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Proc Natl Acad Sci U S A. Oct 25, 2005; 102(43): 15629–15634.
Published online Oct 14, 2005. doi:  10.1073/pnas.0507850102
PMCID: PMC1255738
Microbiology

Changes in energy metabolism of Mycobacterium tuberculosis in mouse lung and under in vitro conditions affecting aerobic respiration

Abstract

Transcription profiling of genes encoding components of the respiratory chain and the ATP synthesizing apparatus of Mycobacterium tuberculosis was conducted in vivo in the infected mouse lung, and in vitro in bacterial cultures subjected to gradual oxygen depletion and to nitric oxide treatment. Transcript levels changed dramatically as infection progressed from bacterial exponential multiplication (acute infection) to cessation of bacterial growth (chronic infection) in response to host immunity. The proton-pumping type-I NADH dehydrogenase and the aa3-type cytochrome c oxidase were strongly down-regulated. Concurrently, the less energy-efficient cytochrome bd oxidase was transiently up-regulated. The nitrate transporter NarK2 was also up-regulated, indicative of increased nitrate respiration. The reduced efficiency of the respiratory chain was accompanied by decreased expression of ATP synthesis genes. Thus, adaptation of M. tuberculosis to host immunity involves three successive respiratory states leading to decreased energy production. Decreased bacterial counts in mice infected with a cydC mutant (defective in the cytochrome bd oxidase-associated transporter) at the transition to chronic infection provided initial evidence that the bd oxidase pathway is required for M. tuberculosis adaptation to host immunity. In vitro, NO treatment and hypoxia caused a switch from transcription of type I to type II NADH dehydrogenase. Moreover, cytochrome bd oxidase expression increased, but cytochrome c oxidase expression decreased slightly (nitric oxide) or not at all (hypoxia). These specific differences in respiratory metabolism during M. tuberculosis growth arrest in vitro and in vivo will guide manipulation of in vitro conditions to model bacterial adaptation to host immunity.

Keywords: nitric oxide treatment, transcriptional profiling, dormancy, hypoxia

Mycobacterium tuberculosis is an airborne bacterial pathogen causing a chronic lung infection that passes through several stages. In most infected persons, host defenses either clear infection or drive it into a chronic latent state that is potentially long-lasting. Weakening of host immunity can result in release from latency and reactivation of disease. It has been suggested that various stages of M. tuberculosis infection are associated with different physiological states of the pathogen (15). Work with murine infection models shows that adaptation of M. tuberculosis to host immunity involves replacement of sugars by fatty acids as a carbon and energy source (6, 7). Respiration may also be an important aspect of energy metabolism involved in M. tuberculosis adaptation to host immunity, because respiration in bacteria is a flexible process that changes as microorganisms respond to environmental stresses (reviewed in refs. 8 and 9). However it is not known whether bacterial pathogens, including M. tuberculosis, reroute electron flow during the infection of a host animal.

One model for studying M. tuberculosis adaptation to host immunity involves infecting mice with tubercle bacilli by the respiratory route. The resulting lung infection exhibits both an acute phase (≈20 days of exponential bacterial growth) and a subsequent chronic phase (stabilization of bacterial counts) (10). The latter is thought to result from arrest of bacterial growth (11, 12) caused by expression of host adaptive T helper 1-mediated immunity and the resulting activation of infected macrophages that produce inducible nitric oxide (NO) synthase and consequently NO (reviewed in refs. 13 and 14). NO acts as a potent oxidant and inhibitor of cellular respiration (15, 16). Cultures of M. tuberculosis exposed to bacteriostatic concentrations of NO in vitro exhibit transcriptional changes, such as the induction of an ≈50-gene set regulated by the dosS/dosT/dosR system (17). This regulon (1820) is also induced in hypoxic cultures undergoing growth arrest (19), following activation of infected macrophages with IFN-γ (21), and upon expression of Th1 immunity in the lungs of infected mice (22). Thus bacterial adaptation to mouse lung immunity shares transcriptional similarities with the bacterial response to agents that modulate cellular respiration, such as NO and hypoxia.

