Cooperative Roles of Nitric Oxide-Metabolizing Enzymes To Counteract Nitrosative Stress in Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) has at least three enzymes, NorV, Hmp, and Hcp, that act independently to lower the toxicity of nitric oxide (NO), a potent antimicrobial molecule. This study aimed to reveal the cooperative roles of these defensive enzymes in EHEC against nitrosative stress. Under anaerobic conditions, combined deletion of all three enzymes significantly increased the NO sensitivity of EHEC determined by the growth at late stationary phase; however, the expression of norV restored the NO resistance of EHEC. On the other hand, the growth of Δhmp mutant EHEC was inhibited after early stationary phase, indicating that NorV and Hmp play a cooperative role in anaerobic growth. Under microaerobic conditions, the growth of Δhmp mutant EHEC was inhibited by NO, indicating that Hmp is the enzyme that protects cells from NO stress under microaerobic conditions. When EHEC cells were exposed to a lower concentration of NO, the NO level in bacterial cells of Δhcp mutant EHEC was higher than those of the other EHEC mutants, suggesting that Hcp is effective at regulating NO levels only at a low concentration. These findings of a low level of NO in bacterial cells with hcp indicate that the NO consumption activity of Hcp was suppressed by Hmp at a low range of NO concentrations. Taken together, these results show that the cooperative effects of NO-metabolizing enzymes are regulated by the range of NO concentrations to which the EHEC cells are exposed.

Functional roles of norCB in Pseudomonas aeruginosa ATCC 9027 under aerobic conditions.

Nitric oxide (NO) reductase (NorCB) of Pseudomonas aeruginosa is an essential enzyme that metabolizes NO and alleviates anaerobic NO toxicity during denitrification processes under anaerobic conditions. However, the molecular functions of norCB in the presence of oxygen are poorly understood. This study utilized norCB knockout from P. aeruginosa ATCC 9027 to analyze the resulting phenotypic changes of ΔnorCB in comparison to the wild-type parental strain (WT) and the complementary strain (ΔnorCB-com). The results demonstrated an increase in planktonic growth and biofilm formation by ΔnorCB compared to WT and ΔnorCB-com in the presence of isothiazolones under aerobic conditions. Deletion of norCB led to increased swimming ability and decreased pyocyanin production. Inactivation of norCB also led to an increase of cellular H2 O2 concentration due to decreased activity of its catalases. In addition, the deletion of norCB also influenced the relative expressions of several other genes, including norD, nirS, hmgA, and hpd. These findings provide preliminary evidence that norCB in P. aeruginosa plays an essential role in bacterial life process under aerobic conditions and improves the application of denitrification in the next step.

Modulation of protein function in membrane mimetics: Characterization of P. denitrificans cNOR in nanodiscs or liposomes.

For detailed functional characterization, membrane proteins are usually studied in detergent. However, it is becoming clear that detergent micelles are often poor mimics of the lipid environment in which these proteins function. In this work we compared the catalytic properties of the membrane-embedded cytochrome c-dependent nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans in detergent, lipid/protein nanodiscs, and proteoliposomes. We used two different lipid mixtures, an extract of soybean lipids and a defined mix of synthetic lipids mimicking the original P. denitrificans membrane. We show that the catalytic activity of detergent-solubilized cNOR increased threefold upon reconstitution from detergent into proteoliposomes with the P. denitrificans lipid mixture, and above two-fold when soybean lipids were used. In contrast, there was only a small activity increase in nanodiscs. We further show that binding of the gaseous ligands CO and O2 are affected differently by reconstitution. In proteoliposomes the turnover rates are affected much more than in nanodiscs, but CO-binding is more significantly accelerated in liposomes with soybean lipids, while O2-binding is faster with the P. denitrificans lipid mix. We also investigated proton-coupled electron transfer during the reaction between fully reduced cNOR and O2, and found that the pKa of the internal proton donor was increased in proteoliposomes but not in nanodiscs. Taking our results together, the liposome-reconstituted enzyme shows significant differences to detergent-solubilized protein. Nanodiscs show much more subtle effects, presumably because of their much lower lipid to protein ratio. Which of these two membrane-mimetic systems best mimics the native membrane is discussed.

