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.

Secreted Flavin Cofactors for Anaerobic Respiration of Fumarate and Urocanate by Shewanella oneidensis: Cost and Role.

Shewanella oneidensis strain MR-1, a facultative anaerobe and model organism for dissimilatory metal reduction, uses a periplasmic flavocytochrome, FccA, both as a terminal fumarate reductase and as a periplasmic electron transfer hub for extracellular respiration of a variety of substrates. It is currently unclear how maturation of FccA and other periplasmic flavoproteins is achieved, specifically in the context of flavin cofactor loading, and the fitness cost of flavin secretion has not been quantified. We demonstrate that deletion of the inner membrane flavin adenine dinucleotide (FAD) exporter Bfe results in a 23% slower growth rate than that of the wild type during fumarate respiration and an 80 to 90% loss in fumarate reductase activity. Exogenous flavin supplementation does not restore FccA activity in a Δbfe mutant unless the gene encoding the periplasmic FAD hydrolase UshA is also deleted. We demonstrate that the small Bfe-independent pool of FccA is sufficient for anaerobic growth with fumarate. Strains lacking Bfe were unable to grow using urocanate as the sole electron acceptor, which relies on the periplasmic flavoprotein UrdA. We show that periplasmic flavoprotein maturation occurs in careful balance with periplasmic FAD hydrolysis, and that the current model for periplasmic flavin cofactor loading must account for a Bfe-independent mechanism for flavin transport. Finally, we determine that the metabolic burden of flavin secretion is not significant during growth with flavin-independent anaerobic electron acceptors. Our work helps frame the physiological motivations that drove evolution of flavin secretion by ShewanellaIMPORTANCEShewanella species are prevalent in marine and aquatic environments, throughout stratified water columns, in mineral-rich sediments, and in association with multicellular marine and aquatic organisms. The diversity of niches shewanellae can occupy are due largely to their respiratory versatility. Shewanella oneidensis is a model organism for dissimilatory metal reduction and can respire a diverse array of organic and inorganic compounds, including dissolved and solid metal oxides. The fumarate reductase FccA is a highly abundant multifunctional periplasmic protein that acts to bridge the periplasm and temporarily store electrons in a variety of respiratory nodes, including metal, nitrate, and dimethyl sulfoxide respiration. However, maturation of this central protein, particularly flavin cofactor acquisition, is poorly understood. Here, we quantify the fitness cost of flavin secretion and describe how free flavins are acquired by FccA and a homologous periplasmic flavoprotein, UrdA.

On the relationship between long-distance and heterogeneous electron transfer in electrode-grown Geobacter sulfurreducens biofilms.

The ability of certain microorganisms to live in a multi-cell thick, electrode-grown biofilm by utilizing the electrode as a metabolic electron acceptor or donor requires electron transfer across cell membranes, through the biofilm, and across the biofilm/electrode interface. Even for the most studied system, anode-grown Geobacter sulfurreducens, the mechanisms underpinning each process and how they connect is largely unresolved. Here we report on G. sulfurreducens biofilms grown across the gap separating two electrodes by maintaining one electrode at 0.300V vs. Ag/AgCl (0.510V vs. SHE) to act as a sustained metabolic electron acceptor while the second electrode was at open circuit. The poised electrode exhibited the characteristic current-time profile for electrode-dependent G. sulfurreducens biofilm growth. The open circuit potential (OCP) of the second electrode however increased after initially decreasing for 1.5-2days. The increase in OCP is taken to indicate the point at which the growing biofilm bridged the gap between the electrodes, enabling cells in contact with the open circuit electrode to utilize the poised electrode as an electron acceptor. After but not prior to reaching this point, the second electrode was able to act as a sustainable electron acceptor immediately after being placed under potential control without requiring further time to develop. These results indicate that heterogeneous ET (H-ET) across the biofilm/electrode interface and long-distance ET (LD-ET) through the biofilm are highly correlated, if not inseparable, and may share many common components.

Formate Metabolism in Shewanella oneidensis Generates Proton Motive Force and Prevents Growth without an Electron Acceptor.