In the present work, we used bacterial transcription profiling to determine whether the transition from acute to chronic infection of the mouse lung entails changes in M. tuberculosis respiratory metabolism, and whether in vitro exposure of tubercle bacilli to NO or to hypoxia models these changes. In vivo, we define a complex transcriptional pattern for M. tuberculosis indicative of three successive respiratory states associated with gradually decreasing energy conservation and of a concomitant decreased expression of the ATP synthesizing apparatus. In vitro, we find that the response of respiratory metabolism to NO treatment and to hypoxia is also characterized by a transcriptional switch from highly coupled to less energy-efficient pathways, but that differences exist with the situation in vivo. Defining the respiratory pathways used by M. tuberculosis over the course of infection has had particular relevance to the control of tuberculosis since the discovery of new antitubercular drugs that target respiratory components and ATP synthesis (2325). Moreover, identifying in vitro conditions that adequately model the adaptation of tubercle bacilli to lung immunity facilitates mechanistic investigations of bacterial physiology that are exceedingly difficult to perform in a living animal.

Materials and Methods

Mouse Infection. M. tuberculosis strain H37Rv and its mutant derivatives were grown as a suspension culture, as described (26). C57BL/6 mice at 8–10 weeks of age were infected with ≈2 × 102 bacterial colony-forming units (cfu) per mouse, as described (22). At selected times, lungs were harvested from four mice per time point. Enumeration of cfu was obtained by spreading 10-fold serial dilutions of homogenates from half of the lung (attached to the left bronchus) on enriched Middlebrook 7H11 agar (Difco) plates followed by counting bacterial colonies after 3 weeks of incubation at 37°C. The half of the lung attached to the right bronchus was snap-frozen in liquid nitrogen for subsequent RNA extraction.

Gradual Oxygen Depletion in Vitro. M. tuberculosis H37Rv was grown in liquid culture at 37°C in Dubos Tween-albumin broth (Becton Dickinson), as described (27). Gradual oxygen depletion was obtained by subjecting bacterial cultures to slow magnetic stirring in sealed tubes with a head/space ratio of 0.5 as described (27). At selected times, cells from 2-ml aliquots were harvested by centrifugation and frozen in a dry-ice/alcohol bath for subsequent RNA extraction. Bacterial cfu were enumerated by plating 10-fold serial dilutions of liquid cultures on Dubos oleic albumin agar.

Treatment with Nitric Oxide in Vitro. Cultures of M. tuberculosis H37Rv were grown at 37°C in Dubos Tween-albumin broth to mid-log phase and treated with increasing concentrations (between 25 and 500 μM) of 2,2′-(hydroxynitrosohydrazono)bisethanimine (DETA/NO) (Sigma). After 30 and 60 min, 4 ml of each culture was centrifuged and the cell pellet frozen in a dry-ice/alcohol bath. Each DETA/NO concentration was tested in triplicate in three independent cultures.

Measurements of Bacterial Transcript Copy Numbers. RNA extraction, RT-PCR, and real-time PCR were performed according to our published protocol (22). Reverse transcription primers, PCR primers, and molecular beacons are listed in Table 2, which is published as supporting information on the PNAS web site. Copy numbers of M. tuberculosis 16S rRNA were used as a normalization factor to enumerate bacterial transcripts per cell, because 16S rRNA levels correlate well with cfu during the course of lung infection (22) and during hypoxic incubation (28), regardless of growth stage.

Construction of cydC Mutant of M. tuberculosis. A cydC::aph mutant of M. tuberculosis H37Rv was constructed by allelic exchange mutagenesis and genotypically confirmed by Southern blot analysis (Supporting Text, which is published as supporting information on the PNAS web site). The complementing vector pHTBCYD was constructed by cloning a 6,536-bp EcoRI/EcoRV fragment from pOTBCYD (29) containing 605 bp of DNA upstream of cydA, the entire cydABDC gene cluster, and 74 bp of DNA downstream of cydC in the integrative vector, pHINT. The construct was electroporated into the cydC::aph mutant strain, and transformants were selected on media containing 50 μg/ml hygromycin.

Results and Discussion

Expression of M. tuberculosis Genes Encoding Components of Respiratory Complexes and the ATP Synthesizing Apparatus in the Mouse Lung. Transcriptional profiling was used to identify respiratory pathways used by M. tuberculosis during mouse lung infection. Transcript levels were measured for M. tuberculosis genes involved in electron flow generated from NADH (Fig. 1). Results for multisubunit enzymes are presented for one representative transcript.

Fig. 1.
Architecture of selected respiratory pathways in M. tuberculosis. Analysis of the annotated bacterial genome (http://genolist.pasteur.fr/TubercuList) and assignment of putative gene functions based on sequence similarities with other microorganisms suggest ...

