Engineering a novel c-di-GMP-binding protein for biofilm dispersal

Bacteria prefer to grow attached to themselves or an interface, and it is important for an array of applications to make biofilms disperse. Here we report simultaneously the discovery and protein engineering of BdcA (formerly YjgI) for biofilm dispersal using the universal signal 3,5-cyclic diguanylic acid (c-di-GMP). The bdcA deletion reduced biofilm dispersal, and production of BdcA increased biofilm dispersal to wild-type level. Since BdcA increases motility and extracellular DNA production while decreasing exopolysaccharide, cell length and aggregation, we reasoned that BdcA decreases the concentration of c-di-GMP, the intracellular messenger that controls cell motility through flagellar rotation and biofilm formation through synthesis of curli and cellulose. Consistently, c-di-GMP levels increase upon deleting bdcA, and purified BdcA binds c-di-GMP but does not act as a phosphodiesterase. Additionally, BdcR (formerly YjgJ) is a negative regulator of bdcA. To increase biofilm dispersal, we used protein engineering to evolve BdcA for greater c-di-GMP binding and found that the single amino acid change E50Q causes nearly complete removal of biofilms via dispersal without affecting initial biofilm formation.