An integrated network analysis reveals that nitric oxide reductase prevents metabolic cycling of nitric oxide by Pseudomonas aeruginosa.

Nitric oxide (NO) is a chemical weapon within the arsenal of immune cells, but is also generated endogenously by different bacteria. Pseudomonas aeruginosa are pathogens that contain an NO-generating nitrite (NO2-) reductase (NirS), and NO has been shown to influence their virulence. Interestingly, P. aeruginosa also contain NO dioxygenase (Fhp) and nitrate (NO3-) reductases, which together with NirS provide the potential for NO to be metabolically cycled (NO→NO3-→NO2-→NO). Deeper understanding of NO metabolism in P. aeruginosa will increase knowledge of its pathogenesis, and computational models have proven to be useful tools for the quantitative dissection of NO biochemical networks. Here we developed such a model for P. aeruginosa and confirmed its predictive accuracy with measurements of NO, O2, NO2-, and NO3- in mutant cultures devoid of Fhp or NorCB (NO reductase) activity. Using the model, we assessed whether NO was metabolically cycled in aerobic P. aeruginosa cultures. Calculated fluxes indicated a bottleneck at NO3-, which was relieved upon O2 depletion. As cell growth depleted dissolved O2 levels, NO3- was converted to NO2- at near-stoichiometric levels, whereas NO2- consumption did not coincide with NO or NO3- accumulation. Assimilatory NO2- reductase (NirBD) or NorCB activity could have prevented NO cycling, and experiments with ΔnirB, ΔnirS, and ΔnorC showed that NorCB was responsible for loss of flux from the cycle. Collectively, this work provides a computational tool to analyze NO metabolism in P. aeruginosa, and establishes that P. aeruginosa use NorCB to prevent metabolic cycling of NO.

Role of the Nitric Oxide Reductase NorVW in the Survival and Virulence of Enterohaemorrhagic Escherichia coli during Infection.

Enterohaemorrhagic Escherichia coli (EHEC) are bacterial pathogens responsible for life-threatening diseases in humans, such as hemolytic and uremic syndrome. It has been previously demonstrated that the interplay between EHEC and nitric oxide (NO), a mediator of the host immune innate response, is critical for infection outcome, since NO affects both Shiga toxin (Stx) production and adhesion to enterocytes. In this study, we investigated the role of the NO reductase NorVW in the virulence and fitness of two EHEC strains in a murine model of infection. We determined that the deletion of norVW in the strain O91:H21 B2F1 has no impact on its virulence, whereas it reduces the ability of the strain O157:H7 620 to persist in the mouse gut and to produce Stx. We also revealed that the fitness defect of strain 620 ΔnorVW is strongly attenuated when mice are treated with an NO synthase inhibitor. Altogether, these results demonstrate that the NO reductase NorVW participates in EHEC resistance against NO produced by the host and promotes virulence through the modulation of Stx synthesis. The contribution of NorVW in the EHEC infectious process is, however, strain-dependent and suggests that the EHEC response to nitrosative stress is complex and multifactorial.

EPR spectroscopy of putative enzyme intermediates in the NO reductase and the auto-nitrosylation reaction of Desulfovibrio vulgaris hybrid cluster protein.

The hybrid cluster protein (Hcp) contains a unique 4Fe cluster that is a hybrid of µ-S and µ-O bridges. Escherichia coli Hcp has recently been found to carry NO reductase activity as well as S-nitrosylation activity in NO-based signaling. In other species, the physiological activity has not been established. No reaction mechanism of any Hcp has been proposed. Here, we show that Desulfovibrio vulgaris (Hildenborough) Hcp has nitric oxide reductase activity with benzyl viologen as electron donor. With EPR spectroscopy, we identify three unexpected putative reaction intermediates: both in reduced and oxidized Hcp, dinitrosyl iron complexes are formed. Also, the hybrid cluster in reduced Hcp, but not in oxidized Hcp, binds the product N2 O. Possible implications for a reaction mechanism are discussed.

Nitric Oxide Stress as a Metabolic Flux.

