•P. aeruginosa possess enzymes capable of forming an NO→NO3−→NO2−→NO cycle.•A kinetic model of P. aeruginosa NO stress was built and experimentally validated.•NO metabolism did not proceed beyond NO3− under O2-rich conditions.•Under low-O2 conditions, NO reductase (NorCB) removed flux from the NO cycle.•Assimilatory NO2− reductase (NirB) had a negligible impact on the NO metabolic cycle.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.

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 O2, 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 O2 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 O2 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 O2 environments. This work provides insight into how bacterial NO· defenses function under the low O2 conditions that are likely to be encountered within host environments.

•Sensitizing bacteria to immune attack is a promising anti-virulence strategy.•Complex biochemical reaction networks exist within the phagosomes of immune cells.•Modeling can improve understanding of bacterial responses to phagosomal stresses.The efficacies of antibiotic treatments have been compromised due to the emergence of (multi)drug-resistant pathogens, and the need for new treatment options is pressing. Within hosts, pathogens are bombarded with combinations of toxic compounds by immune cells, and bacteria have evolved numerous strategies to survive those antimicrobial assaults. Disruption of those defenses could sensitize bacteria to immune attacks and lead to new anti-infective modalities. To realize such therapies, deep understanding of how bacteria cope with those toxic cocktails is desirable. We propose that methods from biochemical engineering can help provide such knowledge and serve as complementary approaches to those that directly use phagocytes. Here, we summarize the rationale for pursuing immune-potentiating anti-infectives, review recent efforts that employ engineering approaches to examine phagosomal stressors and their antibacterial activity, and discuss how biochemical engineering can contribute further to this exciting field.

In nongrowing microbes, proteome turnover is reduced and identification of newly synthesized, low-abundance proteins is challenging. Babin and colleagues recently utilized bio-orthogonal noncanonical amino acid tagging (BONCAT) to identify actively synthesized proteins in nongrowing Pseudomonas aeruginosa, discovering a regulator whose influences range from biofilm formation to secondary metabolism.

•Antimicrobial activity of NO● was predicted to depend strongly on delivery rate.•Fast NO● delivery rates were more effective for low NO● payloads.•Slow NO● delivery rates were more effective for high NO● payloads.•Kinetic modeling of NO● metabolism correctly predicted the observed dependencies.The antimicrobial properties of nitric oxide (NO●) have motivated the design of NO●-releasing materials for the treatment and prevention of infection. The biological activity of NO● is dependent on its delivery rate, suggesting that variable antimicrobial effects can result from identical NO● payloads dosed at different rates. Using a kinetic model of the Escherichia coli NO● biochemical network, we investigated the relationship between NO● delivery rate, payload, and cytotoxicity, as indicated by the duration of respiratory inhibition. At low NO● payloads, the model predicted greater toxicity with rapid delivery, while slower delivery was more effective at higher payloads. These predictions were confirmed experimentally, and exhibited quantitative agreement with measured O2 and NO● concentrations, and durations of respiratory inhibition. These results provide important information on key design parameters in the formulation of NO●-based therapeutics, and highlight the utility of a model-based approach for the analysis of dosing regimens.

•Carbon source transitions stimulate persister formation•Pathway from initial stress to source of bistability and antibiotic tolerance•ppGpp-SpoT forms a metabolic TA moduleBacterial persisters are phenotypic variants that form from the action of stress response pathways triggering toxin-mediated antibiotic tolerance. Although persisters form during normal growth from native stresses, the pathways responsible for this phenomenon remain elusive. Here we have discovered that carbon source transitions stimulate the formation of fluoroquinolone persisters in Escherichia coli. Further, through a combination of genetic, biochemical, and flow cytometric assays in conjunction with a mathematical model, we have reconstructed a molecular-level persister formation pathway from initial stress (glucose exhaustion) to the activation of a metabolic toxin-antitoxin (TA) module (the ppGpp biochemical network) resulting in inhibition of DNA gyrase activity, the primary target of fluoroquinolones. This pathway spans from initial stress to antibiotic target and demonstrates that TA behavior can be exhibited by a metabolite-enzyme interaction (ppGpp-SpoT), in contrast to classical TA systems that involve only protein and/or RNA.

In this issue, Nelson and colleagues (2017) determined that guanidine, the prevalent protein denaturant, is the long-lost ligand sensed by the ykkC class of riboswitches, and identified that members of its regulon are involved in guanidine detoxification and export.