6 research outputs found
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
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Functional and Mechanistic Characterization of Bacterial Nitric Oxide Signaling Pathways
Nitric oxide (NO) is a well-established signaling molecule and cytotoxic agent in mammals. NO is synthesized by nitric oxide synthase (NOS) by macrophages at high concentrations as a key part of the host immune response, and at low concentrations in endothelial and neuronal cells as a signaling agent. In endothelial cells, the primary NO receptor is soluble guanylate cyclase (sGC), which contains a heme-nitric oxide/oxygen binding domain (H-NOX). Selective binding of NO to the H-NOX domain is responsible for activation of sGC. Thus, the mammalian NO signaling system involves NO synthesis by NOS, and NO sensing by the H-NOX domain of sGC. NOS and H-NOX proteins have also been identified in a number of bacterial species, including pathogens. Putative roles for bacterial NOS proteins include protection against oxidative stress and antibiotics, while bacterial H-NOX proteins have been shown to govern processes such as biofilm formation and bioluminescence via interactions with signaling proteins such as diguanylate cyclases (DGC) or histidine kinases (HK). Here, various aspects of NO signaling from three different organisms are characterized: the marine alphaproteobacterium Silicibacter sp. TrichCH4B; the soil-dwelling gammaproteobacterium Shewanella oneidensis, and the marine cyanobacterium Synechococcus sp. PCC 7335. This work and other recent studies seek to understand not only the diverse roles for NO in bacteria, but also the molecular mechanisms of bacterial NO signaling. Silicibacter sp. TrichCH4B is the first bacterial organism discovered to contain both an NOS and H-NOX, thus capable of both NO synthesis and sensing, analogous to mammalian systems. The H-NOX protein from Silicibacter is found in an operon adjacent to an HK, forming part of a two-component phospho-relay signaling network. The response regulator of the network was identified to be a diguanylate cyclase (DGC), which is inactivated upon phosphorylation and establishes the link between NO and intracellular cyclic-di-GMP levels, and consequently biofilm formation. It was also determined that Silicibacter NOS activity is stimulated by a signaling protein from an algal symbiont, Trichodesmium erythraeum, which is a major marine nitrogen fixer. Thus, in the presence of Trichodesmium, the increase in NOS activity results in Silicibacter biofilm formation and poising the two species for nutrient exchange, revealing a novel role for NO in interspecies communication and symbiosis.Given the diverse processes governed by NO/H-NOX signaling, it is crucial to understand the molecular mechanism by which H-NOX regulates HK autophosphorylation activity, the most common outcome of a NO-bound H-NOX. Here, the interaction and signal transduction between the H-NOX-HK signaling pair from Shewanella oneidensis are characterized. Binding kinetics measurements and analytical gel filtration revealed that NO-bound H-NOX has a tighter affinity for the HK, compared with H-NOX in the unliganded state, correlating binding affinity with kinase inhibition. Kinase activity assays with a panel of binding-deficient H-NOX mutants further reveal that while formation of the H-NOX-HK protein complex is required to stabilize the HK, H-NOX conformational changes upon NO binding are necessary for HK inhibition. Characterization of H-NOX proteins has led to an increased understanding of bacterial NO sensing. However, NO production in bacteria is less well-understood, and here the NOS protein from Synechococcus sp. PCC 7335 is characterized. Mammalian NOS proteins are comprised of a P450-like heme/oxidase domain responsible for catalysis, and a reductase domain responsible for electron transfer. While most bacterial NOS proteins discovered to date contain only the heme/oxidase domain, Synechococcus NOS contains both the oxidase and reductase domains, and additionally contains a predicted globin domain resembling bacterial flavohemoglobins. Spectroscopic and biochemical characterization of the globin indicated a possible role in redox communication in this novel class of bacterial NOS enzymes
Nitric Oxide-Induced Conformational Changes Govern H‑NOX and Histidine Kinase Interaction and Regulation in <i>Shewanella oneidensis</i>
Nitric
oxide (NO) is implicated in biofilm regulation in several
bacterial families via heme-nitric oxide/oxygen binding (H-NOX) protein
signaling. Shewanella oneidensis H-NOX
(<i>So</i> H-NOX) is associated with a histidine kinase
(<i>So</i> HnoK) encoded on the same operon, and together
they form a multicomponent signaling network whereby the NO-bound
state of <i>So</i> H-NOX inhibits <i>So</i> HnoK
autophosphorylation activity, affecting the phosphorylation state
of three response regulators. Although the conformational changes
of <i>So</i> H-NOX upon NO binding have been structurally
characterized, the mechanism of HnoK inhibition by NO-bound <i>So</i> H-NOX remains unclear. In the present study, the molecular
details of <i>So</i> H-NOX and <i>So</i> HnoK
interaction and regulation are characterized. The N-terminal domain
in <i>So</i> HnoK was determined to be the site of H-NOX
interaction, and the binding interface on <i>So</i> H-NOX
was identified using a combination of hydrogen–deuterium exchange
mass spectrometry and surface-scanning mutagenesis. Binding kinetics
measurements and analytical gel filtration revealed that NO-bound <i>So</i> H-NOX has a tighter affinity for <i>So</i> HnoK
compared that of H-NOX in the unliganded state, correlating binding
affinity with kinase inhibition. Kinase activity assays with binding-deficient
H-NOX mutants further indicate that while formation of the H-NOX-HnoK
complex is required for HnoK to be catalytically active, H-NOX conformational
changes upon NO-binding are necessary for HnoK inhibition