Nitric oxide regulation of bacterial biofilms

Biofilms, surface-adhered bacterial communities, are widespread, persistent, and extremely resistant to antibiotics. Not surprisingly, they constitute a significant public health hazard; new strategies for biofilm prevention are desperately needed. Our group has taken the approach that the best way to eradicate biofilm infections is not to try to kill bacteria in the biofilm form, but to trick them into dispersing from biofilm, and thus restoring their susceptibility to antibiotics. When we began our work, several reports had documented nitric oxide (NO) dispersion of biofilms through an unknown pathway. We observed that in many bacteria, hnoX genes neighbor genes encoding cyclic-di-guanidinemonophosphate (cyclic-di-GMP) processing enzymes and/or histidine kinases. We therefore hypothesized that two well-established biofilm regulatory mechanisms, intracellular cyclic-di-GMP signaling and intercellular quorum sensing, might be regulated by NO binding to hnoX-encoded proteins, i.e., H-NOXs. We have shown that H-NOX is a modular NO sensor that negatively affects biofilm formation, either:

(1) by directly regulating cyclic-di-GMP processing enzymes;

• Nisbett, L.M.; Binnenkade, L.; Bacon, B.A.; Hossain, S.; Kotloski, N.J.; Brutinel, E.D.; Hartmann, R.; Drescher, K.; Arora, D.P.; Muralidharan, S.; Thormann, K.; Gralnick, J.A.; Boon, E.M.* (2019) NosP signaling controls the NO/H-NOX-mediated multicomponent cyclic-di-GMP network and biofilm formation in Shewanella oneidensis. Biochemistry 58, 4827-4841 (DOI: 10.1021/acs.biochem.9b00706; PMCID: PMC7290162). **ACS Editor’s choice selection.
• Nesbitt, N.M.; Arora, D.P.; Johnson, R.A.; Boon, E.M.* (2015) Modification of a bi-functional diguanylate cyclase-phosphodiesterase to efficiently produce cyclic di-guanylate monophosphate. Biotechnology Reports 7, 30-37 (DOI: 10.1016/j.btre.2015.04.008). Policy Exempt – Not resulting from NIH funding.
• Lahiri, T.; Luan, B.; Raleigh, D.P.; Boon, E.M.* (2014) A structural basis for the regulation of an H-NOX-associated cyclic-di-GMP synthase/phosphodiesterase enzyme by nitric oxide-bound H-NOX. Biochemistry 53, 2126-2135 (DOI: 10.1021/bi401597m; PMCID: PMC3985513).
• Liu, N.; Xu, Y.; Hossain, S.; Huang, N.; Coursolle, D.; Gralnick, J.; Boon, E.M.* (2012) Nitric oxide regulation of cyclic di-GMP synthesis and hydrolysis in Shewanella woodyi. Biochemistry 51, 2087-2099 (DOI: 10.1021/bi201753f). Policy Exempt – Not resulting from NIH funding. **Cited by the Faculty of 1000.

(2) by indirectly regulating cyclic-di-GMP processing enzymes through an intervening histidine kinase (we have also significantly contributed to the development of assays for assaying histidine kinases);

• Fischer, J.T.; Heckler, I.; Boon, E.M.* (2020) SDS-PAGE and dot blot autoradiography: Tools for quantifying histidine kinase autophosphorylation. Methods in Molecular Biology 2077, 37-49 (DOI: 10.1007/978-1-4939-9884-5_3). Policy Exempt – Not resulting from NIH funding.
• Ueno, T.B.; Johnson, R.A.; Boon, E.M.* (2015) Optimized assay for the quantification of histidine kinase autophosphorylation. Biochemical and Biophysical Research Communications 465, 331-337 (DOI: 10.1016/j.bbrc.2015.07.121). Policy Exempt – Not resulting from NIH funding.
• Arora, D.P.; Boon, E.M.* (2013) Unexpected biotinylation using ATP-γ-Biotin-LC-PEO-amine as a kinase substrate. Biochemical and Biophysical Research Communications 432, 287-290 (DOI: 10.1016/j.bbrc.2013.01.115). Policy Exempt – Not resulting from NIH funding.
• Arora, D.P.; Boon, E.M.* (2012) Nitric oxide regulated two-component signaling in Pseudoalteromonas atlantica. Biochemical and Biophysical Research Communications 421, 521-526 (DOI: 10.1016/j.bbrc.2012.04.037). Policy Exempt – Not resulting from NIH funding.

