Department of Microbiology, Otago School of Medical Sciences, University of Otago, PO Box 56, Dunedin, New Zealand1
Author for correspondence: Gregory M. Cook. Tel: +64 3 479 7722. Fax: +64 3 479 8540. e-mail: greg.cook{at}stonebow.otago.ac.nz
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ABSTRACT |
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Keywords: pH regulation, mycobacteria, membrane potential, proton ATPase
Abbreviations: CCCP, carbonyl cyanide m-chlorophenylhydrazone; DCCD, N,N'-dicyclohexylcarbodiimide; p, protonmotive force;
pH, transmembrane proton gradient expressed as pH units;
, membrane potential; TPP+, methyl triphenylphosphonium iodide; Z
pH, transmembrane proton gradient expressed in mV
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INTRODUCTION |
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Mycobacteria are likely to encounter both acidic and mildly acidic pH in the environments they inhabit (Sturgill-Koszycki et al., 1994 ; Oh & Straubinger, 1996
; Iivanainen et al., 1999
). For example, acidic conditions (pH 3·54·3) often prevail in soil and aquatic habitats where saprophytic mycobacteria are found (Iivanainen et al., 1999
). In the host environment, M. tuberculosis has been shown to reside in the phagocytic vacuole of host macrophages where the intraphagosomal pH has been shown to be mildly acidic (pH 6·16·5) (Sturgill-Koszycki et al., 1994
; Oh & Straubinger, 1996
). Reports in the 1960s demonstrated that M. tuberculosis H37Rv had a narrow pH range for growth between pH 6·2 and 7·3 with marked attenuation observed at pH 5·0 and 8·4 (Chapman & Bernard, 1962
). Other species of mycobacteria, for exampleMycobacterium fortuitum, Mycobacterium marinum, Mycobacterium scrofulaceum, Mycobacterium avium and Mycobacterium chelonae, grew at pH 6·0 in an unrestricted manner indicating that M. tuberculosis was unique among the mycobacteria in its extreme sensitivity to acid (Chapman & Bernard, 1962
). Portaels & Pattyn (1982)
reported that Mycobacterium smegmatis was capable of growth over a wide pH range with optimum growth observed between pH 5·0 and 7·4, and partial growth at pH 4·6. A more recent study by OBrien et al. (1996)
has demonstrated that exposure of M. smegmatis to sublethal, adaptive acidic pH (e.g. pH 5·0) conferred a significant level of protection against subsequent exposure to lethal pH (e.g. pH 3·0) compared to unadapted cells grown at pH 7·6, but no mechanism for this acid-tolerant response was provided. In contrast to other micro-organisms that exhibit an acid-tolerant response or acid habituation, the magnitude of protection was only a two- to threefold increase in cell viability for M. smegmatis compared to adapted cells of Salmonella typhimurium which are 100 to 1000 times more resistant to strong acid challenge (e.g. pH 3·3) compared to unadapted cells (Foster & Hall, 1990
).
Due to the paucity of basic information that exists on how mycobacteria cope with acidic pH and pH in general, we have studied the effect of external pH on intracellular pH homeostasis in M. smegmatis and Mycobacterium bovis BCG. The results reported in this communication demonstrate that both species of mycobacteria adopt intracellular pH homeostasis and this was essential for survival at acidic pH.
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METHODS |
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Growth and maintenance.
Cultures of M. smegmatis mc2155 (Snapper et al., 1990 ) and M. bovis BCG (Pasteur 1173P2) were used in this study. For liquid culture, cells were grown with gentle agitation at 37 °C in supplemented Middlebrook 7H9 broth (Difco Laboratories) containing sterile Middlebrook ADC enrichment (Becton Dickinson) and 0·05% Tween 80 (Sigma). For solid medium, supplemented Middlebrook 7H11 (1·5% agar) with OADC (Becton Dickinson) and glycerol (0·5%, v/v) was used. All cells used as inocula were washed in saline (0·85% NaCl). To acidify supplemented and non-supplemented 7H9 medium, the pH was adjusted with 2 M HCl. Culture optical density was measured with a Beckman DU-64 spectrophotometer at 600 nm (OD600) using culture samples diluted with saline to bring the OD600 to below 0·7 when measured in cells of 1 cm light path length.