Measurement of bacterial cfu in mouse lungs shows exponential growth for ≈20 days (acute infection) followed by host-immunity-induced stabilization of bacterial counts (chronic infection) (Fig. 2A). Normalized transcript copy numbers for nuoB (type-I NADH dehydrogenase) and ctaD (aa3-type cytochrome c oxidase) decreased from day 12 to day 30 postinfection. This decrease was 70-fold for nuoB and 15-fold for ctaD (Fig. 2B). Transcript levels for qcrC (cytochrome bc1 complex) and ndh (type-II NADH dehydrogenase) decreased slightly during chronic infection (2- to 2.5-fold on day 100 relative to day 15) (Fig. 2C). We also measured levels of the cyd transcripts. As with other bacteria, the M. tuberculosis genome encodes four cyd genes: cydAB encode the bd-type menaquinol oxidase, whereas cydDC encode an ABC-type transporter (reviewed in ref. 9), which is required for cytochrome assembly in Escherichia coli (30). These two gene pairs are located 89 bp apart on the M. tuberculosis genome. Because it has not been established whether the four genes constitute an operon [they are transcribed as a single transcript in Bacillus subtilis (31)], we used probes specific for cydA and cydC sequences for the RT-PCR measurements. We found that the copy number of the cydA transcript was low during acute infection, increased after day 18, peaked on day 30 (7-fold relative to day 15), and dropped back to baseline by day 50 (Fig. 2D). Similar, albeit smaller (2.5-fold), changes were observed with the cydC-specific probe (Fig. 2D).

Fig. 2.
Transcriptional profiles of selected M. tuberculosis genes involved in energy metabolism. (A) The course of M. tuberculosis infection in the mouse lung. In C57BL/6 mice infected via the respiratory route, tubercle bacilli multiplied exponentially in the ...

The down-regulation of both terminal oxidases by day 50 postinfection suggested utilization of alternative electron acceptors in place of O2 for protonmotive gradient formation and energy conservation. Nitrate reduction is the best-characterized anaerobic respiration pathway of M. tuberculosis (3234): M. tuberculosis DNA contains the narGHJI operon, which encodes nitrate reductase, and narK2X, which encodes the nitrate transporter NarK2 and the “fused” inactive nitrate reductase NarX (34). Levels of the narG transcript showed little variation throughout infection (Fig. 2E). In contrast, levels of the narK2 transcript increased >10-fold on day 15 relative to day 12 and remained high throughout infection (>20-fold increase on day 100 relative to day 12) (Fig. 2E). Thus, the adaptation of M. tuberculosis to expression of lung immunity is associated with the transcriptional signature of increased nitrate reduction. As we observed previously (22), transcriptional shifts (as in Fig. 2) preceded control of bacterial infection in the mouse lung, indicating that the transcriptional response takes place before stabilization of the bacterial burden in the lung becomes manifest.

The observed shift to bioenergetically less-efficient respiratory pathways suggested that expression of the bacterial ATP-synthesizing apparatus, which is encoded by the atpA-H gene cluster, also would decrease. We found that levels of the atpD transcript decreased from day 12 through day 30 (7.5-fold) (Fig. 2F). Thereafter, transcript copy numbers remained low. Similar expression levels and transcription patterns were obtained for atpA (Fig. 2F). Thus, the expression levels of ATP synthesis genes in M. tuberculosis are drastically reduced during chronic infection.