(3) or, sometimes, NO-bound H-NOX enters quorum sensing circuits through a histidine kinase;

• Hossain, S.; Heckler, I.; Boon, E.M.* (2018) Discovery of a nitric oxide responsive quorum sensing circuit in Vibrio cholera. American Chemical Society Chemical Biology 13, 1964–1969 (DOI: 10.1021/acschembio.8b00360; PMCID: PMC6195201).
• Ueno, T.; Fischer, J.T.; Boon, E.M.* (2019) Nitric oxide enters quorum sensing via the H-NOX signaling pathway in Vibrio parahaemolyticus. Frontiers in Microbiology 10:2108 (DOI: 10.3389/fmicb.2019.02108; PMCID: PMC6759604).
• Henares, B.M.; Xu, Y.; Boon, E.M.* (2013) A nitric oxide-responsive quorum sensing circuit in Vibrio harveyi regulates flagella production and biofilm formation. The International Journal of Molecular Sciences 14, 16473-16484 (DOI: 10.3390/ijms140816473; PMCID: PMC3759921). 
• Henares, B.M.; Higgins, K.E.; Boon, E.M.* (2012) Discovery of a nitric oxide-responsive quorum sensing circuit in Vibrio harveyi. American Chemical Society Chemical Biology 7, 1331-1336 (DOI: 10.1021/cb300215t). Policy Exempt – Not resulting from NIH funding. **Featured in Chemical & Engineering News (Fellet, M. 2012 May 29. Nitric Oxide Regulates Group Behavior of Bacteria. Chemical & Engineering News, American Chemical Society) and the ACS Chemical Biology podcast (volume 7, episode 8).

(4) We have also pursued understanding the molecular details of NO/H-NOX signaling. 

NO binds H-NOX at the iron atom of a histidine-ligated heme cofactor that is severely distorted from planarity. It has long been assumed that histidine dissociation upon NO ligation is the critical step for signal initiation. However, we established that the iron-histidine bond is retained after NO binding. Instead, we demonstrated that heme flattening upon NO binding is the trigger for signal transduction. 

(5) Furthermore, we observed that NO regulates biofilm in species, including Pseudomonas aeruginosa, that lack hnoX homologs. 

To bridge this knowledge gap, we discovered an additional heme-based NO sensor named NosP. Disruption of nosP in P. aeruginosa results in significantly less biofilm, and loss of the NO phenotype. Further, we have demonstrated that NO-bound NosP regulates downstream histidine kinases and cyclic-di-GMP synthesis/hydrolysis enzymes in many species. 

• Mendoza, A.G.; Guercio, D.; Smiley, M.K.; Sharma, G.K.; Withorn, J.M.; Hudson-Smith, N.V.; Ndukwe, C.; Dietrich, L.E.P; Boon, E.M.* (2024) The Histidine Kinase NahK Regulates Pyocyanin Production through the PQS system. Journal of Bacteriology, in press (DOI: 10.1128/jb.00276-23; PMID: 38169296).
• Fu, J.; Nisbett, L.M.; Guo, Y.; Boon, E.M.* (2023) NosP detection of heme modulates Burkholderia thailandensis biofilm formation. Biochemistry, 62, 2426-2441 (DOI: 10.1021/acs.biochem.3c00187).
• Fischer, J.T.; Hossain, S.; Boon, E.M.* (2019) NosP modulates cyclic-di-GMP signaling in Legionella pneumophila. Biochemistry58, 4325-4334 (DOI: 10.1021/acs.biochem.9b00618; PMCID: PMC6812680).
• Hossain, S.; Boon, E.M.* (2017) NosP: Discovery of a nitric oxide binding protein and nitric oxide-responsive signaling pathway in Pseudomonas aeruginosa. American Chemical Society Infectious Diseases 3, 454-461 (DOI: 10.1021/acsinfecdis.7b00027; PMCID: PMC5468770). **Cited by the Faculty of 1000; ACS Editor’s choice selection; Featured on the cover of American Chemical Society Infectious Diseases and was its 3^rd most read article in 2017.

In summary, we have discovered the mechanisms underlying NO regulation of biofilms. 

Our work is the starting point for deeper investigations into the role of NO in bacteria and bacterial/host interactions. These fundamental investigations will result in novel strategies for biofilm regulation with widespread application.