Protonmotive force (p) measurements.
Mid-exponential phase cultures were harvested by centrifugation (8000 g, 15 min, 10 °C) and washed in 100 mM sodium citrate/phosphate buffer (pH 7·0). Cells were resuspended to a final OD600 of 1·0 in a volume of 2 ml (glass tubes). Where the external pH was varied (pH 4·0 to 7·0), initial experiments were carried out using non-supplemented 7H9 medium, but there was no detectable difference in pH values between using this medium and one of the following buffers; 100 mM sodium citrate/phosphate buffer, 100 mM potassium- and sodium-phosphate buffer at the external pH being studied. Citrate/phosphate buffer has been used extensively for measuring internal pH and acid survival in other bacteria (Baronofsky et al., 1984
; Terracciano & Kashket, 1986
; McGowan et al., 1998
) and this was the buffer routinely used in this study. Cells were energized with 20 mM glucose for 15 min followed by the addition of [3H]TPP+ (5 µM final concentration), [7-14C]benzoate (11 µM, pKa 4·2) or [14C]salicylic acid (10 µM, pKa 3·0) at pH values below 5·0. [1,2-3H]taurine (50 µM) and 3H2O (25 mM) were used to determine intracellular volume. Taurine has been shown to be non-metabolizable by M. tuberculosis (Zhang et al., 1999
) and this was confirmed for the mycobacteria used in this study. After incubation for 10 min at 37 °C, the cultures were centrifuged through 0·35 ml silicon oil (BDH Laboratory Supplies) in 1·5 ml microcentrifuge tubes (13000 g, 5 min, 22 °C) and 20 µl samples of supernatant were removed. The tubes and contents were frozen (-20 °C), and cell pellets removed with dog nail clippers. Supernatant and cell pellets were dissolved in scintillation fluid and counted. The silicon oil mix used in this study was a 40% mixture of phthalic acid bis(2-ethylhexyl ester) and 60% silicone oil (40% part mixture of DC200/200 silicone oil and 60% DC 550). Silicone oils were left overnight at room temperature to equilibrate.
The intracellular volume [3·45±0·59 µl (mg protein)-1] was estimated from the difference between the partitioning of 3H2O and [14C]taurine. The electrical potential across the cell membrane (membrane potential; ) was calculated from the uptake of [3H]TPP+ according to the Nernst relationship. Non-specific TPP+ binding was estimated from cells which had been treated with valinomycin and nigericin (10 µM each) for 25 min. These inhibitors have been used previously with M. smegmatis (De Rossi et al., 1998
; Choudhuri et al., 1999
). The
pH was determined from the distribution of [14C]benzoate or [14C]salicylic acid using the HendersonHasselbalch equation (Reibeling et al., 1975
) and Z
pH was calculated as 62 mVx
pH.
Determination of lethal internal pH for M. smegmatis and M. bovis BCG.
The determination of lethal internal pH for M. smegmatis and M. bovis BCG was as reported by Foster & Hall (1991) . Mycobacteria were grown to the exponential phase (OD600 0·51·0) in supplemented 7H9 broths (pH 7·0) and harvested by centrifugation (8000 g, 15 min, 10 °C). Cells were resuspended in non-supplemented 7H9 medium and 20 mM glucose at pH 5·0 or 5·5. Preliminary experiments indicated that 20 min exposure to 500 µM CCCP prior to the measurement of intracellular pH was required to reduce the internal pH from 6·7 to 5·5, but CCCP did not eliminate
pH completely. An exposure time of 4 h to 250 µM CCCP at pH 5·0 was required to cause a 68% decline in viability of M. smegmatis; this was the time chosen for cell-survival experiments. To reduce the level of CCCP associated with the cells prior to plating, cell suspensions were harvested by centrifugation (13000 g, 5 min, 22 °C) and washed twice in 1·5 ml sterile 0·9% NaCl. Cell viability (survival) was determined as the number of bacteria remaining as a percentage of the starting count. All samples were diluted in sterile saline (pH 7·0) and three 100 µl volumes of each dilution (10-410-6) were spread plated on 7H11 agar plates in duplicate. Cell viability, as measured by c.f.u., was determined after 23 d for M. smegmatis and 23 weeks for M. bovis BCG, or after colonies were visible at 37 °C. The minimum detection limit was 100 c.f.u. ml-1. The results are expressed as log percentage survival and represent the mean values of two independent experiments.
