Influence of Calcium in Extracellular DNA Mediated Bacterial Aggregation and Biofilm Formation

Abstract

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 Ca2+ 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 (ΔG) 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.

The Impact of Biofilms upon Surfaces Relevant to an Intermediate Level Radioactive Waste Geological Disposal Facility under Simulated Near-Field Conditions

“…In environmental isolates, eDNA has been shown to have a role in the initiation of biofilm development [30] and the promotion of floc formation in marine [31] and alkaliphilic microorganisms [16]. The anionic nature of eDNA results in strong interactions between eDNA and metal cations such as Ca 2+ ions, such interactions between eDNA and Ca 2+ ions have been shown to promote aggregation and biofilm formation [32]. In the experiments reported here the precipitation of calcium carbonate onto the steel surface may be promoting the accumulation of eDNA.…”

Floc Formation Reduces the pH Stress Experienced by Microorganisms Living in Alkaline Environments

“…The results obtained from the calcium binding assay support this conclusion. The eDNA component of the EPS, although acidic in nature as a result of the linkage of nucleotides by the 3=-to-5= phosphodiester bonds, most likely plays a role in calcium sequestering due to its thermodynamically favorable interaction with calcium ions (32). Calcium has been shown to promote bioflocculation (33), as evidenced by the production of flocs up to 240 m in size.…”

Fluconazole-Pyridoxine Bis-Triazolium Compounds with Potent Activity against Pathogenic Bacteria and Fungi Including Their Biofilm-Embedded Forms

“…Antibacterial activity of compound 4b has also been tested on six bacterial strains from the same panel of pathogens in the presence of CaCl 2 [38]. The MIC values of 4b in the presence of Ca 2+ ions were significantly increased for both Gram-positive and Gram-negative strains (>64 g/mL as compared to 1-32 g/mL under Ca 2+ -free conditions).…”

“…To gain insight into possible mechanism of action of compound 4b, its activity has been tested on six bacterial strains from the same panel of pathogens in the presence of CaCl 2 [38]. The mechanism of action of the membrane damaging drugs is often related to removal of Ca 2+ from the cellular membranes.…”