Nitric oxide (NO) is an antimicrobial metabolite produced by immune cells to prohibit infection. Due to its reactivity, NO has numerous reaction routes available to it in biological systems with some leading to cellular damage and others producing innocuous compounds. Pathogens have evolved resistance mechanisms toward NO, and many of these take the form of enzymes that chemically passivate the molecule. In essence, bacteria have channeled NO flux toward useful or harmless compounds, and away from pathways that damage cellular components. Pathogens devoid of detoxification enzymes have been found to have compromised survival in different infection models, which suggests that diverting flux away from NO defenses could be a viable antiinfective strategy. From this perspective, potentiation of NO stress mirrors challenges in metabolic engineering where researchers endeavor to divert flux away from endogenous pathways and toward those that produce desirable biomolecules. In this review, we cast NO stress as a metabolic flux and discuss how the tools and methodologies of metabolic engineering are well suited for analysis of this bacterial stress response. We provide examples of such interdisciplinary applications, discuss the benefits of considering NO stress from a flux perspective, as well as the pitfalls, and offer a vision for how metabolic engineering analyses can assist in deciphering the economics underlying bacterial responses to multistress conditions that are characteristic of the phagosomes of immune cells.

Nitric oxide-enhanced Shiga toxin production was regulated by Fur and RecA in enterohemorrhagic Escherichia coli O157.

Enterohemorrhagic Escherichia coli (EHEC) produces Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). Nitric oxide (NO), which acts as an antimicrobial defense molecule, was found to enhance the production of Stx1 and Stx2 in EHEC under anaerobic conditions. Although EHEC O157 has two types of anaerobic NO reductase genes, an intact norV and a deleted norV, in the deleted norV-type EHEC, a high concentration of NO (12-29 µmol/L, maximum steady-state concentration) is required for enhanced Stx1 production and a low concentration of NO (~12 µmol/L, maximum steady-state concentration) is sufficient for enhanced Stx2 production under anaerobic conditions. These results suggested that different concentration thresholds of NO elicit a discrete set of Stx1 and Stx2 production pathways. Moreover, the enhancement of Shiga toxin production in the intact norV-type EHEC required treatment with a higher concentration of NO than was required for enhancement of Shiga toxin production in the deleted norV-type EHEC, suggesting that the specific NorV type plays an important role in the level of enhancement of Shiga toxin production in response to NO. Finally, Fur derepression and RecA activation in EHEC were shown to participate in the NO-enhanced Stx1 and Stx2 production, respectively.

The evolution of respiratory O2/NO reductases: an out-of-the-phylogenetic-box perspective.

Complex life on our planet crucially depends on strong redox disequilibria afforded by the almost ubiquitous presence of highly oxidizing molecular oxygen. However, the history of O2-levels in the atmosphere is complex and prior to the Great Oxidation Event some 2.3 billion years ago, the amount of O2 in the biosphere is considered to have been extremely low as compared with present-day values. Therefore the evolutionary histories of life and of O2-levels are likely intricately intertwined. The obvious biological proxy for inferring the impact of changing O2-levels on life is the evolutionary history of the enzyme allowing organisms to tap into the redox power of molecular oxygen, i.e. the bioenergetic O2 reductases, alias the cytochrome and quinol oxidases. Consequently, molecular phylogenies reconstructed for this enzyme superfamily have been exploited over the last two decades in attempts to elucidate the interlocking between O2 levels in the environment and the evolution of respiratory bioenergetic processes. Although based on strictly identical datasets, these phylogenetic approaches have led to diametrically opposite scenarios with respect to the history of both the enzyme superfamily and molecular oxygen on the Earth. In an effort to overcome the deadlock of molecular phylogeny, we here review presently available structural, functional, palaeogeochemical and thermodynamic information pertinent to the evolution of the superfamily (which notably also encompasses the subfamily of nitric oxide reductases). The scenario which, in our eyes, most closely fits the ensemble of these non-phylogenetic data, sees the low O2-affinity SoxM- (or A-) type enzymes as the most recent evolutionary innovation and the high-affinity O2 reductases (SoxB or B and cbb3 or C) as arising independently from NO-reducing precursor enzymes.