Department of Veterans Affairs Medical Center, Long Beach, CA 90822, and Department of Microbiology and Molecular Genetics, University of California, Irvine, CA 92717, USA
Correspondence
Charles D. Sohaskey
chuck{at}sohaskey.com
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ABSTRACT |
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A graph showing the effects of DETA/NO and c-PTIO on nitrite synthesis is available as supplementary data with the online version of this paper.
Present address: Tuberculosis Research Laboratory (151), Department of Veterans Affairs Medical Center, 5901 East Seventh Street, Long Beach, CA 90822, USA.
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INTRODUCTION |
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In the human host, tubercle bacilli reside in granulomas, structures which may limit the availability of oxygen. Although M. tuberculosis is classified as an obligate aerobe, in vitro it displays a complicated response to oxygen deprivation, with the induction of over 100 genes (Sherman et al., 2001). When grown in sealed tubes with slow stirring, tubercle bacilli cease replicating when oxygen decreases to the microaerobic level and they enter a non-replicating persistence (NRP) state, NRP-1. With continued incubation, the anaerobic NRP-2 state follows (Wayne & Hayes, 1996
). Many similarities in gene expression between chronic infection of mice and in vitro NRP cultures have been identified (Shi et al., 2005
).
One of the activities induced in M. tuberculosis by hypoxia is the dissimilatory reduction of nitrate to nitrite, which serves to provide energy as the bacteria adapt to anaerobiosis (Wayne & Hayes, 1999; Wayne & Sohaskey, 2001
). Cultures in NRP-1 synthesize greater levels of nitrite than do actively growing (AG) aerobic cultures, but in both cases the nitrite is produced by the nitrate reductase enzyme encoded by narGHJI (Weber et al., 2000
; Sohaskey & Wayne, 2003
). Expression of this operon is constitutive and not induced by hypoxia, although transcription of the narK2 nitrate transporter is. This transporter is a main point of regulation, and is responsible for the upregulation of nitrate reductase activity seen in NRP cultures (Sohaskey & Wayne, 2003
). Aerobic cultures are thought to rely on the slow diffusion of nitrate into the cells to provide substrate for the NarGHJI enzyme. Neither narGHJI nor narK2 shows induction by either nitrite or nitrate.
One source of nitrate in vivo is the breakdown of nitric oxide (NO). Both hypoxia and NO can act as signals to M. tuberculosis that result in the initiation of the NRP state (Voskuil et al., 2002). It has been suggested that oxygen limitation may be the important signal for NRP in humans, but in mice it may be NO (Boshoff & Barry, 2005
). NO is essential in the control of M. tuberculosis in the murine model (Adams et al., 1997
; Chan et al., 1995
; MacMicking et al., 1997
) and humans (Nathan, 2002
; Chan et al., 2001
). NO is synthesized by activated macrophages in mice when the cell-mediated immune system is activated, so there is an increase in available nitrate at the same time that the tubercle bacilli cease replicating.
A set of 48 genes, called the dormancy regulon, is induced by either hypoxia or low levels of NO (Voskuil et al., 2002; Ohno et al., 2003
). The narGHJI operon is not induced by these stimuli, while narK2 is induced by both (Sohaskey & Wayne, 2003
; Ohno et al., 2003
; Voskuil et al., 2002
). We analysed the effect of oxygen and NO on nitrate reduction in both aerobic and NRP cultures of M. tuberculosis.
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METHODS |
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Chemicals.
Kanamycin was used at 25 µg ml1 for M. tuberculosis. All chemicals were from Sigma. Potassium ferricyanide (III) [K3Fe(CN)6], Triton X-100 and hydroxylamine hydrochloride were dissolved in water and filter-sterilized. The sources of nitric oxide were N-(2-aminoethyl)-N-(2-hydroxy-2-nitrosohydrazino)-1,2-ethylene diamine (DETA/NO) and S-nitrosoglutathione (GSNO), and the NO trap was 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO), all of which were dissolved in sterile water.
Measurement of nitrite in culture.
Cultures were inoculated to contain approximately 2·5x106 cells ml1, and were grown either in 10 ml DTA with shaking, for aerobic (AG) cultures, or in 17 ml DTA in sealed tubes with slow magnetic stirring, for NRP cultures. After 112 h of incubation, aerobic cultures were in the mid-exponential phase of growth (OD580 0·4), whereas the NRP cultures had by then been in microaerobic NRP-1 for approximately 45 h (Wayne & Hayes, 1996
).
