Current Research

When this work began, low concentrations of nitric oxide (NO) were known to promote biofilm dispersal, but the molecular mechanisms connecting NO detection to changes in biofilm behavior were poorly understood. My laboratory established that bacterial H-NOX proteins connect NO sensing to c-di-GMP, a second messenger controlling the transition between motile and biofilm-associated states. In Shewanella woodyi, we demonstrated that H-NOX regulates a bifunctional c-di-GMP synthase and phosphodiesterase: unligated H-NOX promotes c-di-GMP synthesis, whereas NO-bound H-NOX enhances c-di-GMP hydrolysis. We subsequently defined the protein-interaction surface and structural mechanism through which H-NOX regulates this enzyme. We also showed that H -NOX proteins can regulate associated histidine kinases and multicomponent phosphorelay systems, establishing that bacterial NO sensors employ multiple biochemical architectures to control c-di-GMP concentrations and community behavior. This body of work provided a mechanistic foundation for understanding how NO can remodel bacterial biofilms.

Selected publications:

• Liu N, Xu Y, Hossain S, Huang N, Coursolle D, Gralnick JA, Boon EM. (2012) Nitric oxide regulation of cyclic di-GMP synthesis and hydrolysis in Shewanella woodyi. Biochemistry. 2012;51:2087–2099. PMID: 22360279.

• Arora DP, Boon EM. Nitric oxide-regulated two-component signaling in Pseudoalteromonas atlantica. Biochemical and Biophysical Research Communications. 2012;421:521–526. PMID: 22521885.

• Lahiri T, Luan B, Raleigh DP, Boon EM. A structural basis for the regulation of an H-NOX-associated cyclic-di-GMP synthase/phosphodiesterase enzyme by nitric oxide-bound H-NOX. Biochemistry. 2014;53:2126–2135. PMID: 24628400.

• Nisbett LM, Boon EM. Nitric oxide regulation of H-NOX signaling pathways in bacteria. Biochemistry. 2016;55:4873–4884. PMID: 27479081.

Quorum sensing (QS) was traditionally viewed primarily as a mechanism through which bacteria estimate population density. My laboratory demonstrated that quorum-sensing networks also integrate NO as information about the chemical environment. In Vibrio harveyi, we discovered an NO-responsive quorum-sensing pathway in which NO-bound H-NOX inhibits an associated histidine kinase, thereby altering phosphate transfer through the central LuxU/LuxO quorum-sensing phosphorelay. We showed that this pathway controls quorum-sensing outputs, including bioluminescence, flagellar production, and biofilm formation. We subsequently identified related NO-responsive quorum-sensing circuits in Vibrio parahaemolyticus and demonstrated that NosP can regulate quorum sensing in Vibrio cholerae. These studies broadened the conceptual definition of quorum sensing by demonstrating that bacterial collective decisions integrate population-density signals with information about environmental or host-derived NO.

Selected publications:

• Henares BM, Higgins KE, Boon EM. Discovery of a nitric oxide-responsive quorum-sensing circuit in Vibrio harveyi. ACS Chemical Biology. 2012;7(8):1331–1336. PMID: 22606970.

• Henares BM, Xu Y, Boon EM. A nitric oxide-responsive quorum-sensing circuit in Vibrio harveyi regulates flagella production and biofilm formation. International Journal of Molecular Sciences. 2013;14(8):16473–16484. PMID: 23965964.

• Hossain S, Heckler I, Boon EM. Discovery of a nitric oxide-responsive quorum-sensing circuit in Vibrio cholerae. ACS Chemical Biology. 2018;13(8):1964–1969. PMID: 30060647.

• Ueno T, Fischer JT, Boon EM. Nitric oxide enters quorum sensing via the H-NOX signaling pathway in Vibrio parahaemolyticus. Frontiers in Microbiology. 2019;10:2108. PMID: 31620101.

Many clinically important bacteria respond to low concentrations of NO but lack an identifiable H-NOX protein, indicating that additional bacterial NO receptors remained undiscovered. My laboratory identified NosP, a previously unrecognized family of heme-binding proteins that is more broadly distributed among bacteria than H-NOX. We established that NosP binds NO and regulates associated histidine kinases and c-di-GMP-processing enzymes. In Pseudomonas aeruginosa, we showed that NosP controls an NO-responsive signaling pathway required for normal biofilm formation and NO-dependent biofilm dispersal. Spectroscopic studies subsequently defined the heme coordination and ligand-binding properties of NosP, supporting its function as a biologically relevant gas sensor. The discovery of NosP substantially expanded the known repertoire of bacterial NO-sensing proteins and provided a molecular explanation for NO responsiveness in pathogens lacking H-NOX.

Selected publications:

• Hossain S, Boon EM. Discovery of a novel nitric oxide-binding protein and nitric-oxide-responsive signaling pathway in Pseudomonas aeruginosa. ACS Infectious Diseases. 2017;3(6):454–461. PMID: 28238256.

• Hossain S, Nisbett LM, Boon EM. Discovery of two bacterial nitric oxide-responsive proteins and their roles in bacterial biofilm regulation. Accounts of Chemical Research. 2017;50(7):1633–1639. PMID: 28605194.