Measurement of oxygen consumption by washed cell suspensions, and other analyses.
For oxygen consumption measurements, cells were harvested from exponentially growing cultures (OD600 0·5) by centrifugation (8000 g, 15 min, 10 °C), washed in 100 mM sodium citrate/phosphate buffer (pH 7·0) and resuspended in the same buffer to give protein concentrations of 510 mg protein ml-1. Respiration rates were measured in a Rank Bros Clark-type oxygen electrode at 37 °C as described by Carneiro de Melo et al. (1996)
. Glucose (20 mM) was added as an oxidizable carbon source. The oxygen electrode was calibrated with air-saturated sodium citrate/phosphate buffer (220 nmol dissolved O2 ml-1 at 37 °C). Protein from NaOH-hydrolysed cells (0·2 M NaOH, 100 °C, 20 min) and cell membranes was assayed by the method of Markwell et al. (1978)
.
Preparation of bacterial cell membranes and ATPase assays.
Cell membranes of M. smegmatis were prepared as previously described (Basu et al., 1992 ). Membrane-bound ATPase activity was determined in triplicate by the colourimetric assay of inorganic phosphate liberated from ATP hydrolysis as described by Kobayashi & Anraku (1972)
. The incubation time and concentration of membrane protein was adjusted so that the assay was linear with time and less than 50% of the ATP hydrolysed. Non-enzymic degradation of ATP under these conditions was less than 10% of the total phosphate. ATPase activity was expressed as µmol inorganic phosphate liberated min-1 (mg protein)-1 at 37 °C.
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RESULTS |
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The use of radioactive probes to measure the total p requires corrections for non-specific binding of probes such as TPP+. Since the cell wall of mycobacteria is unlike that of conventional eubacteria, we first tested the effect of ionophores and protonophores on the growth of these bacteria. The following inhibitors at the concentrations listed completely arrested growth of exponentially growing cells of M. smegmatis at pH 7·0: CCCP, 100 µM; DCCD, 200 µM; monensin, 100 µM; nigericin/valinomycin, 10 µM each. At pH 5·0, the following concentrations were required to inhibit growth of M. smegmatis; CCCP, 50 µM; DCCD, 300 µM; monensin, 10 µM; nigericin/valinomycin, 10 µM each. These inhibitors were also effective against M. bovis BCG at similar concentrations (data not shown). These results show that the cell wall of M. smegmatis and M. bovis BCG did not pose a barrier to these compounds.
The effect of extracellular pH on intracellular pH regulation was studied over the pH range 4·0 to 7·0 (Fig. 2). The
of M. smegmatis was approximately -178 mV at pH 7·0 and decreased with declining pH (Fig. 2a
). As the
decreased, the total
p remained greater than -180 mV and there was an increase in the Z
pH from -8 mV at pH 7·0 to -155 mV at pH 4·0 (Fig. 2a
). These results indicated that M. smegmatis was interconverting
to Z
pH to maintain the
p constant. The internal pH as a function of the external pH is shown in Fig. 2b
. The maximum
pH (2·3 units) was observed at pH 4·5. Identical experiments were carried out with M. bovis BCG (Fig. 3
). As the external pH declined, the
decreased to approximately -50 mV and the Z
pH increased to -130 mV, but the
p did not remain constant (Fig. 3a
). The internal pH as a function of the external pH is shown in Fig. 3b
and the maximal
pH was 2·1 units observed at an external pH of 4·0.
