CONSPECTUS Bacterial biofilms form when bacteria adhere to a surface and produce an exopolysaccharide matrix (Costerton et al. Science 1999, 284, 1318; Davies et al. Science 1998, 280, 295; Flemming et al. Nat. Rev. Microbiol. 2010, 8, 623). Because biofilms are resistant to antibiotics, they are problematic in many aspects of human health and welfare, causing, for instance, persistent fouling of medical implants such as catheters and artificial joints (Brunetto et al. Chimia 2008, 62, 249). They are responsible for chronic infections in the lungs of cystic fibrosis patients and in open wounds, such as those associated with burns and diabetes. They are also a major contributor to hospital-acquired infections (Sievert et al. Infec. Control Hosp. Epidemiol. 2013, 34, 1; Tatterson et al. Front. Biosci. 2001, 6, D890). It has been hypothesized that effective methods of biofilm control will have widespread application (Landini et al. Appl. Microbiol. Biotechnol. 2010, 86, 813). A promising strategy is to target the mechanisms that drive biofilm dispersal, because dispersal results in biofilm removal and in the restoration of antibiotic sensitivity. First documented in Nitrosomonas europaea (Schmidt et al. J. Bacteriol. 2004, 186, 2781) and the cystic fibrosis-associated pathogen Pseudomonas aeruginosa (Barraud et al. J. Bacteriol. 2006, 188, 7344; J. Bacteriol. 2009, 191, 7333), regulation of biofilm formation by nanomolar levels of the diatomic gas nitric oxide (NO) has now been documented in numerous bacteria (Barraud et al. Microb. Biotechnol. 2009, 2, 370; McDougald et al. Nat. Rev. Microbiol. 2012, 10, 39; Arora et al. Biochemistry 2015, 54, 3717; Barraud et al. Curr. Pharm. Des. 2015, 21, 31). NO-mediated pathways are, therefore, promising candidates for biofilm regulation. Characterization of the NO sensors and NO-regulated signaling pathways should allow for rational manipulation of these pathways for therapeutic applications. Several laboratories, including our own, have shown that a class of NO sensors called H-NOX (heme-nitric oxide or oxygen binding domain) affects biofilm formation by regulating intracellular cyclic di-GMP concentrations and quorum sensing (Arora et al. Biochemistry 2015, 54, 3717; Plate et al. Trends Biochem. Sci. 2013, 38, 566; Nisbett et al. Biochemistry 2016, 55, 4873). Many bacteria that respond to NO do not encode an hnoX gene, however. My laboratory has now discovered an additional family of bacterial NO sensors, called NosP (nitric oxide sensing protein). Importantly, NosP domains are widely conserved in bacteria, especially Gram-negative bacteria, where they are encoded as fusions with or in close chromosomal proximity to histidine kinases or cyclic di-GMP synthesis or phosphodiesterase enzyme, consistent with signaling. In this Account, we briefly review NO and HNOX signaling in bacterial biofilms, describe our discovery of the NosP family, and provide support for its role in biofilm regulation in Pseudomonas aeruginosa, Vibrio cholerae, Legionella pneumophila, and Shewanella o...

Nitric oxide (NO) is a freely diffusible, radical gas that has now been established as an integral signaling molecule in eukaryotes and bacteria. It has been demonstrated that NO signaling is initiated upon ligation to the heme iron of an H-NOX domain in mammals and in some bacteria. Bacterial H-NOX proteins have been found to interact with enzymes that participate in signaling pathways and regulate bacterial processes such as quorum sensing, biofilm formation and symbiosis. Here, we review the biochemical characterization of these signaling pathways, and where available, describe how NO ligation to H-NOX specifically regulates the activity of these pathways and their associated bacterial phenotypes.

The molecular mechanisms of NO regulation in many bacteria remain to be established. Future studies are required to gain knowledge about the mechanism of NosP signaling. Advancements on structural and molecular understanding of heme-based sensors in bacteria could lead to strategies to alleviate or control bacterial biofilm formation or persistent biofilm-related infections.

Biofilms form when bacteria aggregate in a self-secreted exopolysaccharide matrix; they are resistant to antibiotics and implicated in disease. Nitric oxide (NO) is known to mediate biofilm formation in many bacteria via ligation to H-NOX (heme-NO/oxygen binding) domains. Most NO-responsive bacteria, however, lack H-NOX domain-containing proteins. We have identified another NO-sensing protein (NosP), which is predicted to be involved in two-component signaling and biofilm regulation in many species. Here, we demonstrate that NosP participates in the previously described H-NOX/NO-responsive multicomponent c-di-GMP signaling network in Shewanella oneidensis. Strains lacking either nosP or its co-cistronic kinase nahK (previously hnoS) produce immature biofilms, while hnoX and hnoK (kinase responsive to NO/H-NOX) mutants result in wild-type biofilm architecture. We demonstrate that NosP regulates the autophosphorylation activity of NahK as well as HnoK. HnoK and NahK have been shown to regulate three response regulators (HnoB, HnoC, and HnoD) that together comprise a NO-responsive multicomponent c-di-GMP signaling network. Here, we propose that NosP/NahK adds regulation on top of H-NOX/HnoK to modulate this c-di-GMP signaling network, and ultimately biofilm formation, by governing the flux of phosphate through both HnoK and NahK. In addition, it appears that NosP and H-NOX act to counter each other in a push–pull mechanism; NosP/NahK promotes biofilm formation through inhibition of H-NOX/HnoK signaling, which itself reduces the extent of biofilm formation. Addition of NO results in a reduction of c-di-GMP and biofilm formation, primarily through disinhibition of HnoK activity.

Low concentrations of nitric oxide (NO) modulate varied behaviours in bacteria including biofilm dispersal and quorum sensing-dependent light production. H-NOX (haem-nitric oxide/oxygen binding) is a haem-bound protein domain that has been shown to be involved in mediating these bacterial responses to NO in several organisms. However, many bacteria that respond to nanomolar concentrations of NO do not contain an annotated H-NOX domain. Nitric oxide sensing protein (NosP), a newly discovered bacterial NO-sensing haemoprotein, may fill this role. The focus of this review is to discuss structure, ligand binding, and activation of H-NOX proteins, as well as to discuss the early evidence for NO sensing and regulation by NosP domains. Further, these findings are connected to the regulation of bacterial biofilm phenotypes and symbiotic relationships.