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

Abstract

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 (NO) reductase (NirS), and NO has been shown to influence their virulence. Interestingly, P. aeruginosa also contain NO dioxygenase (Fhp) and nitrate (NO) reductases, which together with NirS provide the potential for NO to be metabolically cycled (NO→NO→NO→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, O, NO, and NO 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 NO, which was relieved upon O depletion. As cell growth depleted dissolved O levels, NO was converted to NO at near-stoichiometric levels, whereas NO consumption did not coincide with NO or NO accumulation. Assimilatory NO 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.

Nitric Oxide-Mediated Induction of Dispersal in Pseudomonas aeruginosa Biofilms Is Inhibited by Flavohemoglobin Production and Is Enhanced by Imidazole

“…The NO-sensing and protective mechanisms appear to be highly diverse in bacteria, and in E. coli, NorR regulates a flavorubredoxin, NorV, rather than the Fhp homologue flavohemoglobin Hmp (40), the expression of which is regulated by the Fe-S cluster regulator NsrR (41). In E. coli and P. aeruginosa, the interplay between NO-producing and NO-scavenging systems was recently shown to generate oscillating cycles of NO levels, thus ensuring that intracellular NO remains present at nontoxic levels (42,43). In several bacterial species, the molecular mechanisms of NO-mediated dispersal have been shown to involve bis-(3=-5=)-cyclic dimeric GMP (c-di-GMP), a soluble second messenger, which is the key element of a genetic network governing motile-to-sessile transitions and is conserved in Gram-negative bacteria.…”

Core regulon of the global anaerobic regulator Anr targets central metabolism functions in Pseudomonas species

“…These functions include the presence of high oxygen affinity cytochromes, the utilization of nitrate as alternative electron acceptor and arginine and pyruvate fermentation 2,4,8,31 that have been described as controlled by Anr. However, anaerobic metabolism is not conserved among Pseudomonas species, while P. aeruginosa PAO1 can carry out a complete denitrification process 32 , P. extremaustralis is only able to reduce nitrate to nitrite, and both species can perform arginine and pyruvate fermentation 16,25 . On the contrary, P. putida KT2440 is not able to use nitrate as electron acceptor and neither carry out pyruvate fermentation 33 .…”

Electroanalytical characterization of the direct Marinobacter hydrocarbonoclasticus nitric oxide reductase-catalysed nitric oxide and dioxygen reduction

“…cNOR, the first class, is a membrane enzyme with two different subunits, a NorB (the catalytic center) and a NorC (responsible for electron transfer) [5,6]. cNOR can be extracted from Paracoccus denitrificans [7][8][9], Pseudomonas nautica (also designated as Marinobacter hydrocarbonoclasticus) [5,10,11], Pseudomonas aeruginosa [12,13], Halomonas halodenitrificans [14], Roseobacter denitrificans [15] and Thermus thermophilus [16]. Several methods have been applied to study the NO reduction by different NORs, which included density functional theory (DFT) calculations [7,12,17], fluorescence [18], Raman [19,20] and UV/Vis spectroscopy [21,22].…”