The histidine kinase NahK regulates pyocyanin production through the PQS system.

Many bacterial histidine kinases work in two-component systems that combine into larger multi-kinase networks. NahK is one of the kinases in the GacS Multi-Kinase Network (MKN), which is the MKN that controls biofilm regulation in the opportunistic pathogen Pseudomonas aeruginosa. This network has also been associated with regulating many virulence factors P. aeruginosa secretes to cause disease. However, the individual role of each kinase is unknown. In this study, we identify NahK as a novel regulator of the phenazine pyocyanin (PYO). Deletion of nahK leads to a fourfold increase in PYO production, almost exclusively through upregulation of phenazine operon two (phz2). We determined that this upregulation is due to mis-regulation of all P. aeruginosa quorum-sensing (QS) systems, with a large upregulation of the Pseudomonas quinolone signal system and a decrease in production of the acyl-homoserine lactone-producing system, las. In addition, we see differences in expression of quorum-sensing inhibitor proteins that align with these changes. Together, these data contribute to understanding how the GacS MKN modulates QS and virulence and suggest a mechanism for cell density-independent regulation of quorum sensing. IMPORTANCE Pseudomonas aeruginosa is a Gram-negative bacterium that establishes biofilms as part of its pathogenicity. P. aeruginosa infections are associated with nosocomial infections. As the prevalence of multi-drug-resistant P. aeruginosa increases, it is essential to understand underlying virulence molecular mechanisms. Histidine kinase NahK is one of several kinases in P. aeruginosa implicated in biofilm formation and dispersal. Previous work has shown that the nitric oxide sensor, NosP, triggers biofilm dispersal by inhibiting NahK. The data presented here demonstrate that NahK plays additional important roles in the P. aeruginosa lifestyle, including regulating bacterial communication mechanisms such as quorum sensing. These effects have larger implications in infection as they affect toxin production and virulence.

Negative regulation of biofilm formation by nitric oxide sensing proteins.

Biofilm-based infections pose a serious threat to public health. Biofilms are surface-attached communities of microorganisms, most commonly bacteria and yeast, residing in an extracellular polymeric substance (EPS). The EPS is composed of several secreted biomolecules that shield the microorganisms from harsh environmental stressors and promote antibiotic resistance. Due to the increasing prominence of multidrug-resistant microorganisms and a decreased development of bactericidal agents in clinical production, there is an increasing need to discover alternative targets and treatment regimens for biofilm-based infections. One promising strategy to combat antibiotic resistance in biofilm-forming bacteria is to trigger biofilm dispersal, which is a natural part of the bacterial biofilm life cycle. One signal for biofilm dispersal is the diatomic gas nitric oxide (NO). Low intracellular levels of NO have been well documented to rapidly disperse biofilm macrostructures and are sensed by a widely conserved NO-sensory protein, NosP, in many pathogenic bacteria. When bound to heme and ligated to NO, NosP inhibits the autophosphorylation of NosP's associated histidine kinase, NahK, reducing overall biofilm formation. This reduction in biofilm formation is regulated by the decrease in secondary metabolite bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP). The NosP/NahK signaling pathway is also associated with other major regulatory systems in the maturation of bacterial biofilms, including virulence and quorum sensing. In this review, we will focus on recent discoveries investigating NosP, NahK and NO-mediated biofilm dispersal in pathogenic bacteria.

NosP Detection of Heme Modulates Burkholderia thailandensis Biofilm Formation.

