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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

Nitric Oxide Regulation of Bacterial Biofilms.

Biofilms are surface-associated, multicellular communities of bacteria. Once established, they are extremely difficult to eradicate by antimicrobial treatment. It has been demonstrated in many species that biofilm formation may be regulated by the diatomic signaling molecule nitric oxide (NO). Although this is still a relatively new area of research, we review here the literature reporting an effect of NO on bacterial biofilm formation, emphasizing dose-dependent responses to NO concentrations when possible. Where it has been investigated, the underlying NO sensors or signaling pathways are also discussed. Most of the examples of NO-mediated biofilm regulation have been documented with exogenously applied NO, but we also survey possible natural sources of NO in biofilm regulation, including endogenously generated NO. Finally, because of the apparent broad-spectrum, antibiofilm effects of NO, NO-releasing materials and prodrugs have also been explored in this minireview.

Optimized assay for the quantification of histidine kinase autophosphorylation.

Although two-component signaling systems, comprising a sensory histidine kinase and a response regulator, are a primary means by which bacteria detect and respond to environmental stimuli, they are poorly characterized. Here we report optimized conditions for detecting histidine phosphorylation using a facile medium-throughput filter paper-binding assay. Employing this assay we report the kinetic parameters of previously uncharacterized histidine kinases from Vibrio haveyi, Vibrio parahaemolytius, Shewanella oneidensis, and Legionella pneumophila. In characterizing these kinases, we effectively double the number of kinetically characterized histidine kinases that have been reported in the literature.

A structural basis for the regulation of an H-NOX-associated cyclic-di-GMP synthase/phosphodiesterase enzyme by nitric oxide-bound H-NOX.

Biofilms are surface-attached communities of bacteria enclosed in a polysaccharide matrix. Bacteria in a biofilm are extremely resistant to antibiotics. Several recent reports have linked the signaling molecule nitric oxide (NO) with biofilm dispersal. We have previously reported that an H-NOX (heme-nitric oxide/oxygen binding) protein in the biofilm-dwelling bacterium Shewanella woodyi mediates NO-induced biofilm dispersal. In S. woodyi, H-NOX (SwH-NOX) is cocistronic with a gene encoding a dual-functioning diguanylate cyclase/phosphodiesterase enzyme, designated here as HaCE (H-NOX-associated cyclic-di-GMP processing enzyme). Enzymes such as these are responsible for regulating the intracellular concentrations of cyclic-di-GMP, a secondary signaling molecule essential to biofilm formation in bacteria. We have demonstrated that NO-bound SwH-NOX regulates both enzymatic activities of SwHaCE, resulting in decreased cellular cyclic-di-GMP levels and disruption of biofilm formation. Thus, H-NOX/HaCE represents a potential drug target for regulating biofilm formation. In this work, the SwH-NOX surface residues critical for the formation of a protein complex with SwHaCE are identified using nuclear magnetic resonance, fluorescence quenching, and cosedimentation. Enzyme assays confirm this protein-protein interface and its importance for H-NOX/HaCE function.

Unexpected biotinylation using ATP-γ-Biotin-LC-PEO-amine as a kinase substrate.

Protein phosphorylation is the most widely studied post-translational modification. Reversible protein phosphorylation is implicated in the regulation of a broad range of cellular processes. As such, there is extensive interest in simple and sensitive procedures for the isolation and detection of phosphorylated proteins. Synthetic analogues of ATP, with a biotin linked to the gamma-phosphate of ATP, have been reported to biotinylate kinase substrates in a kinase-catalyzed reaction. This could be an extremely attractive and versatile method for affinity enrichment of phosphorylated proteins. However, as we report here, the commercially available biotin-ATP analogue, ATP-γ-Biotin-LC-PEO-amine, is capable of biotinylating proteins independent of kinase activity. In fact, we demonstrate that this reagent is capable of non-specifically biotinylating any protein. Although the mechanism of biotinylation is not known, this report uncovers a flaw in a commercially available reagent and also highlights the importance of control experiments when developing new biochemical tools to study enzyme activity.

A nitric oxide-responsive quorum sensing circuit in Vibrio harveyi regulates flagella production and biofilm formation.

Cell signaling plays an important role in the survival of bacterial colonies. They use small molecules to coordinate gene expression in a cell density dependent manner. This process, known as quorum sensing, helps bacteria regulate diverse functions such as bioluminescence, biofilm formation and virulence. In Vibrio harveyi, a bioluminescent marine bacterium, four parallel quorum-sensing systems have been identified to regulate light production. We have previously reported that nitric oxide (NO), through the H-NOX/HqsK quorum sensing pathway contributes to light production in V. harveyi through the LuxU/LuxO/LuxR quorum sensing pathway. In this study, we show that nitric oxide (NO) also regulates flagellar production and enhances biofilm formation. Our data suggest that V. harveyi is capable of switching between lifestyles to be able to adapt to changes in the environment.