A Model of M. tuberculosis Respiratory Flexibility During Infection. Based on the in vivo data presented above, we propose that M. tuberculosis undergoes three successive respiratory states during infection of the mouse lung (Fig. 3). During exponential growth in the mouse lung, M. tuberculosis utilizes the respiratory pathway terminating in the aa3-type oxidase (respiratory state I, Fig. 3). Then expression of Th1-mediated immunity causes activated macrophages to produce NO, which inhibits cellular respiration by competing with O2 for the binding site on cytochrome c oxidase (15, 35). Concurrently, the highly coupled respiratory pathway is strongly down-regulated (up to 70-fold) (Fig. 2B), leading to arrest of bacterial replication. Collapse of electron flow, which would disrupt membrane potential and redox homeostasis, is prevented by establishing a new bacterial respiratory state (respiratory state II, Fig. 3) that is characterized by up-regulation of the less energy-efficient cytochrome bd oxidase (7-fold) (Fig. 2D) and of the nitrate transporter NarK2 (>20-fold) (Fig. 2E). Because NarK2 up-regulation reflects increased nitrate reduction in M. tuberculosis (34), the transcription profile of respiratory state II suggests that cytochrome bd oxidase-dependent aerobic respiration and respiratory nitrate reduction operate in parallel, as described for other microorganisms (8). Due to its high affinity for O2, cytochrome bd oxidase may additionally serve an antioxidant function by scavenging cellular O2, as seen in many bacteria (9, 36). As cytochrome bd oxidase is down-regulated, presumably because of dissipation of the inducing signal as sufficient electron flow and redox balance are restored, respiratory metabolism shifts to nitrate as the preferred terminal electron acceptor (respiratory state III, Fig. 3). A role for nitrate reduction during mouse infection is supported by in vivo growth defects observed with narG deletion mutants of Mycobacterium bovis bacillus Calmette–Guérin (37). Respiratory nitrate reduction contributes greatly to redox balance (8, 38), perhaps reflecting a high requirement for recycling of reducing equivalents when energy is produced primarily from β-oxidation of fatty acids (6). Concurrently, expression levels of ATP synthesis genes decrease (Fig. 2F). This reshaping of M. tuberculosis energy metabolism is part of the definition of M. tuberculosis dormancy.

Fig. 3.
Respiratory states of M. tuberculosis during infection of mouse lung. Presented are events we propose are generated from the high-output production of NO by macrophages activated by Th1-mediated immunity. Respiratory state I: bacteria are actively replicating ...

The changes in energy metabolism in M. tuberculosis during infection inferred from transcriptional profiling need independent support. One line of investigation involves use of bacterial mutants to identify genes required for tubercle bacilli to survive the expression of lung immunity, and another involves biochemical assays to determine the nature and extent of the proposed energy metabolism changes. These measurements are best performed with pure bacterial cultures in vitro, making it important to establish in vitro culture conditions that accurately model the adaptation of M. tuberculosis to mouse lung immunity. Data from both lines of investigation are presented below.

Analysis of the M. tuberculosis cydC Mutant During Mouse Lung Infection. The transient nature of cytochrome bd oxidase up-regulation during the course of lung infection suggests that this enzyme serves an adaptive function in the gradual transition of tubercle bacilli from highly coupled aerobic respiration to less-energy-conserving anaerobic respiration. We examined this possibility by determining the ability of a M. tuberculosis cydC knockout mutant to respond to the expression of lung immunity. When we measured bacterial cfu in the lungs of C57BL/6 mice infected with the M. tuberculosis mutant, the mutant complemented with the cydABDC gene cluster, and wild-type tubercle bacilli, similar cfu counts were observed 20 days postinfection. Thus the cydC mutation did not impair bacillary growth during acute infection (Table 1). In contrast, lung cfu in mice infected with the mutant were 10-fold lower at day 50 relative to day 20 (log10 6.5 vs. log10 5.5), indicating a reduced ability to survive the transition from acute to chronic infection. Complementation of the cydC mutant with cydABDC largely (but not fully) restored bacterial growth. Consistent with a requirement of cyd genes during immunity-induced chronic infection, cydC-deficient bacteria continued to grow to log10 7.6 to day 42 in inducible NO synthase-deficient (NOS2–/–) mice, at which time the animals died (data not shown). No direct evidence currently exists that the cydC mutant strain is impaired in cytochrome bd oxidase function. However, in E. coli, cydDC mutants share all of the phenotypes of cydAB mutants (9). Moreover, the adjacent location of cydAB and cydDC on the M. tuberculosis chromosome suggests a functional association between bd oxidase and the ABC transporter, as seen in many bacteria (39). Further, the M. tuberculosis genome encodes >80 predicted ABC transporters (40), making it reasonable to postulate that the lack of redundancy of cydDC function in vivo is linked to impaired cytochrome bd oxidase function. Thus, the data provided in Table 1 give strong, albeit indirect, evidence that cytochrome bd oxidase is required for adaptation of M. tuberculosis to conditions generated by expression of lung immunity.

