INTRODUCTION

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M. tuberculosis grows within the phagocytic vacuoles of macrophages (2), where it is exposed to a relatively hostile environment. The interior of the phagosome may be limited in nutrients (11, 12) as well as acidic in pH. Although M. tuberculosis limits acidification of phagosomes by exclusion of the vesicular proton ATPase (19, 20), the vacuole is moderately acidic with the pH measured at values between 6.1 and 6.5 (14, 17, 19, 20).

An acidic environment is one of the most stressful conditions encountered by living cells. In order for enzymes and proteins to function normally, bacteria must maintain an internal pH close to pH 7 (4). The importance of maintaining a constant internal pH is underscored by the multiple systems utilized by bacteria during growth under acidic conditions. These include transport systems which exchange protons for cations, systems which transport protons out of cells in association with ATP hydrolysis, and the production of cytoplasmic macromolecules which function as internal buffers (5, 10). Although mycobacterial species have been reported to grow under acidic conditions in a complex media (7, 18), their response to pH stress in a defined medium has not been examined. The stress induced by acidic pH is known to be more severe in a nutrient-limited environment (10). In addition, nothing is known about specific factors which may facilitate the growth of mycobacteria under acidic conditions.

During our study of M. tuberculosis genes that are required for growth within macrophages, we noticed an association between bacterial growth in acidic medium and the requirement for Mg²⁺. Our experiments demonstrated that an M. tuberculosis mutant in the mgtC gene was attenuated for growth in media with reduced Mg²⁺ levels but only when the pH of the media was mildly acidic (6). The mgtC mutant of M. tuberculosis was also attenuated for growth in human macrophages and for growth in vivo in the lungs and spleens of mice. This suggested that during infection of the host M. tuberculosis grows in a moderately acidic environment with limiting levels of Mg²⁺. We therefore wanted to study the relationship between the growth of mycobacteria, including virulent M. tuberculosis, in acidic media and relate this to the requirement for Mg²⁺ or other divalent cations. In this report we demonstrate that M. tuberculosis is very restricted for growth by acid pH by using a defined medium and that any growth in moderately acidic media requires elevated levels of Mg²⁺. The requirement for Mg²⁺ during growth in acidic medium could not be replaced with the divalent cations Ca²⁺, Mn²⁺, or Zn²⁺. We also observed a great diversity among eight mycobacterial species in their ability to grow in media with acidic pH and low levels of Mg²⁺, with M. tuberculosis being among the most restricted for growth under these conditions.

	MATERIALS AND METHODS

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Bacterial strains. The bacterial strains used in this study include M. tuberculosis Erdman (ATCC 35801), M. smegmatis (ATCC 607), M. chelonae subsp. chelonae (ATCC 35752), M. fortuitum subsp. peregrinum (ATCC 14467), M. kansasii (clinical isolate), M. scrofulaceum (clinical isolate), M. marinum (ATCC 927), and M. avium (clinical isolate). The bacteria were routinely grown in Middlebrook 7H9 supplemented with ADC and 0.05% Tween 80 (15).

Growth assays under variable pH levels and divalent cation concentrations. Growth of mycobacteria under conditions of variable pH and divalent cation levels was studied using a defined medium (Sauton medium) that was modified so that the pH and divalent cation concentrations could be varied. Sauton medium (1) was modified by omitting the magnesium sulfate, which was the sole Mg²⁺ source, and replacing the sulfate with 28 mM potassium sulfate. To limit clumping during growth, 0.05% Tween 80 was added along with 10% ADC (15). Media were buffered with either 100 mM morpholineethanesulfonic acid (MES) or 100 mM morpholinepropanesulfonic acid (MOPS) (16), adjusted to the appropriate pH, filter sterilized, and then supplemented with various levels of magnesium chloride, calcium chloride, manganese chloride, or zinc chloride. For each assay, bacteria were grown to an optical density at 580 nm (OD₅₈₀) of 0.3 to 0.5 in Middlebrook 7H9, washed once in saline, and diluted 1/4,000 in 10 ml of the appropriate modified Sauton medium. Cultures were incubated at 37°C with 5% CO₂ for 3 to 4 weeks. The assays were terminated before bacterial clumping was evident. Growth was monitored by measuring the OD₅₈₀. Data are expressed as the mean and standard error of the mean from triplicate flasks.

	RESULTS

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The growth of M. tuberculosis is very restricted by acidic pH. A modified Sauton medium was used to evaluate the influence of pH and divalent cation concentration on the growth of mycobacteria. The Sauton medium was chosen because it is a defined medium in which divalent cation levels can be readily manipulated. To modify the Sauton medium, the sole divalent cation source (magnesium sulfate) was omitted and was replaced by potassium sulfate (as the sulfate source). Various levels of magnesium chloride, calcium chloride, zinc chloride, or manganese chloride were then added. In addition, all media were buffered with either 100 mM MES or 100 mM MOPS (16) to prevent the pH from changing during the course of the experiment.