Aggregated bacteria embedded within self-secreted extracellular polymeric substances, or biofilms, are resistant to antibiotics and cause chronic infections. As such, they are a significant public health threat. Heme is an abundant iron source for pathogenic bacteria during infection; many bacteria have systems to detect heme assimilated from host cells, which is correlated with the transition between acute and chronic infection states. Here, we investigate the heme-sensing function of a newly discovered multifactorial sensory hemoprotein called NosP and its role in biofilm regulation in the soil-dwelling bacterium Burkholderia thailandensis, the close surrogate of Bio-Safety-Level-3 pathogen Burkholderia pseudomallei. The NosP family protein has previously been shown to exhibit both nitric oxide (NO)- and heme-sensing functions and to regulate biofilms through NosP-associated histidine kinases and two-component systems. Our in vitro studies suggest that BtNosP exhibits heme-binding kinetics and thermodynamics consistent with a labile heme-responsive protein and that the holo-form of BtNosP acts as an inhibitor of its associated histidine kinase BtNahK. Furthermore, our in vivo studies suggest that increasing the concentration of extracellular heme decreases B. thailandensis biofilm formation, and deletion of nosP and nahK abolishes this phenotype, consistent with a model that BtNosP detects heme and exerts an inhibitory effect on BtNahK to decrease the biofilm.

H-NOX Regulates Biofilm Formation in Agrobacterium Vitis in Response to NO.

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.

Self-Patterned Nanoscale Topography of Thin Copolymer Films Prepared by Evaporative Assembly-Resist Early-Stage Bacterial Adhesion.

Biofilm formation on the surfaces of indwelling medical devices has become a growing health threat due to the development of antimicrobial resistance to infection-causing bacteria. For example, ventilator-associated pneumonia caused by Pseudomonas and Staphylococci species has become a significant concern in treatment of patients during COVID-19 pandemic. Nanostructured surfaces with antifouling activity are of interest as a promising strategy to prevent bacterial adhesion without triggering drug resistance. In this study, we report a facile evaporative approach to prepare block copolymer film coatings with nanoscale topography that resist bacterial adhesion. The initial attachment of the target bacterium Pseudomonas aeruginosa PAO1 to copolymer films as well as homopolymer films was evaluated by fluorescence microscopy. Significant reduction in bacterial adhesion (93-99% less) and area coverage (>92% less) on the copolymer films was observed compared with that on the control and homopolymer films [poly(methacrylic acid) (PMAA)─only 40 and 23% less, respectively]. The surfaces of poly(styrene)-PMAA copolymer films with patterned nanoscale topography that contains sharp peaks ranging from 20 to 80 nm spaced at 30-50 nm were confirmed by atomic force microscopy and the corresponding surface morphology analysis. Investigation of the surface wettability and surface potential of polymer films assists in understanding the effect of surface properties on the bacterial attachment. Comparison of bacterial growth studies in polymer solutions with the growth studies on coatings highlights the importance of physical nanostructure in resisting bacterial adhesion, as opposed to chemical characteristics of the copolymers. Such self-patterned antifouling surface coatings, produced with a straightforward and energy-efficient approach, could provide a convenient and effective method to resist bacterial fouling on the surface of medical devices and reduce device-associated infections.

Nitric oxide stimulates type IV MSHA pilus retraction in Vibrio cholerae via activation of the phosphodiesterase CdpA.

Bacteria use surface appendages called type IV pili to perform diverse activities including DNA uptake, twitching motility, and attachment to surfaces. The dynamic extension and retraction of pili are often required for these activities, but the stimuli that regulate these dynamics remain poorly characterized. To address this question, we study the bacterial pathogen Vibrio cholerae, which uses mannose-sensitive hemagglutinin (MSHA) pili to attach to surfaces in aquatic environments as the first step in biofilm formation. Here, we use a combination of genetic and cell biological approaches to describe a regulatory pathway that allows V. cholerae to rapidly abort biofilm formation. Specifically, we show that V. cholerae cells retract MSHA pili and detach from a surface in a diffusion-limited, enclosed environment. This response is dependent on the phosphodiesterase CdpA, which decreases intracellular levels of cyclic-di-GMP to induce MSHA pilus retraction. CdpA contains a putative nitric oxide (NO)-sensing NosP domain, and we demonstrate that NO is necessary and sufficient to stimulate CdpA-dependent detachment. Thus, we hypothesize that the endogenous production of NO (or an NO-like molecule) in V. cholerae stimulates the retraction of MSHA pili. These results extend our understanding of how environmental cues can be integrated into the complex regulatory pathways that control pilus dynamic activity and attachment in bacterial species.