• Bacon BA, Liu Y, Kincaid JR, Boon EM. Spectral characterization of a novel NO-sensing protein in bacteria: NosP. Biochemistry. 2018;57(44):6187–6200. PMID: 30272959.

• Williams DE, Boon EM. Towards understanding the molecular basis of nitric oxide-regulated group behaviors in pathogenic bacteria. Journal of Innate Immunity. 2019;11(3):205–215. PMID: 30557874.

Following the discovery of NosP, my laboratory established that these proteins are multifunctional environmental sensors whose regulatory activities extend beyond a single organism or signaling output. In Legionella pneumophila, we demonstrated that NosP regulates an associated c-di-GMP phosphodiesterase and thereby controls intracellular c-di-GMP concentrations. In Shewanella oneidensis, we showed that NosP and H-NOX feed into the same multicomponent c-di-GMP network and act through a push-pull mechanism to regulate biofilm development. We further discovered that heme binding can inhibit a NosP-associated phosphodiesterase and that NosP detects labile heme to regulate biofilm formation in Burkholderia thailandensis. These findings established that NosP proteins can integrate NO and heme availability with two-component and c-di-GMP signaling. This work broadened the significance of bacterial heme-sensor proteins from dedicated gas receptors to multifunctional regulators of environmental adaptation and community behavior.

Selected publications:

• Fischer JT, Hossain S, Boon EM. NosP modulates cyclic-di-GMP signaling in Legionella pneumophila. Biochemistry. 2019;58(42):4325–4334. PMID: 31576744.

• Nisbett LM, Binnenkade L, Bacon B, et al. NosP signaling modulates the NO/H-NOX-mediated multicomponent cyclic-di-GMP network and biofilm formation in Shewanella oneidensis. Biochemistry. 2019;58(48):4827–4841. PMID: 31682418.

• Heckler I, Hossain S, Boon EM. Heme inhibits the activity of a c-di-GMP phosphodiesterase in Vibrio cholerae. Biochemical and Biophysical Research Communications. 2020;529(4):1112-1116. PMID: 32819573.

• Fu J, Hall S, Boon EM. NosP detection of heme modulates Burkholderia thailandensis biofilm formation. Biochemistry. 2023;62(16):2426-2441PMID: 37498555.

My laboratory discovered that the NosP-associated histidine kinase NahK is embedded within the highly interconnected GacS multikinase network of P. aeruginosa. We demonstrated that NO-bound NosP inhibits NahK autophosphorylation and that NahK signaling controls biofilm architecture, exopolysaccharide production, motility, c-di-GMP signaling, and virulence-associated phenotypes. We established that NahK regulates pyocyanin production primarily through the PQS quorum-sensing system and subsequently showed that NahK controls denitrification, endogenous NO accumulation, and anaerobic physiology through the global post-transcriptional regulator RsmA. More recently, we demonstrated that NahK and RetS provide distinct regulatory inputs that tune RsmA activity and permit rapid transitions between motile and biofilm-associated states. Together, these studies reveal that NosP–NahK is not a simple linear dispersal pathway but a central signaling module through which P. aeruginosa integrates NO, respiration, quorum sensing, nutrient status, and stress to select among persistent, virulent, motile, and dispersal-associated states.

Selected publications

• Hossain S, Boon EM. Discovery of a novel nitric oxide-binding protein and nitric-oxide-responsive signaling pathway in Pseudomonas aeruginosa. ACS Infectious Diseases. 2017;3:454–461. PMID: 28238256.

•  Mendoza AG, et al. The histidine kinase NahK regulates pyocyanin production through the PQS system. Journal of Bacteriology. 2024;206. PMID: 38169296.

• Guercio D, Boon EM. The histidine kinase NahK regulates denitrification and nitric oxide accumulation through RsmA in Pseudomonas aeruginosa. Journal of Bacteriology. 2025;207. PMID: 39660891.

• Withorn JM, et al. Pseudomonas aeruginosa uses kinases NahK and RetS to control the motile–biofilm switch. 2025. PMID: 41040157.

Start with these review articles:

• Anantharaman, Guercio, Mendoza, WIthorn, Boon (2023) Biochem Soc Trans, 51, 1447-1458.

• Hossain, Nisbett, Boon (2017) Accounts Chem Res, 50, 1633-1639.

• Bacon, Nisbett, Boon (2017) Adv Microb Physiol, 70, 1-36.

• Nisbett, Boon (2016) Biochemistry, 55, 4873-4884.

• Arora, Hossain, Xu, Boon (2015) Biochemistry, 54, 3717-3728.

We are grateful for generous funding from these sources:

• Stony Brook University

• New York State Foundation for Science, Technology, and Innovation

• The Office of Naval Research

• PECASE

• The National Science Foundation

• The Stony Wold-Herbert Fund

• The National Institutes of Health

We are affiliated with the following graduate training programs:

• Chemistry

• Chemical Biology

• Biochemistry & Structural Biology

• Molecular & Cellular Biology

• Microbiology & Immunology

• Molecular Pharmacology

• Molecular and Cell Biology of Infectious Diseases