Calcium Prevents Biofilm Dispersion in Bacillus subtilis.

Biofilm dispersion is the final stage of biofilm development, during which biofilm cells actively escape from biofilms in response to deteriorating conditions within the biofilm. Biofilm dispersion allows cells to spread to new locations and form new biofilms in better locations. However, dispersal mechanisms have been elucidated only in a limited number of bacteria. Here, we investigated biofilm dispersion in Bacillus subtilis. Biofilm dispersion was clearly observed when B. subtilis was grown under static conditions in modified LB medium containing glycerol and manganese. Biofilm dispersion was synergistically caused by two mechanisms: decreased expression of the epsA operon encoding exopolysaccharide synthetases and the induction of sporulation. Indeed, constitutive expression of the epsA operon in the sporulation-defective ΔsigK mutant prevented biofilm dispersion. The addition of calcium to the medium prevented biofilm dispersion without significantly affecting the expression of the epsA operon and sporulation genes. In synthetic medium, eliminating calcium did not prevent the expression of biofilm matrix genes and, thereby, biofilm formation, but it attenuated biofilm architecture. These results indicate that calcium structurally stabilizes biofilms and causes resistance to biofilm dispersion mechanisms. Sporulation-dependent biofilm dispersion required the spoVF operon, encoding dipicolinic acid (DPA) synthase. During sporulation, an enormous amount of DPA is synthesized and stored in spores as a chelate with calcium. We speculate that, during sporulation, calcium bound to biofilm matrix components may be transported to spores as a calcium-DPA complex, which weakens biofilm structure and leads to biofilm dispersion. IMPORTANCE Bacteria growing as biofilms are notoriously difficult to eradicate and sometimes pose serious threats to public health. Bacteria escape from biofilms by degrading them when biofilm conditions deteriorate. This process, called biofilm dispersion, has been studied as a promising strategy for safely controlling biofilms. However, the regulation and mechanism of biofilm dispersion has been elucidated only in a limited number of bacteria. Here, we identified two biofilm dispersion mechanisms in the Gram-positive, spore-forming bacterium Bacillus subtilis. The addition of calcium to the medium stabilized biofilms and caused resistance to dispersal mechanisms. Our findings provide new insights into biofilm dispersion and biofilm control.