Discovery of a Novel Nitric Oxide Binding Protein and Nitric-Oxide-Responsive Signaling Pathway in Pseudomonas aeruginosa

[Image: see text] Nitric oxide (NO) is a radical diatomic gas molecule that, at low concentrations, plays important signaling roles in both eukaryotes and bacteria. In recent years, it has become evident that bacteria respond to low levels of NO in order to modulate their group behavior. Many bacteria respond via NO ligation to a well-established NO sensor called H-NOX (heme-nitric oxide/oxygen binding domain). Many others, such as Pseudomonas aeruginosa, lack an annotated hnoX gene in their genome yet are able to respond to low levels of NO to disperse their biofilms. This suggests the existence of a previously uncharacterized NO sensor. In this study, we describe the discovery of a novel nitric oxide binding protein (NosP; NO-sensing protein), which is much more widely conserved in bacteria than H-NOX, as well as a novel NO-responsive pathway in P. aeruginosa. We demonstrate that biofilms of a P. aeruginosa mutant lacking components of the NosP pathway lose the ability to disperse in response to NO. Upon cloning, expressing, and purifying NosP, we find it binds heme and ligates to NO with a dissociation rate constant that is comparable to that of other well-established NO-sensing proteins. Moreover, we show that NO-bound NosP is able to regulate the phosphorelay activity of a hybrid histidine kinase that is involved in biofilm regulation in P. aeruginosa. Thus, here, we present evidence of a novel NO-responsive pathway that regulates biofilm in P. aeruginosa.