NRP-1 cultures, which contained approximately 108 cells ml1, were opened, vortexed to introduce air and then pooled. Aerobic cultures were pooled and diluted with DTA to the same approximate cell concentration based on optical density (OD580 0·1=6·25x107 c.f.u. ml1). Chloramphenicol to 20 µg ml1 and NaNO3 to 5 mM were added where indicated. The cultures were then dispensed into tubes (in triplicate). Loosely capped tubes were incubated with shaking for aerobic conditions. For anaerobic incubation, Oxyrase (Oxyrase Inc., Mansfield, OH) was added to tightly capped tubes to completely remove dissolved oxygen. Attainment of anaerobiosis was monitored by the decolourization of the redox indicator methylene blue present in independent control tubes. In every experiment, control tubes of AG and NRP cultures were incubated both aerobically and anaerobically.
At intervals, samples were removed to determine nitrite concentrations by the Griess reaction (Wayne & Doubek, 1965). In experiments with DETA/NO, this level had to be measured in control tubes containing no cells and subtracted as background. In experiments with GSNO or hydroxylamine, spontaneous conversion to nitrite was measured, and subtracted as background. M. tuberculosis did not produce nitrite from hydroxylamine under the conditions used here (data not shown).
Measurement of oxygen utilization.
Mid-exponential-phase cultures containing approximately 2·5x108 cells ml1 (OD580 0·4) were opened, vortexed to introduce air and pooled. Methylene blue was added to 0·0003 % and cultures were aliquoted to 8 ml screw-capped culture tubes. DETA/NO, GSNO, hydroxylamine, ethanol or water was added to samples in triplicate, which were then sealed with Parafilm and incubated at 37 °C. Decolourization of methylene blue indicating utilization of oxygen was monitored by the measurement of absorbance at 665 nm.
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RESULTS |
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To investigate the effect of oxygen on nitrate reduction, cultures of M. tuberculosis were grown either aerobically (AG) to mid-exponential phase or to microaerobic phase (NRP). Subsequently, chloramphenicol and nitrate were added, the cultures were aerated by vigorous vortexing, and the cell densities adjusted so that all cultures contained similar numbers of cells in the tested volumes. Both cultures were split into identical samples which were then incubated either aerobically or anaerobically. Oxyrase was used to establish anaerobiosis rapidly, and it permitted multiple readings from a single tube. Samples were withdrawn from each tube at 3 h intervals and nitrite levels measured. An AG culture incubated aerobically had a low rate of increase of 1 µM h1 (Fig. 1
), while one incubated anaerobically had greater activity, creating nitrite at the rate of 3 µM
h1. An NRP culture incubated anaerobically with Oxyrase showed a linear increase in nitrite concentration for at least 12 h at a rate of 75 µM
h1. In contrast, the NRP culture incubated aerobically showed low activity of 1 µM
h1. The rate was linear for at least 12 h, indicating no decrease in nitrate-reduction capacity during the course of the experiment. One set of NRP tubes was incubated anaerobically, and after 6 h the samples were opened and vortexed to aerate and inhibit Oxyrase activity. Incubation was then continued aerobically in loosely capped tubes. The rate of nitrite production decreased abruptly from anaerobic levels to aerobic levels (Fig. 1
). Another set of NRP cultures was initially incubated aerobically, and after 6 h Oxyrase was added and the tubes sealed, resulting in anaerobiosis. This set showed an increase in the rate of nitrite synthesis approaching that of other NRP samples that had been incubated anaerobically. Results were similar when rifampicin was used in place of chloramphenicol (data not shown). An aerobic culture incubated anaerobically but without the chloramphenicol used in the other samples showed an increasing rate of nitrite production at each time point.
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In aerobically grown cells incubated aerobically, the addition of Triton X-100 increased the rate of nitrite production approximately fourfold (Table 1). This supports previous work showing that diffusion of nitrate into the cell is the limiting factor in the reduction of nitrate in aerobic cultures (Sohaskey & Wayne, 2003
). NRP cultures incubated aerobically showed very low activity that was also increased threefold by Triton X-100, suggesting that increasing nitrate transport across the membrane partially relieved the inhibition caused by oxygen. An NRP culture incubated anaerobically showed decreased nitrite synthesis from 63 to 40 µM
h1 with the addition of Triton X-100, possibly due to increased diffusion of nitrate out of the cell.
