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.

Pathogenic bacteria have many strategies for causing disease in humans. One such strategy is the ability to live both as single-celled motile organisms or as part of a community of bacteria called a biofilm. Biofilms are frequently adhered to biotic or abiotic surfaces and are extremely antibiotic resistant. Upon biofilm dispersal, bacteria become more antibiotic susceptible but are also able to readily infect another host. Various studies have shown that low, nontoxic levels of nitric oxide (NO) may induce biofilm dispersal in many bacterial species. While the molecular details of this phenotype remain largely unknown, in several species, NO has been implicated in biofilm-to-planktonic cell transitions via ligation to 1 of 2 characterized NO sensors, NosP or H-NOX. Based on the data available to date, it appears that NO binding to H-NOX or NosP triggers a downstream response based on changes in cellular cyclic di-GMP concentrations and/or the modulation of quorum sensing. In order to develop applications for control of biofilm infections, the identification and characterization of biofilm dispersal mechanisms is vital. This review focuses on the efforts made to understand NO-mediated control of H-NOX and NosP pathways in the 3 pathogenic bacteria Legionella pneumophila, Vibrio cholerae, and Pseudomonas aeruginosa.

Nitric oxide (NO) detection and signalling are widely mediated by haemoproteins in eukaryotes and bacteria. This review highlights the ligand-binding properties, activation mechanisms, and structures of six proteins that have been classified as haem-based NO-sensing proteins: sGC, H-NOX, YybT, E75, NosP, and DNR. sGC is a eukaryotic haem-based sensor that responds to NO to catalyse the production of the ubiquitous secondary messaging signalling molecule cGMP. Much of the progress toward elucidating the NO activation mechanism of sGC has been achieved through the study of bacterial haem-nitric oxide and oxygen (H-NOX) binding proteins. H-NOX proteins are capable of influencing downstream signal transduction in several bacterial species; however, many bacteria that respond to nanomolar concentrations of NO do not contain an annotated H-NOX domain. Of all bacterial species, NO signalling has been most frequently investigated in Pseudomonas aeruginosa, which do not encode an H-NOX domain, and so several receptors of NO have been suggested in this species. Most recently, a newly discovered family of NO-sensing proteins (NosP) was demonstrated to be a mediator of a histidine kinase signal-transduction pathway in P. aeruginosa. NosP proteins are widely conserved in bacteria but have thus far only been characterized in P. aeruginosa. Additionally, a transcriptional regulator called DNR (dissimilative nitrate respiration regulator) has been shown to be a haem-based NO receptor that controls anaerobic denitrification in P. aeruginosa. Another putative bacterial haem-based NO sensor, the cyclic-di-AMP-specific phosphodiesterase YybT is widely distributed across the firmicutes phylum and has been implicated in bacterial survival. Finally, a putative NO sensor in insects, E75, is a haem-based transcriptional regulator. sGC, H-NOX, YybT, E75, NosP, and DNR are discussed in more detail.

Transitions between motile and biofilm lifestyles are highly regulated and fundamental to microbial pathogenesis. H-NOX (heme-nitric oxide/oxygen-binding domain) is a key regulator of bacterial communal behaviors, such as biofilm formation. A predicted bifunctional cyclic di-GMP metabolizing enzyme, composed of diguanylate cyclase and phosphodiesterase (PDE) domains (avi_3097), is annotated downstream of an hnoX gene in Agrobacterium vitis S4. Here, we demonstrate that avH-NOX is a nitric oxide (NO)-binding hemoprotein that binds to and regulates the activity of avi_3097 (avHaCE; H-NOX-associated cyclic di-GMP processing enzyme). Kinetic analysis of avHaCE indicates a ∼four-fold increase in PDE activity in the presence of NO-bound avH-NOX. Biofilm analysis with crystal violet staining reveals that low concentrations of NO reduce biofilm growth in the wild-type A. vitis S4 strain, but the mutant ΔhnoX strain has no NO phenotype, suggesting that H-NOX is responsible for the NO biofilm phenotype in A. vitis. Together, these data indicate that avH-NOX enhances cyclic di-GMP degradation to reduce biofilm formation in response to NO in A. vitis.