UNLABELLED: Shewanella oneidensis strain MR-1 is a facultative anaerobe that thrives in redox-stratified environments due to its ability to utilize a wide array of terminal electron acceptors. Conversely, the electron donors utilized by S. oneidensis are more limited and include products of primary fermentation such as lactate, pyruvate, formate, and hydrogen. Lactate, pyruvate, and hydrogen metabolisms inS. oneidensis have been described previously, but little is known about the role of formate oxidation in the ecophysiology of these bacteria. Formate is produced by S. oneidensis through pyruvate formate lyase during anaerobic growth on carbon sources that enter metabolism at or above the level of pyruvate, and the genome contains three gene clusters predicted to encode three complete formate dehydrogenase complexes. To determine the contribution of each complex to formate metabolism, strains lacking one, two, or all three annotated formate dehydrogenase gene clusters were generated and examined for growth rates and yields on a variety of carbon sources. Here, we report that formate oxidation contributes to both the growth rate and yield of S. oneidensis through the generation of proton motive force. Exogenous formate also greatly accelerated growth on N-acetylglucosamine, a carbon source normally utilized very slowly by S. oneidensis under anaerobic conditions. Surprisingly, deletion of all three formate dehydrogenase gene clusters enabled growth of S. oneidensis using pyruvate in the absence of a terminal electron acceptor, a mode of growth never before observed in these bacteria. Our results demonstrate that formate oxidation is a fundamental strategy under anaerobic conditions for energy conservation inS. oneidensis. IMPORTANCE: Shewanella species have garnered interest in biotechnology applications for their ability to respire extracellular terminal electron acceptors, such as insoluble iron oxides and electrodes. While much effort has gone into studying the proteins for extracellular electron transport, how electrons generated through the oxidation of organic carbon sources enter this pathway remains understudied. Here, we quantify the role of formate oxidation in the anaerobic physiology of Shewanella oneidensis Formate oxidation contributes to both the growth rate and yield on a variety of carbon sources through the generation of proton motive force. Advances in our understanding of the anaerobic metabolism of S. oneidensis are important for our ability to utilize and engineer this organism for applications in bioenergy, biocatalysis, and bioremediation.

Oxygen allows Shewanella oneidensis MR-1 to overcome mediator washout in a continuously fed bioelectrochemical system.

Many bioelectrochemical systems (BESs) harness the ability of electrode-active microbes to catalyze reactions between electrodes and chemicals, often to perform useful functions such as wastewater treatment, fuel production, and biosensing. A microbial fuel cell (MFC) is one type of BES, which generates electric power through microbial respiration with an anode as the electron acceptor, and typically with oxygen reduction at the cathode to provide the terminal electron acceptor. Oxygen intrusion into MFCs is typically viewed as detrimental because it competes with anodes for electrons and lowers the coulombic efficiency. However, recent evidence suggests that it does not necessarily lead to lower performances­particularly for the model organism Shewanella oneidensis MR-1. Because flavin-mediated electron transfer is important for Shewanella species, which can produce this electron shuttle endogenuously, we investigated the role of flavins in the performance of pure-culture BESs with S. oneidensis MR-1 with and without oxygen. We found that oxygen increases current production more than twofold under continuously fed conditions, but only modestly increases current production under batch-fed conditions.We hypothesized that oxygen is more beneficial under continuously fed conditions because it allows S. oneidensis to grow and produce flavins at a faster rate, and thus lowers flavin washout. Our conclusions were supported by experiments with a flavin-secretion deficient mutant of S. oneidensis.

Flavin electron shuttles dominate extracellular electron transfer by Shewanella oneidensis.

UNLABELLED: Shewanella oneidensis strain MR-1 is widely studied for its ability to respire a diverse array of soluble and insoluble electron acceptors. The ability to breathe insoluble substrates is defined as extracellular electron transfer and can occur via direct contact or by electron shuttling in S. oneidensis. To determine the contribution of flavin electron shuttles in extracellular electron transfer, a transposon mutagenesis screen was performed with S. oneidensis to identify mutants unable to secrete flavins. A multidrug and toxin efflux transporter encoded by SO_0702 was identified and renamed bfe (bacterial flavin adenine dinucleotide [FAD] exporter) based on phenotypic characterization. Deletion of bfe resulted in a severe decrease in extracellular flavins, while overexpression of bfe increased the concentration of extracellular flavins. Strains lacking bfe had no defect in reduction of soluble Fe(III), but these strains were deficient in the rate of insoluble Fe(III) oxide reduction, which was alleviated by the addition of exogenous flavins. To test a different insoluble electron acceptor, graphite electrode bioreactors were set up to measure current produced by wild-type S. oneidensis and the Δbfe mutant. With the same concentration of supplemented flavins, the two strains produced similar amounts of current. However, when exogenous flavins were not supplemented to bioreactors, bfe mutant strains produced significantly less current than the wild type. We have demonstrated that flavin electron shuttling accounts for ~75% of extracellular electron transfer to insoluble substrates by S. oneidensis and have identified the first FAD transporter in bacteria. IMPORTANCE: Extracellular electron transfer by microbes is critical for the geochemical cycling of metals, bioremediation, and biocatalysis using electrodes. A controversy in the field was addressed by demonstrating that flavin electron shuttling, not direct electron transfer or nanowires, is the primary mechanism of extracellular electron transfer employed by the bacterium Shewanella oneidensis. We have identified a flavin adenine dinucleotide transporter conserved in all sequenced Shewanella species that facilitates export of flavin electron shuttles in S. oneidensis. Analysis of a strain that is unable to secrete flavins demonstrated that electron shuttling accounts for ~75% of the insoluble extracellular electron transfer capacity in S. oneidensis.