Recent evidence for multifactorial biofilm regulation by heme sensor proteins NosP and H-NOX.

Heme is involved in signal transduction by either acting as a cofactor of heme-based gas/redox sensors or binding reversely to heme-responsive proteins. Bacteria respond to low concentrations of nitric oxide (NO) to modulate group behaviors such as biofilms through the well-characterized H-NOX family and the newly discovered heme sensor protein NosP. NosP shares functional similarities with H-NOX as a heme-based NO sensor; both regulate two-component systems and/or cyclic-di-GMP metabolizing enzymes, playing roles in processes such as quorum sensing and biofilm regulation. Interestingly, aside from its role in NO signaling, recent studies suggest that NosP may also sense labile heme. In this Highlight Review, we briefly summarize H-NOX-dependent NO signaling in bacteria, then focus on recent advances in NosP-mediated NO signaling and labile heme sensing.

Heme inhibits the activity of a c-di-GMP phosphodiesterase in Vibrio cholerae.

Heme, a complex of iron and protoporphyrin IX, plays an essential role in numerous biological processes including oxygen transport, oxygen storage, and electron transfer. The role of heme as a prosthetic group in bacterial hemoprotein gas sensors, which utilize heme as a cofactor for the binding of diatomic gas molecules, has been well studied. Less well known is the role of protein sensors of heme. In this report, we characterize the heme binding properties of a phosphodiesterase, CdpA, from Vibrio cholerae. We demonstrate that the N-terminal domain of CdpA is a NosP domain capable of heme binding, which consequently inhibits the c-di-GMP hydrolysis activity of the C-terminal phosphodiesterase domain. Further evidence for CdpA as a heme responsive sensor is supported by a relatively fast rate of heme dissociation. This study provides insight into an emerging class of heme-responsive sensor proteins.

SDS-PAGE and Dot Blot Autoradiography: Tools for Quantifying Histidine Kinase Autophosphorylation.

Histidine kinases play a vital role in bacterial signal transduction. However, methods for studying the activity of histidine kinases in vitro are limited in comparison to those for investigating serine, threonine, and tyrosine kinases, largely due to the lability of the phosphoramidate (P-N) bond. Here, we describe two useful methods for quantifying histidine kinase autophosphorylation: SDS-PAGE autoradiography and dot blot autoradiography/scintillation counting.

NosP Signaling Modulates the NO/H-NOX-Mediated Multicomponent c-Di-GMP Network and Biofilm Formation in Shewanella oneidensis.

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.

NosP Modulates Cyclic-di-GMP Signaling in Legionella pneumophila.

Biofilms form when bacteria adhere to a surface and secrete an extracellular polymeric substance. Bacteria embedded within a biofilm benefit from increased resistance to antibiotics, host immune responses, and harsh environmental factors. Nitric oxide (NO) is a signaling molecule that can modulate communal behavior, including biofilm formation, in many bacteria. In many cases, NO-induced biofilm dispersal is accomplished through signal transduction pathways that ultimately lead to a decrease in intracellular cyclic-di-GMP levels. H-NOX (heme nitric oxide/oxygen binding domain) proteins are the best characterized bacterial NO sensors and have been implicated in NO-mediated cyclic-di-GMP signaling, but we have recently discovered a second family of NO-sensitive proteins in bacteria named NosP (NO sensing protein); to date, a clear link between NosP signaling and cyclic-di-GMP metabolism has not been established. Here we present evidence that NosP (Lpg0279) binds to NO and directly affects cyclic-di-GMP production from two-component signaling proteins Lpg0278 and Lpg0277 encoded within the NosP operon. Lpg0278 and Lpg0277 are a histidine kinase and cyclic-di-GMP synthase/phosphodiesterase, respectively, that have already been established as being important in regulating Legionella pneumophila cyclic-di-GMP levels; NosP is thus implicated in regulating cyclic-di-GMP in L. pneumophila.