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Effect of protonophores, ionophores and DCCD on intracellular pH regulation in M. smegmatis
To gain a better understanding of how mycobacteria regulate their internal pH, experiments were performed with M. smegmatis and known inhibitors of key proteins (e.g. proton-translocating F1F0-ATPase) and the generation of ionic gradients (e.g. H+, K+, Na+) across the cell membrane that have been implicated in pH homeostasis. The addition of either the protonophore CCCP or the ionophore nigericin (K+/H+ antiporter) in combination with valinomycin (K+ uniporter), or monensin alone (Na+/H+ antiporter) to cells at pH 5·0 almost completely dissipated the pH gradient (Table 1
). Valinomycin alone had no effect on the
pH gradient. When DCCD (1 mM), an inhibitor of the F1F0-ATPase, was added to cells at pH 5·0, the internal pH was 5·70 (Table 1
). At neutral pH, under all conditions tested, the internal pH was alkaline with respect to the external pH (7·287·60) in the presence of the inhibitors used (Table 1
).
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The F1F0-ATPase has been shown to be essential for the survival of Escherichia coli at external pH values below 4·0 (Bearson et al., 1997 ). Further experiments were conducted to determine the effect of DCCD on the survival of M. smegmatis at both acidic and neutral pH. M. smegmatis cells were incubated in sodium citrate/phosphate buffer at pH 3·0, 5·0 and 7·0 in the presence and absence of 1 mM DCCD, a concentration shown to lower the internal pH at external pH 5·0 (Table 1
). In the absence of DCCD, M. smegmatis survived at each of the pH values tested (Fig. 5
). The addition of 1 mM DCCD caused a 60% reduction in viable cell numbers of M. smegmatis at pH 7·0 and a 72% reduction at pH 5·0 (Fig. 5
). At an external pH of 3·0, DCCD caused a 99% decrease in cell viability. These results demonstrate that ATPase activity is required for survival of M. smegmatis at all pH values tested, but appears to be more essential for survival at low pH values (e.g. pH 3·0).
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DISCUSSION |
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The maintenance of intracellular pH near neutrality when faced with decreasing external pH requires changes in the ZpH that is a component of the
p. One mechanism bacteria employ to modify their Z
pH while maintaining
p constant is to make compensatory changes in
. This may be accomplished by the use of various cation transport systems (Booth, 1985
). For example, Enterococcus faecalis and E. coli are able to interconvert Z
pH for
by electrogenic K+ transport (Bakker & Mangerich, 1981
; Booth, 1985
). Results here indicated that M. smegmatis interconverted
for Z
pH to maintain the
p constant with declining external pH; the
p values were in good agreement with those published for other respiring neutrophiles (Kashket, 1985
). In contrast to M. smegmatis, the
p for M. bovis BCG varied over the pH range studied. The differences in
p were largely reflected in the
values that varied significantly between the two species. M. tuberculosis has been shown to express a multidrug efflux pump, Mmr, that confers resistance to TPP+, ethidium bromide and erythromycin (De Rossi et al., 1998
). Importantly, M. smegmatis does not contain the mmr gene and accumulates [3H]TPP+ with no efflux observed (De Rossi et al., 1998
). M. bovis has been shown to contain the mmr gene (De Rossi et al., 1998
) and therefore we cannot rule out the operation of a TPP+ efflux pump in M. bovis BCG; this may explain the low levels of TPP+ accumulation (e.g. low
) observed in this species and therefore the large fluctuations in the
p observed with external pH.