An essential role for UshA in processing of extracellular flavin electron shuttles by Shewanella oneidensis.

The facultative anaerobe Shewanella oneidensis can reduce a number of insoluble extracellular metals. Direct adsorption of cells to the metal surface is not necessary, and it has been shown that S. oneidensis releases low concentrations flavins, including riboflavin and flavin mononucleotide (FMN), into the surrounding medium to act as extracellular electron shuttles. However, the mechanism of flavin release by Shewanella remains unknown. We have conducted a transposon mutagenesis screen to identify mutants deficient in extracellular flavin accumulation. Mutations in ushA, encoding a predicted 5'-nucleotidase, resulted in accumulation of flavin adenine dinucleotide (FAD) in culture supernatants, with a corresponding decrease in FMN and riboflavin. Cellular extracts of S. oneidensis convert FAD to FMN, whereas extracts of ushA mutants do not, and fractionation experiments show that UshA activity is periplasmic. We hypothesize that S. oneidensis secretes FAD into the periplasmic space, where it is hydrolysed by UshA to FMN and adenosine monophosphate (AMP). FMN diffuses through outer membrane porins where it accelerates extracellular electron transfer, and AMP is dephosphorylated by UshA and reassimilated by the cell. We predict that transport of FAD into the periplasm also satisfies the cofactor requirement of the unusual periplasmic fumarate reductase found in Shewanella.

Role of IL-17, transforming growth factor-beta, and IL-6 in the development of arthritis and production of anti-outer surface protein A borreliacidal antibodies in Borrelia-vaccinated and -challenged mice.

We showed recently that the adaptive immune events leading to the development of arthritis in Borrelia burgdorferi isolate 297-vaccinated and Borrelia bissettii-challenged mice involve IL-17. Here, we show in Borrelia-vaccinated and -challenged mice that two cytokines known to induce the production of IL-17, IL-6 and transforming growth factor (TGF)-beta, are also involved in the development of arthritis. Vaccinated and challenged mice administered either anti-TGF-beta or anti-IL-6 antibodies developed histopathologic changes of the hind paws similar to or greater than untreated control mice. By contrast, simultaneous blockage of these cytokines reduced the severity of arthritis in Borrelia-vaccinated and -challenged mice. Moreover, administration of anti-IL-17 antibodies to these dual-antibody-treated mice completely prevented the development of histopathologic changes of the ankle joints, significantly reduced edema of the hind paws, and prevented the production of anti-outer surface protein A borreliacidal antibodies. These findings demonstrate a role for the combined effects of IL-17, IL-6, and TGF-beta in the adaptive immune events leading to the development of Borrelia-induced arthritis.

Interleukin-23 is required for development of arthritis in mice vaccinated and challenged with Borrelia species.

We recently hypothesized that T helper 17 (Th17) cells and their associated cytokines are involved in the development of arthritis following infection with Borrelia burgdorferi. Here, we show that interleukin-23 (IL-23), a survival factor for Th17 cells, is required for the induction of arthritis in mice vaccinated with B. burgdorferi strain 297 and challenged with "Borrelia bissettii." When Borrelia-vaccinated and -challenged mice were given antibodies to the p19 subunit of IL-23, they failed to develop the histopathological changes observed in untreated vaccinated and challenged mice. In addition, viable B. bissettii organisms stimulated the secretion of IL-17 from Borrelia-immune lymph node cells during in vitro culture. When anti-IL-23 p19 antibody was included in cultures of B. bissettii organisms and Borrelia-immune lymph node cells, the production of IL-17 was reduced to levels observed in cultures containing immune cells alone. Taken together, these results support the hypothesis that Th17 cell-associated cytokines are involved in the development of Borrelia-mediated arthritis. These findings provide insight into previously overlooked immune mechanisms responsible for the development of Lyme arthritis.