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When 100 µM DETA/NO was added to chloramphenicol-treated AG cultures incubated both aerobically and anaerobically, only a slight inhibition of nitrite production was seen (Fig. 3A). The addition of 100 µM DETA/NO had little effect on an NRP culture incubated anaerobically. However, when NRP cultures were incubated aerobically with DETA/NO, there was an 18-fold increase in nitrite synthesis.
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To show that this increase in nitrite synthesis was due to NO, the NO-specific trap c-PTIO was used (Goldstein et al., 2004). Addition of c-PTIO at the same time as DETA/NO resulted in no change in the rate of nitrite production (Supplementary Fig. S1). When c-PTIO was added to an NRP culture previously treated with DETA/NO, the rate of nitrite production decreased immediately.
Effect of inhibition of the electron transport chain
Nitric oxide inhibits terminal oxidases (Clementi et al., 1999; Brunori et al., 2004
). The effect of inhibiting these oxidases by other means was determined. Either cyanide or hydroxylamine was added to chloramphenicol-treated AG and NRP cultures. Cyanide at a low level inhibited nitrate reduction in AG cultures. M. tuberculosis under these conditions does not express narK2, so this effect is probably due to inhibition of the nitrate reductase enzyme itself. Therefore no additional work was done with cyanide. Hydroxylamine at 5 mM inhibited growth of M. tuberculosis (data not shown), but did not interfere with nitrite production in AG cultures incubated either aerobically or anaerobically (Fig. 4A
). NRP cultures incubated anaerobically showed some decrease in nitrite synthesis. However, when incubated aerobically with hydroxylamine, these NRP cultures showed a 15-fold increase in the rate of nitrate reduction.
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The effect of Triton X-100 on the RVW3 strain was also tested. NRP cultures were used and the rate of nitrite production was 0·7±0·2 µM h1. This was boosted approximately 10-fold to 10·3±0·4 µM
h1 by the addition of Triton X-100. When incubated anaerobically the increase was twofold from 4·0±0·2 to 8·4±1·4 µM
h1. Therefore, the effect of Triton X-100 was independent of the presence of NarK2.
Effect of ethanol
Transcription of narK2 is regulated by the hypoxic regulator DosR/DevR. Ethanol induced the transcription of dosR/devR under aerobic conditions (Kendall et al., 2004). Therefore, the effect of ethanol on nitrite synthesis was determined. At 5 % (800 mM), the level shown to cause a strong induction of dosR/devR, there was little effect on nitrite production by an AG culture (Fig. 4B
). An NRP culture incubated anaerobically also showed only a slight inhibition by ethanol. The same NRP culture incubated aerobically produced an almost 40-fold increase in nitrite production (Fig. 4B
).
Inhibition of respiration
Although NO, hydroxylamine and ethanol can have many effects, one they have in common is the ability to inhibit respiration. To determine if the concentrations used here resulted in inhibition of respiration in M. tuberculosis, oxygen consumption was followed with the redox indicator methylene blue. Methylene blue was added to mid-exponential phase AG cultures which were incubated in sealed tubes. The presence of oxygen was monitored by decolourization of the methylene blue (Fig. 6). The untreated control tubes faded in 7 h, while treated samples remained blue.
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DISCUSSION |
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(1) Microaerobic NRP cultures had greater activity than aerobically growing ones. This difference has been previously described (Sohaskey & Wayne, 2003; Wayne & Hayes, 1999
). In the paradigm system, Escherichia coli, the large difference in activity between aerobic and anaerobic cultures is mainly due to differences in the level of the nitrate reductase enzyme NarGHJI. In contrast, in M. tuberculosis, there is no change in transcription of narGHJI in response to hypoxia. The genome of M. tuberculosis encodes four genes with homology to nitrate transporters (Cole et al., 1998
). Transcription of narK2 increases markedly in response to hypoxia and exposure to NO, while none of the other three are induced by these stimuli (Sohaskey & Wayne, 2003
; Voskuil et al., 2002
; Ohno et al., 2003
). Their role, if any, in nitrate reduction is unknown. The presence of NarK2 in NRP cultures, not the level of nitrate reductase enzyme, was responsible for the approximately 75-fold difference in the rate of nitrite synthesis between AG and NRP cultures (Fig. 1
) (Sohaskey & Wayne, 2003
). The narK2 mutant, RVW3, lacked the strong hypoxic induction of activity (Fig. 5
). Without chloramphenicol, the rate of nitrite synthesis increased with each time point, probably due to induction and synthesis of narK2 (Fig. 1
).