Studies with the protonophore CCCP, used to equilibrate internal pH with external pH, indicated that the lethal internal pH for mycobacteria was less than pH 6·0 and this was associated with a rapid decrease in cell survival and viability suggesting that in mycobacteria, acid death is related to internal pH rather than external pH. However, it should be pointed out that at the high concentrations of CCCP used, not only may H+ be moved but also other ions (e.g. internal K+) and therefore the effect on viability may not only be due to acidification of the cytoplasm. Foster & Hall (1991) showed that high concentrations of the protonophore dinitrophenol (200400 µM) were required to collapse the
pH of S. typhimurium. At an external pH of 4·0, non-growing cells of both mycobacteria had a significant
p (greater than -150 mV) and the internal pH was greater than pH 6·0, but neither species of mycobacteria could grow at this external pH. The reason why these bacteria are unable to grow under conditions where the
p and internal pH appear to be favourable for growth is unknown. Perhaps the act of maintaining a high internal pH under conditions of low growth rate and ATP generation decreases the amount of energy available for growth. The mycobacteria may stop growing at acidic pH in order to direct energy towards maintenance of their internal pH, which is crucial for their survival. Alternatively, there may be a pH-sensitive element (e.g. membrane-bound protein) that is essential for the growth of M. smegmatis and M. bovis BCG at acidic pH.
The basic mechanism(s) that mycobacteria use to cope with acidic pH is not known, but the pH gradient of M. smegmatis at pH 5·0 was dissipated by the protonophore CCCP and the ionophores monensin and nigericin (Pressman, 1976
), indicating that permeability of the cytoplasmic membrane to protons plays a key role in maintaining internal pH near neutral. Harold et al. (1970)
demonstrated that the generation and maintenance of the pH gradient of Ent. faecalis was energy dependent, and could be prevented by incubation with the F1F0-ATPase inhibitor DCCD. Furthermore, Ent. faecalis has been shown to increase the amount of F1F0-ATPase twofold when the internal pH is lowered artificially by gramicidin D or growth at low pH (Kobayashi et al., 1984
, 1986
). The ability of low pH to increase the amount and activity of proton ATPases has also been reported in other bacteria (Nannen & Hutkins, 1991
; Miwa et al., 1997
; Amachi et al., 1998
; Kullen et al., 1999
). In E. coli and Bacillus subtilis, environmental pH does not influence expression of the atp operon (Santana et al., 1994
; Kasimoglu et al., 1996
). In this study, DCCD caused a reduction in the
pH of M. smegmatis and there was an increase in the level of membrane-bound ATPase activity in cells that were grown at pH 5·0, implying a potential role for this enzyme in intracellular pH homeostasis. Piddington et al. (2000)
have demonstrated a role for Mg2+ in the adaptation of M. tuberculosis to mildly acidic growth conditions, but the role of Mg2+ was unknown. The authors hypothesized that Mg2+ may play a role in the maintenance of neutral pH, perhaps by influencing the Mg2+-dependent proton translocating ATP synthase. Inspection of the M. tuberculosis genome sequence reveals homologues of ATP synthase genes (e.g. atpBFEADHCG) (Cole et al., 1998
).
Datta & Benjamin (1997) have demonstrated that DCCD at 1 mM inhibits the survival of Listeria monocytogenes at pH 3·0 but had no effect on the survival of L. monocytogenes at pH 7·3, indicating that the effect of DCCD was due to low-pH-induced lethality. Our results demonstrated that M. smegmatis exhibited a striking sensitivity towards DCCD at acidic pH, suggesting that the ATPase may be more essential for survival at acidic pH. E. coli mutants defective in the F1F0-ATPase (e.g. atp::Tn10 or
atp) are extremely acid sensitive, but only to external pH values below 4·0. To test the hypothesis that the ATPase is essential for survival of M. smegmatis at low pH values and rule out potential non-specific effects of DCCD, further work is required using atp mutants of M. smegmatis.
In conclusion, our data demonstrate that both species of mycobacteria studied here adopt intracellular pH homeostasis to maintain their internal pH near neutral at acidic pH. The permeability of the membrane to protons and the activity of the membrane-bound proton F1F0-ATPase play important roles in this process. Current studies are aimed at determining the molecular responses of mycobacteria to pH stress.
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ACKNOWLEDGEMENTS |
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Received 20 September 2000;
revised 27 November 2000;
accepted 22 December 2000.