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.

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Phytocannabinoids, such as the principal bioactive component of marijuana, ⌬ 9 -tetrahydrocannabinol, have been used for thousands of years for medical and recreational purposes. ⌬ 9 -Tetrahydrocannabinol and endogenous cannabinoids (e.g., anandamide) initiate their agonist properties by stimulating the cannabinoid family of G protein-coupled receptors (CB 1 and CB2). The biosynthesis and physiology of anandamide is well understood, but its mechanism of uptake (resulting in signal termination by fatty acid amide hydrolase) has been elusive. Mounting evidence points to the existence of a specific anandamide transport protein; however, no direct evidence for this protein has been provided. Here, we use a potent, competitive small molecule inhibitor of anandamide uptake (LY2318912, IC 50 7.27 ؎ 0.510 nM) to identify a high-affinity, saturable anandamide transporter binding site (LY2318912; K d ‫؍‬ 7.62 ؎ 1.18 nM, Bmax ‫؍‬ 31.6 ؎ 1.80 fmol͞mg protein) that is distinct from fatty acid amide hydrolase. Systemic administration of the inhibitor into rodents elevates anandamide levels 5-fold in the brain and demonstrates efficacy in the formalin paw-licking model of persistent pain with no obvious adverse effects on motor function. Identification of the anandamide transporter binding site resolves a missing mechanistic link in endocannabinoid signaling, and in vivo results suggest that endocannabinoid transporter antagonists may provide a strategy for positive modulation of cannabinoid receptors.anandamide ͉ fatty acid amide hydrolase ͉ cannabinoid ͉ marijuana ͉ transporter E ndocannabinoids are recognized as significant intracellular lipid signaling molecules in the central nervous system with extensive control of physiological and behavioral mood and affect. Increases in endocannabinoid neurotransmission have broad therapeutic potential, including reduction of nausea and emesis (1), appetite stimulation (2), analgesia (3), anxiolytic activity (4), antispasmodic activity (5), and lowering of intraocular pressure in glaucoma (6). Identification of a specific binding site for the phytocannabinoid, ⌬ 9 -tetrahydrocannabinol (⌬ 9 -THC) (7), cloning of the cannabinoid receptors (CB 1 and CB 2 ) (8, 9), and the identification of an endogenous ligand, anandamide (N-arachidonoylethanolamide) (10), provided evidence of an endogenous cannabinoid system. Anandamide represents a class of lipid neurotransmitters that stimulate not only presynaptic and postsynaptic CB 1 receptors but also TRPV1 ion channels (11, 12), 5-hydroxytryptamine receptors (13-16), and possibly other receptors, as well as CB 2 receptors in the periphery (10,(17)(18)(19). More recently, the enzymes that are responsible for anandamide synthesis (phospholipase D) and catabolism (fatty acid amide hydrolase, FAAH) have been identified and characterized (20,21). Unlike typical neurotransmitter molecules, anandamide is synthesized in the membrane bilayer, resulting in the phospholipid precursor of anandamide, Narachidonoylphosphatidylethanolamine (22-25). Calciumacti...

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.

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.