Biofilm formation of Pseudomonas fluorescens induced by a novel diguanylate cyclase modulated c-di-GMP promotes spoilage of large yellow croaker (Larimichthys crocea).

Pseudomonas as major agents cause the microbial spoilage in aerobically stored seafoods due to the strong biofilm forming ability, resulting in significant economic losses. C-di-GMP regulates the transition to biofilm states in numerous bacteria, however, its function in biofilm and spoilage of Pseudomonas fluorescens has still been scarce. Here, in a fish spoiler P. fluorescens PF07 strain, 26 proteins of diguanylate cyclase (DGC) containing a GGDEF domain were characterized, and both intracellular c-di-GMP and biofilm formation consistently decreased in the constructed 12 deletion mutants of DGC domain. Compared to wild type (WT) strain, both swimming and swarming in these mutants remarkably enhanced, while the secretion of siderophore, protease activity, and the production of total volatile basic nitrogen (TVB-N) were decreased in several mutants, indicating the different modulating effects among these DGC mutants. Furthermore, correlation analysis of these six phenotypes, PF07_04309 exhibited the most significant alteration, which was identified a novel functional DGC enzyme. Moreover, the GGAAA mutation of PF07_04309 induced the down-regulation of Psl and Alg operons and increased flagellar related gene, resulting in forming the sparser and thinner biofilms. Two mutants of 04309 induced by low c-di-GMP significantly declined the accumulation of TVB-N, thiobarbituric acid, extracellular protease activity and spoilage flavor compounds, especially methylamine and carbon disulfide, in the fillets of large yellow croaker stored at 4 °C. Thus, our results indicated that a novel DGC 04309 modulated the polysaccharide secretion, flagellar, and iron carrier by synthesis of c-di-GMP, positively regulating the spoilage potential of P. fluorescens, which expanded the original insights of DGC and c-di-GMP function on microbial food spoilage.