Nitric oxide regulation of cyclic di-GMP synthesis and hydrolysis in Shewanella woodyi.

Although several reports have documented nitric oxide (NO) regulation of biofilm formation, the molecular basis of this phenomenon is unknown. In many bacteria, an H-NOX (heme-nitric oxide/oxygen-binding) gene is found near a diguanylate cyclase (DGC) gene. H-NOX domains are conserved hemoproteins that are known NO sensors. It is widely recognized that cyclic di-GMP (c-di-GMP) is a ubiquitous bacterial signaling molecule that regulates the transition between motility and biofilm. Therefore, NO may influence biofilm formation through H-NOX regulation of DGC, thus providing a molecular-level explanation for NO regulation of biofilm formation. This work demonstrates that, indeed, NO-bound H-NOX negatively affects biofilm formation by directly regulating c-di-GMP turnover in Shewanella woodyi strain MS32. Exposure of wild-type S. woodyi to a nanomolar level of NO resulted in the formation of thinner biofilms, and less intracellular c-di-GMP, than in the absence of NO. Also, a mutant strain in the gene encoding SwH-NOX showed a decreased level of biofilm formation (and a decreased amount of intracellular c-di-GMP) with no change observed upon NO addition. Furthermore, using purified proteins, it was demonstrated that SwH-NOX and SwDGC are binding partners. SwDGC is a dual-functioning DGC; it has diguanylate cyclase and phosphodiesterase activities. These data indicate that NO-bound SwH-NOX enhances c-di-GMP degradation, but not synthesis, by SwDGC. These results support the biofilm growth data and indicate that S. woodyi senses nanomolar NO with an H-NOX domain and that SwH-NOX regulates SwDGC activity, resulting in a reduction in c-di-GMP concentration and a decreased level of biofilm growth in the presence of NO. These data provide a detailed molecular mechanism for NO regulation of c-di-GMP signaling and biofilm formation.

Heme flattening is sufficient for signal transduction in the H-NOX family.

The H-NOX family of nitric oxide (NO) sensing proteins has received considerable attention because its members include the mammalian NO sensor, soluble guanylate cyclase. Despite this attention, the mechanism of signal transduction has not been elucidated. Structural studies of bacterial members of the family have revealed that the H-NOX heme cofactor is extremely distorted from planarity. Furthermore, it has been determined that heme distortion is maintained primarily by a conserved proline residue located in the proximal heme pocket. It has been suggested that changes in heme planarity may contribute to signal transduction. Here we demonstrate that heme flattening is, indeed, sufficient for signal transduction in the H-NOX family. Using our previously described H-NOX/diguanylate cyclase functional partners from Shewanella woodyi, we demonstrate that mutation of the conserved proline (P117 in SwH-NOX) to alanine, which results in heme flattening, has the same affect on phosphodiesterase activity as NO binding to wildtype SwH-NOX. This study demonstrates, for the first time, that heme flattening mimics the activated, NO-bound state of H-NOX and suggests that NO binding induces heme flattening as part of the signal transduction mechanism in the H-NOX family.

Nitric oxide regulated two-component signaling in Pseudoalteromonas atlantica.

Bacteria employ two-component signaling to detect and respond to environmental stimuli. In essence, two-component signaling relies on a protein called a response regulator that can elicit a change in gene expression or protein function in response to phosphoryl transfer from a histidine kinase. Phosphorylation of the associated histidine kinase is regulated by detection of an environmental signal, thus linking sensing to cellular response. Recently, it has been suggested that H-NOX (Heme-nitric oxide/oxygen binding) proteins may act as nitric oxide (NO) sensors in two-component signaling systems. NO/H-NOX regulated histidine kinases have been reported, but their cognate response regulators have yet to be identified. In this work we provide biochemical characterization of a complete NO/H-NOX-regulated two-component signaling pathway in the biofilm-dwelling marine bacterium, Pseudoalteromonas atlantica. In P. atlantica, as is typical for bacteria that code for H-NOX, an hnoX gene is found in the same operon as a gene coding for a two-component signaling histidine kinase (H-NOX-associated histidine kinase; HahK). We find that HahK is capable of autophosphorylation in vitro and that NO-bound H-NOX inhibits HahK activity, implicating H-NOX as a selective NO sensor. The cognate response regulator, a protein annotated as a cyclic-di-GMP processing enzyme that we have named HarR (H-NOX-associated response regulator), was identified using bioinformatics tools. Phosphoryl transfer from HahK to HarR has been established. This report reveals the first biochemical characterization of an H-NOX-associated response regulator and contributes to a deeper understanding of NO/H-NOX signaling in bacteria.