Calcium chloride induces dose-dependent architectural, mechanical, and transcriptional remodeling in Pseudomonas putida biofilms: an exploratory multi-scale study

Calcium ions (Ca 2+ ) are known to enhance biofilm structural integrity in many bacteria, including Pseudomonas aeruginosa , by cross-linking exopolysaccharides. Still, their role in the soil bacterium P. putida remains largely unexplored. Here, we investigated the dose-dependent effects of calcium chloride on P. putida KT2442 biofilm architecture, matrix properties, nanomechanics, and global gene expression. Contrary to the stabilizing role observed in P. aeruginosa , calcium induced complex biphasic responses in P. putida . Calcium elicited complex, non-monotonic effects: while 1.5 mM CaCl₂ increased biofilm biovolume, a moderate 3 mM concentration limited architectural expansion and corresponded with the lowest measured biofilm stiffness in exploratory AFM assays. At 15 mM, biofilm thickness increased, but mechanical rigidity did not fully recover, and the molecular weight of matrix polysaccharides decreased. Transcriptomic analysis revealed dose-dependent reprogramming affecting approximately 3,600 genes at 3 mM and 15 mM calcium, with downregulation of genes involved in exopolysaccharide biosynthesis, large adhesins ( lapA , lapF ), and motility, alongside upregulation of stress response and ribosomal biogenesis pathways. These correlative findings suggest that calcium may act not merely as a structural ion but as a potent environmental signal that triggers species-specific adaptive responses correlated with altered biofilm architecture, mechanics, and transcriptional networks in P. putida . The direct causal links and the unique responses of calcium in comparison to other divalent cations have yet to be confirmed.