Table 1.
cfu enumeration of wild-type and mutant M. tuberculosis in lungs of C57BL/6 mice

Transcriptional Response of Respiratory and ATP Synthesis Genes of M. tuberculosis to Hypoxia and NO Treatment in Vitro. The transcriptional response of tubercle bacilli to expression of mouse lung immunity shares similarities with M. tuberculosis cultures undergoing growth arrest caused by gradual O2 depletion or to treatment with bacteriostatic concentrations of NO (17, 19, 22). To determine how well these in vitro culture conditions model M. tuberculosis adaptation to mouse lung immunity with regard to respiratory metabolism and ATP synthesis, we measured transcription profiles in these in vitro models for the same genes examined in vivo.

In the classical Wayne model of gradual oxygen depletion in vitro (27, 33), two phases of M. tuberculosis growth are described. The first, nonreplicating persistence 1 (NRP-1), initiates when the O2 level approaches 1% saturation. This microaerobic phase is marked by the arrest of bacterial replication and DNA synthesis. When the dissolved O2 content of the culture drops to 0.06% saturation, tubercle bacilli shift to an anaerobic stage, NRP-2. We found that the nuoB transcript level decreased in both NRP-1 and NRP-2 (3- and 6-fold) relative to aerated mid-log growth, whereas qcrC and ctaD transcript levels were roughly constant (Fig. 4). Transcript levels for ndh, cydA, and cydC peaked during early NRP-2 (3-fold for ndh, 8-fold for cydA, and 2.5-fold for cydC), with cydA increasing even in NRP-1 (4-fold) (Fig. 4). Induction of these transcripts was also detected in microarray-based analyses (23, 41) but, as expected, the magnitude of the change measured by our quantitative RT-PCR assay was greater. Measurements of transcripts involved in nitrate reduction showed that narG mRNA remained stable throughout culture, whereas narK2X mRNA was strongly up-regulated in both NRP-1 and -2 (≈40- and 120-fold, respectively) (Fig. 4), in accord with previous reports (19, 34). Increased narK2X and constitutive narGHJI expression provide the transcriptional signature of increased nitrate reduction in M. tuberculosis (34). For atpA and atpD transcripts, copy numbers gradually decreased (3-fold in NRP-2) (Fig. 4).

Fig. 4.
Normalized copy number of selected M. tuberculosis transcripts in liquid cultures exposed to gradual O2 depletion. M. tuberculosis was cultured under conditions of gradual O2 depletion established by Wayne and Hayes (ref. 27; see Materials and Methods ...

The same transcripts were measured in cultures of M. tuberculosis exposed to NO. Treatment of mid-log aerobically growing bacterial cultures for 30 min with increasing doses of DETA/NO (25–500 μM; see Materials and Methods) increased both the number of genes affected and the magnitude of the change in transcript level (Fig. 5). The narK2X transcript was up-regulated ≈250-fold in cells treated with 25 μM DETA/NO; no other transcript tested showed copy number changes at the same DETA/NO concentration. Treatment with 100 μM DETA/NO caused a 5-fold decrease of the nuoB transcript and a small increase of ndh and cydA (4- and 3-fold, respectively). Induction of cydC became detectable (3.5- to 12-fold) only when treatment was prolonged to 60 min (see Fig. 5 Inset). Treatment with 500 μM DETA/NO resulted in a further 25-fold decrease of nuoB and a 4-fold decrease of qcrC. Only a minor (2-fold) variation was measured for ctaD transcript levels. In contrast, copy numbers of the ndh and cydA transcripts further increased with the DETA/NO dose, to 6- to 7-fold with 250 μM and to 9-fold with 500 μM DETA/NO (Fig. 5). Although the changes observed in NO-treated cultures for narK2X, which is a dosR-regulated gene, are in accord with previous reports (17, 21), shifts in the levels of ndh, cydA, and cydC transcripts were not revealed in previous analyses (21, 23, 42), presumably because changes of the scale observed (<10-fold) are readily detected by quantitative RT-PCR but not necessarily by microarray-based transcriptome analysis.

Fig. 5.
Normalized copy number of selected M. tuberculosis transcripts in liquid cultures exposed to nitric oxide. M. tuberculosis cultures were treated with increasing concentrations of the NO donor DETA/NO for 30 and 60 min as described under Materials and ...