(2) Oxygen inhibited nitrate reduction in aerobic, AG cultures. An aerobic culture of M. tuberculosis showed an approximately fourfold increase in nitrite production when incubated anaerobically. Aerobically grown cells do not produce NarK2, indicating that this increase is independent of NarK2. As further evidence, an aerobic culture of RVW3 also showed a threefold increase in nitrite production when incubated anaerobically (Fig. 5). The increase in nitrite production could be due to a diversion of electrons from cytochrome oxidase. In both Paracoccus denitrificans and E. coli, competition for electrons between the pathways to oxygen or nitrate has been proposed to be a main influence on nitrate reductase activity (Kucera et al., 1996
; Denis et al., 1990
). Partial inhibition of the nitrate reductase enzyme could also play a role, although it appears to be resistant to oxygen (Sohaskey & Wayne, 2003
).
(3) Oxygen inhibited the activity of NarK2. An NRP culture that was exposed to air did not totally stop nitrate reduction, but continued at a level characteristic of aerobic cultures (Fig. 1). Increasing the diffusion of nitrate into the cell with Triton X-100, even in the narK2 mutant, increased nitrite production. These experiments suggest that NarK2 was inhibited in the presence of oxygen. Inhibition was relatively rapid and was not due to degradation of NarK2, as the effect could be reversed by the removal of oxygen from cultures containing chloramphenicol.
The nitrate reductase system does not appear to sense oxygen levels directly. In the presence of potassium ferricyanide, nitrite production was inhibited in NRP cultures incubated anaerobically (Fig. 2), suggesting that the redox state of some cell component, rather than molecular oxygen, is important.
Hypoxia results in an inactive cytochrome oxidase. NO and hydroxylamine can also inactivate cytochrome oxidase (Clementi et al., 1999; Brunori et al., 2004
; Kucera & Skladal, 1990
; Kucera et al., 1981
). Ethanol can inhibit the NADH dehydrogenase (complex I) (Kayser et al., 2003
). All of these treatments resulted in decreased respiration and an active NarK2 protein at oxygen concentrations that were previously inhibitory. Aerobic cultures did not show this increase in activity, suggesting the involvement of NarK2. This is supported by similar results with RVW3.
Inactivation of cytochrome oxidase either by lack of oxygen, or by the presence of NO or hydroxylamine, would increase the level of reduced components of the electron transport chain. A decreased electron flow may be part of the signal required for NarK2 activity. As a membrane protein, NarK2 is ideally located to sense components of the electron transport chain. A possible candidate is the quinone pool of M. tuberculosis. Quinone/quinol are non-protein components of electron transport and freely diffusible in the membrane. This pool could serve, not only as an electron source for the nitrate reductase enzyme, but also as an indirect indicator of oxygen levels. In P. denitrificans, it has been proposed that transport of nitrate is regulated by the redox level of the ubiquinone pool (Alefounder et al., 1983).
Regulation of nitrate transport by oxygen has been documented in other bacteria (John, 1977; Noji & Taniguchi, 1987
; Hernandez & Rowe, 1988
). It is intriguing that M. tuberculosis, classified as an obligate aerobe, should have such intricate control of an anaerobic enzyme system. Transcription of narK2 is controlled by DosR/DevR, which responds to hypoxia and NO (Ohno et al., 2003
; Voskuil et al., 2002
). Both the transcription and the activity of NarK2 are controlled by similar signals in what appears to be a redundant system, the purpose of which is not entirely clear.
The nitrate reductase system of M. tuberculosis may be important in vivo. The narGHJI operon is important for the survival of Mycobacterium bovis BCG in SCID mice (Weber et al., 2000). A narG knockout mutant of M. bovis BCG shows decreased virulence in these mice in comparison to the wild-type strain. The mutant M. bovis BCG produces smaller granulomas with fewer bacteria. In immunocompetent mice, the mutant shows decreased ability to persist in the lungs, liver and kidneys, but no decrease in the spleen (Fritz et al., 2002
). A role for nitrate reductase in maintaining ATP levels in M. tuberculosis during hypoxia has been demonstrated from work with pyrazinamide (Wade & Zhang, 2004
; Zhang et al., 2003
).
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ACKNOWLEDGEMENTS |
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Received 13 June 2005;
revised 29 July 2005;
accepted 6 August 2005.
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