Biofilm selection activates c-di-GMP synthesis by the bifunctional enzyme MbaA

Abstract Biofilms, in which microbes are encased within a self-produced matrix, represent the primary mode of microbial life. Yet, our understanding of the molecular mechanisms governing biofilm formation remains incomplete, limiting the development of targeted interventions against biofilm-associated infections. By selecting for Vibrio cholerae biofilms, we identified mutations in the polyamine-regulated, bifunctional enzyme MbaA that alter its enzymatic activity in metabolizing the conserved second messenger cyclic diguanylate (c-di-GMP). These mutations occur in both the c-di-GMP-producing GGDEF domain and the c-di-GMP-hydrolyzing EAL domain of MbaA. Mutagenesis and enzyme kinetic analyses revealed regulatory crosstalk between these domains, where mutations in either domain enhanced production of c-di-GMP by MbaA while concurrently reducing its hydrolytic activity. In the wild-type enzyme, c-di-GMP metabolism is post-translationally regulated by specific polyamines. Biofilm-selected mutations not only shift MbaA’s enzymatic activity but also disrupt this regulation, rendering the enzyme insensitive to external polyamine signaling. Our findings illustrate how evolutionary pressure can rewire existing enzymes, promoting a biofilm-associated lifestyle.