Collectively, the in vitro data show that tubercle bacilli treated with NO or under anaerobic incubation (NRP-2) switched from transcription of type I to type II NADH dehydrogenases. This switch represents a significant departure from the E. coli paradigm, in which noncoupling dehydrogenases, such as Ndh, are preferentially expressed during aerobic respiration (43), whereas coupling dehydrogenases, such as NuoA-N, are associated with “energy-limited” anaerobic respiratory pathways (44). Moreover, the bd-type oxidase genes were up-regulated under both sets of conditions, whereas cytochrome c oxidase expression showed a downward tendency in NO-treated cells but not in hypoxic cultures. Overall, these changes were reminiscent of respiratory changes in vivo. However, important differences were observed: ndh was induced in vitro but not in vivo (compare Fig. 2C with Figs. Figs.33 and and4),4), and ctaD exhibited a large down-regulation in vivo but little (NO) or none (hypoxia) in vitro (compare Fig. 2B with Figs. Figs.33 and and4).4). Moreover, cydA induction was transient in the mouse lung (Fig. 2D) but not during hypoxic growth in vitro (Fig. 4). Obviously, the situation in vivo is multifactorial. For example, down-regulation of nuoB and ctaD was not as marked in the NO-treated cells as it was in vivo (compare Fig. 2B with Fig. 5), perhaps because of prolonged exposure to NO in vivo or to additional as-yet-unidentified factors. Moreover, even though NO-treated cells show induced cyd genes, the induction of cytochrome bd oxidase expression seen in mouse lung may also be mediated by proteins sensing integrated signals perhaps derived from changes in respiratory chain efficiency, as previously proposed (23). This latter scenario is supported by the decreased cydAB transcript levels by day 50 postinfection, which may result from dissipation of the inducing signal as sufficient electron flow and redox balance are restored by the combined activity of the bd-type terminal oxidase and nitrate reductase, as suggested above.

Conclusion

Parallel transcriptional profiling of components of respiratory pathways and of the ATP synthesis apparatus of M. tuberculosis during mouse respiratory infection and in two in vitro treatments that affect aerobic respiration have identified important similarities consistent with bacterial growth arrest under all three conditions. These data open the way to modification of the in vitro conditions tested to better model the transcriptional profiles seen in vivo. These observations have the potential of further elucidating the mouse model of infection, which is commonly used for screening new drugs and vaccines against tuberculosis.

The overall implication of the model proposed in Fig. 3 is that respiratory flexibility is an essential component of the adaptation of M. tuberculosis to host immunity. This view is supported by the reduced ability of a cydC deletion mutant of M. tuberculosis to undergo the transition to chronic infection and by in vivo growth defects observed with a narG deletion mutant of M. bovis bacillus Calmette–Guérin (37). Moreover, the proposed role of the bd-type terminal oxidase may explain the classic observations relating virulence and hypoxia tolerance in tubercle bacilli (45, 46), because hypoxia tolerance may be a surrogate marker for the pathogen's ability to adapt to conditions created by host immunity. If our model is correct, it will lead to new targets for drugs and vaccines against tuberculosis.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Lynn Ryan and Ron LaCourse (Trudeau Institute) for mouse infections. We are also grateful to Karl Drlica, Carol Lusty, David Perlin, and Larry Wayne for stimulating discussions and critical reading of this manuscript. Special thanks go to Harvey Penefsky for his help in highlighting the significance of the findings and their implications in the biochemistry of the M. tuberculosis-infected lung. V.M. and B.D.K. also thank Harvey Rubin for stimulating their interest in the field of M. tuberculosis respiration. This work was supported by grants from the Medical Research Services of the U.S. Department of Veterans Affairs (to C.S.), the Medical Research Council of South Africa and the Howard Hughes Medical Institute (International Scholars Grant) (to V.M.), the Futura Foundation (to M.L.G.), and National Institutes of Health [Grants AI-37844 and AI-059557 (to R.J.N.); Grant AI-43420 (to Harvey Rubin, supporting work in V.M.'s laboratory); and AI-36989 (to M.L.G.)].

Notes

Author contributions: L.S., C.D.S., R.J.N., V.M., and M.L.G. designed research; L.S., C.D.S., B.D.K., and R.J.N. performed research; B.D.K. and S.D. contributed new reagents/analytic tools; L.S., R.J.N., V.M., and M.L.G. analyzed data; and L.S., C.D.S., B.D.K., V.M., and M.L.G. wrote the paper.

Abbreviations: NRP, nonreplicating persistence; DETA/NO, 2,2′-(hydroxynitrosohydrazono)bisethanimine; cfu, colony-forming unit.

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