORTANCE: The persistence of bacteria in various biofilms frequently leads to food spoilage and foodborne illnesses. Pseudomonas fluorescens is widely recognized as one of the most prevalent spoilage organisms, with a robust capacity for biofilm formation. Temperature is a critical factor in food processing, distribution, and preservation. This study identifies a novel temperature-responsive c-di-GMP signaling module centered on the novel diguanylate cyclase DebA and the cold-adapted phosphodiesterase BifA, which governs Fap-dependent biofilm formation in P. fluorescens PF07. Our findings expand the known repertoire of c-di-GMP-mediated biofilm regulatory pathways and may inform the development of improved antibiofilm strategies for the food industry.