Nitric Oxide Enters Quorum Sensing via the H-NOX Signaling Pathway in Vibrio parahaemolyticus.

Nitric oxide (NO) plays a major role in the regulation of mammalian biological functions. In recent years, NO has also been implicated in bacterial life cycles, including in the regulation of biofilm formation, and the metabolism of the bacterial second messenger signaling molecule cyclic-di-GMP. In a previous study, we reported the discovery of an NO-responsive quorum sensing (QS) circuit in Vibrio harveyi. Here, we characterize the homologous QS pathway in Vibrio parahaemolyticus. Spectroscopic analysis shows V. parahaemolyticus H-NOX is an NO sensory protein that binds NO in 5/6-coordinated mixed manner. Further, we demonstrate that through ligation to H-NOX, NO inhibits the autophosphorylation activity of an H-NOX-associated histidine kinase (HqsK; H-NOX-associated quorum sensing kinase) that transfers phosphate to the Hpt (histidine-containing phosphotransfer protein) protein LuxU. Indeed, among the three Hpt proteins encoded by V. parahaemolyticus, HqsK transfers phosphate only to the QS-associated phosphotransfer protein LuxU. Finally, we show that NO promotes transcription of the master quorum sensing regulatory gene opaR at low cell density.

Insights Into Nitric Oxide Modulated Quorum Sensing Pathways.

The emerging threat of drug resistant bacteria has prompted the investigation into bacterial signaling pathways responsible for pathogenesis. One such mechanism by which bacteria regulate their physiology during infection of a host is through a process known as quorum sensing (QS). Bacteria use QS to regulate community-wide gene expression in response to changes in population density. In order to sense these changes in population density, bacteria produce, secrete and detect small molecules called autoinducers. The most common signals detected by Gram-negative and Gram-positive bacteria are acylated homoserine lactones and autoinducing peptides (AIPs), respectively. However, increasing evidence has supported a role for the small molecule nitric oxide (NO) in influencing QS-mediated group behaviors like bioluminescence, biofilm production, and virulence. In this review, we discuss three bacteria that have an established role for NO in influencing bacterial physiology through QS circuits. In two Vibrio species, NO has been shown to affect QS pathways upon coordination of hemoprotein sensors. Further, NO has been demonstrated to serve a protective role against staphylococcal pneumonia through S-nitrosylation of a QS regulator of virulence.

Towards Understanding the Molecular Basis of Nitric Oxide-Regulated Group Behaviors in Pathogenic Bacteria.

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.

Spectral Characterization of a Novel NO Sensing Protein in Bacteria: NosP.

A novel family of bacterial hemoproteins named NosP has been discovered recently; its members are proposed to function as nitric oxide (NO) responsive proteins involved in bacterial group behaviors such as quorum sensing and biofilm growth and dispersal. Currently, little is known about molecular activation mechanisms in NosP. Here, functional studies were performed utilizing the distinct spectroscopic characteristics associated with the NosP heme cofactor. NosPs from Pseudomonas aeruginosa ( Pa), Vibrio cholerae ( Vc), and Legionella pneumophila ( Lpg) were studied in their ferrous unligated forms as well as their ferrous CO, ferrous NO, and ferric CN adducts. The resonance Raman (rR) data collected on the ferric forms strongly support the existence of a distorted heme cofactor, which is a common feature in NO sensors. The ferrous spectra exhibit a 213 cm-1 feature, which is assigned to the Fe-Nhis stretching mode. The Fe-C and C-O frequencies in the spectra of ferrous CO NosP complexes are inversely correlated with relatively similar frequencies, consistent with a proximal histidine ligand and a relatively hydrophobic environment. The rR spectra obtained for isotopically labeled ferrous NO adducts provide evidence of formation of a 5-coordinate NO complex, resulting from proximal Fe-Nhis cleavage, which is believed to play a role in biological heme-NO signal transduction. Additionally, we found that of the three NosPs studied, Lpg NosP contains the most electropositive ligand binding pocket, while Pa NosP has the most electronegative ligand binding pocket. This pattern is also observed in the measured heme reduction potentials for these three proteins, which may indicate distinct functions for each.

