Discovery and characterization of NO-responsive hemoproteins that regulate bacterial biofilms

Bacteria colonize most surfaces, forming multicellular, antibiotic-resistant, communities known as biofilms. Biofilms cause chronic infections and persistent biofouling of medical implants, marine vessels, and environmental sensors. Biofilm dispersal by nanomolar nitric oxide (NO) appears to be a general phenomenon, but fundamental questions remain concerning the identity of the NO sensor and mechanism of signal transduction. NO has been reported to disperse bacterial biofilms through regulation of intracellular cyclic-di-guanosine monophosphate concentrations. C-di-GMP is a tightly regulated second messenger-signaling molecule that is tightly correlated with biofilm formation. HNOX proteins are well known NO sensors conserved in many bacteria. Indeed, we have shown that NO/H-NOX signaling disperses bacterial biofilms through a mechanism consistent with c-di-GMP signaling. However, H-NOX proteins are not conserved in most human pathogens. Therefore, an alternate NO sensor must also exist. We have identified a potential alternate NO sensor, a novel hemoprotein we named NosP (nitric oxide sensing protein). NosP domains are conserved in many bacterial genomes, they bind NO, but not molecular oxygen, as expected for a NO-specific sensor, and they are encoded as fusions with, or in close chromosomal proximity to, proteins annotated as cdi-GMP synthesis or hydrolysis enyzmes. We hypothesize that NO generally disperses bacterial biofilms through regulation of intracellular c-di-GMP concentrations, but the sensor varies; both NosP and H-NOX can fill this role.