Increased Osmolarity in Biofilm Triggers RcsB-Dependent Lipid A Palmitoylation in Escherichia coli

Biofilms are often described as protective shelters that preserve bacteria from hostile surroundings. However, biofilm bacteria are also exposed to various stresses and need to adjust to the heterogeneous physicochemical conditions prevailing within biofilms. In Gram-negative bacteria, such adaptations can result in modifications of the lipopolysaccharide, a major component of the outer membrane characterized by a highly dynamic structure responding to environmental changes. We previously showed that Gram-negative biofilm bacteria undergo an increase in lipid A palmitoylation mediated by the PagP enzyme, contributing to increased resistance to host defenses. Here we describe a regulatory pathway leading to transcriptional induction of pagP in response to specific conditions created in the biofilm environment. We show that pagP expression is induced via the Rcs envelope stress system independently of the Rcs phosphorelay cascade and that it requires the GadE auxiliary regulator. Moreover, we identify an increase in osmolarity (i.e., ionic stress) as a signal able to induce pagP expression in an RcsB-dependent manner. Consistently, we show that the biofilm is a hyperosmolar environment and that RcsB-dependent pagP induction can be dampened in the presence of an osmoprotectant. These results provide new insights into the adaptive mechanisms of bacterial differentiation in biofilm.