Calcium-mediated modulation of Pseudomonas fluorescens biofilm formation

Biofilm formation is usually affected by many environmental factors, including divalent cations. The purpose of the current work was to analyze how calcium (Ca²⁺) affects the biofilm formation of dairy Pseudomonas fluorescens isolates by investigating their growth, swarming motility, biofilm-forming capacity, extracellular polymeric substance production, and biofilm structures. Moreover, the regulation mechanism of Ca²⁺ involved in its biofilm formation was explored through RNA-sequencing analysis. This work revealed that supplementation of 5, 10, 15, and 20 mM Ca²⁺ significantly reduced the swarming motility of P. fluorescens strains (P.F₂, P.F₄, and P.F₁₇), but the biofilm-forming ability and polysaccharide production were increased after the supplementation of 5 and 10 mM Ca²⁺. By the supplementation of Ca²⁺, complex structures with more cell clusters glued together in P. fluorescens P.F₄ biofilms were confirmed by scanning electron microscopy, and increased biomass and coverage of P. fluorescens P.F₄ biofilms were observed by confocal laser scanning microscopy. In addition, RNA-sequencing results showed that P. fluorescens P.F₄ showed a transcriptional response to the supplementation of 10 mM Ca²⁺, and a total of 137 genes were significantly expressed. The differential genes were represented in 4 upregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (nonribosomal peptide structures, quorum sensing, biosynthesis of siderophore group nonribosomal peptides, and phenylalanine metabolism), and 4 downregulated KEGG pathways (flagellar assembly, amino sugar and nucleotide sugar metabolism, nitrotoluene degradation, and cationic antimicrobial peptide resistance). The results indicate that Ca²⁺ might serve as an enhancer to substantially trigger the biofilm formation of dairy P. fluorescens isolates in the dairy industry.