Discovery of a Nitric Oxide Responsive Quorum Sensing Circuit in Vibrio cholerae.

Group behavior of the human pathogen Vibrio cholerae, including biofilm formation and virulence factor secretion, is mediated by a process known as quorum sensing. Quorum sensing is a way by which bacteria coordinate gene expression in response to population density through the production, secretion, and detection of small molecules called autoinducers. Four autoinducer-mediated receptor histidine kinases have been implicated in quorum sensing through the phosphotransfer protein LuxU: CqsS, LuxP/Q, CqsR, and VpsS (Vc1445). Of these receptor kinases, VpsS is predicted to be cytosolic, and its cognate autoinducer is currently unknown. In this study, we demonstrate that the nitric oxide-bound complex of a member of the recently discovered family of nitric oxide-responsive hemoproteins called NosP (VcNosP is encoded by Vc1444; this gene product is also known as VpsV) inhibits the autophosphorylation activity of VpsS and thus phosphate flow to LuxU. Therefore, we propose that VpsS contributes to the regulation of quorum sensing in a nitric-oxide-dependent manner through its interaction with NosP.

Discovery of Two Bacterial Nitric Oxide-Responsive Proteins and Their Roles in Bacterial Biofilm Regulation.

Bacterial biofilms form when bacteria adhere to a surface and produce an exopolysaccharide matrix ( Costerton Science 1999 , 284 , 1318 ; Davies Science 1998 , 280 , 295 ; Flemming 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 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 Infec. Control Hosp. Epidemiol. 2013 , 34 , 1 ; Tatterson Front. Biosci. 2001 , 6 , D890 ). It has been hypothesized that effective methods of biofilm control will have widespread application ( Landini 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 J. Bacteriol. 2004 , 186 , 2781 ) and the cystic fibrosis-associated pathogen Pseudomonas aeruginosa ( Barraud 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 Microb. Biotechnol. 2009 , 2 , 370 ; McDougald Nat. Rev. Microbiol. 2012 , 10 , 39 ; Arora Biochemistry 2015 , 54 , 3717 ; Barraud 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 Biochemistry 2015 , 54 , 3717 ; Plate Trends Biochem. Sci. 2013 , 38 , 566 ; Nisbett 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 H-NOX 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 oneidensis.

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

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.

Nitric Oxide Regulation of H-NOX Signaling Pathways in Bacteria.

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 ligation of NO to H-NOX specifically regulates the activity of these pathways and their associated bacterial phenotypes.

Kinetic Control of O2 Reactivity in H-NOX Domains.

Transient absorption, resonance Raman, and vibrational coherence spectroscopies are used to investigate the mechanisms of NO and O2 binding to WT Tt H-NOX and its P115A mutant. Vibrational coherence spectra of the oxy complexes provide clear evidence for the enhancement of an iron-histidine mode near 217 cm(-1) following photoexcitation, which indicates that O2 can be dissociated in these proteins. However, the quantum yield of O2 photolysis is low, particularly in the wild type (≲3%). Geminate recombination of O2 and NO in both of these proteins is very fast (∼1.4 × 10(11) s(-1)) and highly efficient. We show that the distal heme pocket of the H-NOX system forms an efficient trap that limits the O2 off-rate and determines the overall affinity. The distal pocket hydrogen bond, which appears to be stronger in the P115A mutant, may help retard the O2 ligand from escaping into the solvent following either photoinduced or thermal dissociation. This, along with a strengthening of the Fe-O2 bond that is correlated with the significant heme ruffing and saddling distortions, explains the unusually high O2 affinity of WT Tt H-NOX and the even higher affinity found in the P115A mutant.