Discovery and dissection of metabolic oscillations in the microaerobic nitric oxide response network ofEscherichia coli

Abstract

The virulence of many pathogens depends upon their ability to cope with immune-generated nitric oxide (NO · ). In Escherichia coli, the major NO · detoxification systems are Hmp, an NO · dioxygenase (NOD), and NorV, an NO · reductase (NOR). It is well established that Hmp is the dominant system under aerobic conditions, whereas NorV dominates anaerobic conditions; however, the quantitative contributions of these systems under the physiologically relevant microaerobic regime remain ill defined. Here, we investigated NO · detoxification in environments ranging from 0 to 50 μM O 2 , and discovered a regime in which E. coli NO · defenses were severely compromised, as well as conditions that exhibited oscillations in the concentration of NO · . Using an integrated computational and experimental approach, E. coli NO · detoxification was found to be extremely impaired at low O 2 due to a combination of its inhibitory effects on NorV, Hmp, and translational activities, whereas oscillations were found to result from a kinetic competition for O 2 between Hmp and respiratory cytochromes. Because at least 777 different bacterial species contain the genetic requirements of this stress response oscillator, we hypothesize that such oscillatory behavior could be a widespread phenomenon. In support of this hypothesis, Pseudomonas aeruginosa, whose respiratory and NO · response networks differ considerably from those of E. coli, was found to exhibit analogous oscillations in low O 2 environments. This work provides insight into how bacterial NO · defenses function under the low O 2 conditions that are likely to be encountered within host environments.nitrosative stress | E. coli | microaerobic | Pseudomonas aeruginosa | kinetic modeling N itric oxide (NO · ) plays a critical role in mammalian innate immunity as a potent antimicrobial (1-3), where its broad reactivity contributes to a diverse repertoire of cytotoxic effects including respiratory inhibition, thiol nitrosation, iron-sulfur cluster ([Fe-S]) destruction, DNA deamination, and tyrosine nitration/ nitrosylation (4, 5). To cope with this stress, many pathogens harbor defenses to detoxify NO · and its reaction products, and repair NO · -mediated damage to biomolecules (6, 7). Disruption of these defenses has been shown to attenuate virulence in many pathogenic species, such as Neisseria meningitides, Pseudomonas aeruginosa, Yersinia pestis, Mycobacterium tuberculosis, Vibrio cholerae, uropathogenic and enterohemorrhagic Escherichia coli (UPEC and EHEC, respectively), Staphylococcus aureus, and Salmonella enterica serovar Typhimurium (4, 6), which underscores the importance of NO · to immune function and highlights microbial NO · defense networks as a promising source of targets for the development of next-generation antiinfectives (8). This potential has inspired many investigations of bacterial NO · stress, from which major defense systems, such as NO · dioxygenase (NOD) (9, 10) and NO · reductase (NOR) (11, 12), have been identified. NODs are widely distributed among dif...

An integrated network analysis identifies how ArcAB enables metabolic oscillations in the nitric oxide detoxification network of Escherichia coli

“…Previously, computational modeling of the E. coli NO• stress network suggested that loss of respiratory activity would eliminate NO• oscillations and that prediction was confirmed experimentally with use of a triple cytochrome terminal oxidase mutant (∆CYT: ∆ cyoA ∆ appB ∆ cydB ::kanR), which cannot respire on O 2 . Experimentally, ∆ arcA and ∆ arcB eliminated NO• oscillations (Fig.…”

“…A [Cyd] = [Cyd] 0 or 3B [Cyo] = [Cyo] 0 ). This quantitative disagreement has been discussed previously , and attributed to a parsimonious rate expression for translation that is used within the model. In that previous work, we hypothesized that complex translation dynamics during the oscillatory period were not being sufficiently captured by the model.…”

“…Specifically, we quantified [NO•] and [O 2 ] following DPTA treatment of WT, ∆ arcA , and ∆ arcB in media that initially contained 10 µM dissolved O 2 . Consistent with previous observations , WT cultures exhibited oscillations in the [NO•] and [O 2 ] (Fig. A).…”

“…Notably, transcription, translation, or catalytic issues with Hmp would have manifested as significant perturbations to the initial NO• clearance time. Without Hmp, it would have taken many hours for [NO•] to be cleared under these experimental conditions, as we have demonstrated previously . The comparable initial clearance times between WT, Δ arcA , and Δ arcB suggest that Hmp synthesis and catalytic activity are preserved in the absence of ArcAB.…”

“…To assess the accuracies of predictions drawn from the in silico simulations, we measured NO• detoxification in cultures of ∆ cydB and ∆ cyoA , and for completeness, ∆ appB . Each of these mutations eliminates the catalytic activity of their respective complexes , and notably, loss of all three eliminates the capacity of E. coli to respire on O 2 . As predicted by the systems‐level simulations, loss of CydAB activity eliminated oscillations, whereas loss of CyoABCD activity did not (Fig.…”