Influence of calcium in extracellular DNA mediated bacterial aggregation and biofilm formation

Calcium (Ca²⁺) has an important structural role in guaranteeing the integrity of the outer lipopolysaccharide layer and cell walls of bacterial cells. Extracellular DNA (eDNA) being part of the slimy matrix produced by bacteria promotes biofilm formation through enhanced structural integrity of the matrix. Here, the concurrent role of Ca²⁺ and eDNA in mediating bacterial aggregation and biofilm formation was studied for the first time using a variety of bacterial strains and the thermodynamics of DNA to Ca²⁺ binding. It was found that the eDNA concentrations under both planktonic and biofilm growth conditions were different among bacterial strains. Whilst Ca ²⁺ had no influence on eDNA release, presence of eDNA by itself favours bacterial aggregation via attractive acid-base interactions in addition, its binding with Ca²⁺ at biologically relevant concentrations was shown further increase in bacterial aggregation via cationic bridging. Negative Gibbs free energy (DG) values in iTC data confirmed that the interaction between DNA and Ca²⁺ is thermodynamically favourable and that the binding process is spontaneous and exothermic owing to its highly negative enthalpy. Removal of eDNA through DNase I treatment revealed that Ca²⁺ alone did not enhance cell aggregation and biofilm formation. This discovery signifies the importance of eDNA and concludes that existence of eDNA on bacterial cell surfaces is a key facilitator in binding of Ca²⁺ to eDNA thereby mediating bacterial aggregation and biofilm formation.