Nitric Oxide Mediates Biofilm Formation and Symbiosis in Silicibacter sp. Strain TrichCH4B.

UnlabelledNitric oxide (NO) plays an important signaling role in all domains of life. Many bacteria contain a heme-nitric oxide/oxygen binding (H-NOX) protein that selectively binds NO. These H-NOX proteins often act as sensors that regulate histidine kinase (HK) activity, forming part of a bacterial two-component signaling system that also involves one or more response regulators. In several organisms, NO binding to the H-NOX protein governs bacterial biofilm formation; however, the source of NO exposure for these bacteria is unknown. In mammals, NO is generated by the enzyme nitric oxide synthase (NOS) and signals through binding the H-NOX domain of soluble guanylate cyclase. Recently, several bacterial NOS proteins have also been reported, but the corresponding bacteria do not also encode an H-NOX protein. Here, we report the first characterization of a bacterium that encodes both a NOS and H-NOX, thus resembling the mammalian system capable of both synthesizing and sensing NO. We characterized the NO signaling pathway of the marine alphaproteobacterium Silicibacter sp. strain TrichCH4B, determining that the NOS is activated by an algal symbiont, Trichodesmium erythraeum. NO signaling through a histidine kinase-response regulator two-component signaling pathway results in increased concentrations of cyclic diguanosine monophosphate, a key bacterial second messenger molecule that controls cellular adhesion and biofilm formation. Silicibacter sp. TrichCH4B biofilm formation, activated by T. erythraeum, may be an important mechanism for symbiosis between the two organisms, revealing that NO plays a previously unknown key role in bacterial communication and symbiosis.ImportanceBacterial nitric oxide (NO) signaling via heme-nitric oxide/oxygen binding (H-NOX) proteins regulates biofilm formation, playing an important role in protecting bacteria from oxidative stress and other environmental stresses. Biofilms are also an important part of symbiosis, allowing the organism to remain in a nutrient-rich environment. In this study, we show that in Silicibacter sp. strain TrichCH4B, NO mediates symbiosis with the alga Trichodesmium erythraeum, a major marine diazotroph. In addition, Silicibacter sp. TrichCH4B is the first characterized bacteria to harbor both the NOS and H-NOX proteins, making it uniquely capable of both synthesizing and sensing NO, analogous to mammalian NO signaling. Our study expands current understanding of the role of NO in bacterial signaling, providing a novel role for NO in bacterial communication and symbiosis

Nitric oxide: biosynthesis and biological significance

The recent discovery that mammalian cells can synthesize nitric oxide coincided with the identification of this simple gas as a factor involved in cellular communication. Nitric oxide has now been shown to be derived from -arginine in macrophages, endothelial cells and possibly other cell types. Its physiological role in macrophages may be as a cytotoxic agent. However, nitric oxide produced by endothelial cells is thought to trigger vascular smooth muscle relaxation through activation of the enzyme guanylate cyclase.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27667/1/0000049.pd

Regulation of nitric oxide signaling by formation of a distal receptor-ligand complex.

The binding of nitric oxide (NO) to the heme cofactor of heme-nitric oxide/oxygen binding (H-NOX) proteins can lead to the dissociation of the heme-ligating histidine residue and yield a five-coordinate nitrosyl complex, an important step for NO-dependent signaling. In the five-coordinate nitrosyl complex, NO can reside on either the distal or proximal side of the heme, which could have a profound influence over the lifetime of the in vivo signal. To investigate this central molecular question, we characterized the Shewanella oneidensis H-NOX (So H-NOX)-NO complex biophysically under limiting and excess NO conditions. The results show that So H-NOX preferably forms a distal NO species with both limiting and excess NO. Therefore, signal strength and complex lifetime in vivo will be dictated by the dissociation rate of NO from the distal complex and the rebinding of the histidine ligand to the heme

Allosteric activation of the nitric oxide receptor soluble guanylate cyclase mapped by cryo-electron microscopy.

Soluble guanylate cyclase (sGC) is the primary receptor for nitric oxide (NO) in mammalian nitric oxide signaling. We determined structures of full-length Manduca sexta sGC in both inactive and active states using cryo-electron microscopy. NO and the sGC-specific stimulator YC-1 induce a 71° rotation of the heme-binding β H-NOX and PAS domains. Repositioning of the β H-NOX domain leads to a straightening of the coiled-coil domains, which, in turn, use the motion to move the catalytic domains into an active conformation. YC-1 binds directly between the β H-NOX domain and the two CC domains. The structural elongation of the particle observed in cryo-EM was corroborated in solution using small angle X-ray scattering (SAXS). These structures delineate the endpoints of the allosteric transition responsible for the major cyclic GMP-dependent physiological effects of NO

The crystal structure of the catalytic domain of a eukaryotic guanylate cyclase

<p>Abstract</p> <p>Background</p> <p>Soluble guanylate cyclases generate cyclic GMP when bound to nitric oxide, thereby linking nitric oxide levels to the control of processes such as vascular homeostasis and neurotransmission. The guanylate cyclase catalytic module, for which no structure has been determined at present, is a class III nucleotide cyclase domain that is also found in mammalian membrane-bound guanylate and adenylate cyclases.</p> <p>Results</p> <p>We have determined the crystal structure of the catalytic domain of a soluble guanylate cyclase from the green algae <it>Chlamydomonas reinhardtii </it>at 2.55 Å resolution, and show that it is a dimeric molecule.</p> <p>Conclusion</p> <p>Comparison of the structure of the guanylate cyclase domain with the known structures of adenylate cyclases confirms the close similarity in architecture between these two enzymes, as expected from their sequence similarity. The comparison also suggests that the crystallized guanylate cyclase is in an inactive conformation, and the structure provides indications as to how activation might occur. We demonstrate that the two active sites in the dimer exhibit positive cooperativity, with a Hill coefficient of ~1.5. Positive cooperativity has also been observed in the homodimeric mammalian membrane-bound guanylate cyclases. The structure described here provides a reliable model for functional analysis of mammalian guanylate cyclases, which are closely related in sequence.</p

Inactivation of macrophage nitric oxide synthase activity by NG-Methyl-L-arginine

[middle dot]N=O synthase catalyzes the oxidation of one of the two chemically equivalent guanido nitrogens of L-arginine to nitric oxide ([middle dot]N=O). NG-Methyl-L-arginine has been previously characterized as a potent competitive inhibitor of both major types of [middle dot]N=O synthases. Initial rate kinetics were performed with a spectrophotometric assay based on the oxidation of oxy- to methemoglobin by [middle dot]N=O. NG-Methyl-L-arginine was a competitive inhibitor of [middle dot]N=O synthase activity derived from activated murine macrophages with a Ki of 6.2 [mu]M. When the enzyme was pre-incubated in the presence of the required cofactors NADPH and tetrahydrobiopterin, time- and concentration-dependent irreversible inactivation of the activity was observed. At 37[deg] C the kinact was 0.050 min-1. This inactivation process exhibited substrate protection, saturation kinetics and required the cofactors necessary for enzymatic turnover. These data indicate that NG-methyl-L-arginine asts as a mechanism-based enzyme inactivator of murine macrophage [middle dot]N=O synthase.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29284/1/0000343.pd

Redox Couples of Inducible Nitric Oxide Synthase

We report direct electrochemistry of the iNOS heme domain in a DDAB film on the surface of a basal plane graphite electrode. Cyclic voltammetry reveals Fe^(III/II) and Fe^(II/I) couples at −191 and −1049 mV (vs Ag/AgCl). Imidazole and carbon monoxide in solution shift the Fe^(III/II) potential by +20 and +62 mV, while the addition of dioxygen results in large catalytic waves at the onset of Fe^(III) reduction. Voltammetry at higher scan rates (with pH variations) reveals that the Fe^(III/II) cathodic peak can be resolved into two components, which are attributable to Fe^(III/II) couples of five- and six-coordinate hemes. Digital simulation of our experimental data implicates water dissociation from the heme as a gating mechanism for ET in iNOS