View larger version (26K): [in this window] [in a new window]   FIG. 1.   M. tuberculosis requires elevated levels of Mg2+ for growth in acidic media. Growth of M. tuberculosis in modified Sauton media buffered to the indicated pH and containing various levels of Mg2+ is shown. Growth was measured at day 24 by measuring the OD580. Data are expressed as the mean and standard error of the mean from three cultures.

M. tuberculosis requires elevated levels of Mg²⁺ to grow in acidic media. We went on to examine the requirement for Mg²⁺ during growth of M. tuberculosis in both neutral and acidic media. At neutral pH (pH 7.0), the growth rate of M. tuberculosis was similar in cultures with low (10 and 20 µM) and moderate (100 µM) levels of Mg²⁺. In mildly acidic media (pH 6.5), M. tuberculosis grew significantly better in the cultures containing higher levels (100 µM) of Mg²⁺ (OD₅₈₀ 0.315) compared to medium with 10 µM Mg²⁺ (OD₅₈₀ 0.203). In the more acidic media the requirement for Mg²⁺ became more pronounced. At pH 6.25, there was fourfold more growth in the culture with 100 µM Mg²⁺ compared to the culture with 10 µM Mg²⁺. The culture with 20 µM Mg²⁺ produced intermediate levels of growth. At pH 6.0, the only measurable growth of M. tuberculosis was in the culture containing 100 µM Mg²⁺. Thus, M. tuberculosis requires higher levels of Mg²⁺ for growth at acidic pH compared to growth at neutral pH.

Other divalent cations cannot replace Mg²⁺ for growth at acid pH. We next examined whether divalent cations other Mg²⁺ could facilitate the growth of M. tuberculosis in acidic media. Modified Sauton media containing 10 µM Mg²⁺ and buffered to either pH 6.25 or 7.0 served as the base media for these assays. We had previously determined that 10 µM Mg²⁺ was sufficient Mg²⁺ for growth of M. tuberculosis at pH 7.0 (Fig. 1). Aliquots of the base media were supplemented with 100 µM concentrations of the divalent cations Mg²⁺, Ca²⁺, Zn²⁺, or Mn²⁺. Growth of M. tuberculosis in the base medium at pH 6.25 or 7.0 was compared to growth in media supplemented with each of the divalent cations. The amount of growth attained at day 19 is presented in Fig. 2. At pH 6.25, the addition of Mg²⁺ (100 µM) increased the growth from an OD₅₈₀ of 0.126 in the base medium to an OD₅₈₀ of 0.200 with the Mg²⁺. The addition of Ca²⁺ (100 µM) to the culture produced a slight increase in growth compared to the base medium from OD₅₈₀ 0.126 to 0.137. However, growth in the Ca²⁺ culture was significantly less than the level attained with the addition of Mg²⁺ (P < 0.05). Zn²⁺ had no effect on growth, while 100 µM Mn²⁺ was inhibitory. At pH 7.0, the addition of Ca²⁺ enhanced the growth of M. tuberculosis to a level close to that with Mg²⁺. The addition of Zn²⁺ or Mn²⁺ was inhibitory at pH 7.0. Higher levels (1,000 µM) of Ca²⁺, Zn²⁺, or Mn²⁺ or lower (50 µM) levels of Zn²⁺ or Mn²⁺ were unable to replace Mg²⁺ during growth at pH 6.25 (data not shown). Thus, M. tuberculosis specifically requires Mg²⁺ for maximum growth at pH 6.25. 

View larger version (31K): [in this window] [in a new window]   FIG. 2.   Other divalent cations cannot replace Mg2+ during growth of M. tuberculosis at pH 6.25. Growth of M. tuberculosis in modified Sauton media buffered to the indicated pH and supplemented with 100 µM Mg2+, Ca2+, Mn2+, or Zn2+ is shown. Growth was measured at day 19. Data are expressed as the mean and standard error of the mean from three cultures. *, P < 0.05 compared to growth in the Mg2+ supplemented culture.

The limited growth of M. tuberculosis at pH 6.0 is unique among eight species of mycobacteria. We went on to compare the growth rate of M. tuberculosis with mycobacteria that may encounter acidic conditions in soil or aquatic environments. The mycobacteria were grown in media buffered to pH 6.0 or 7.0 and containing either 10 or 100 µM Mg²⁺. As we had previously observed, M. tuberculosis grew very poorly at pH 6.0 and required the higher level of Mg²⁺ for the limited growth that was observed (Fig. 3). Among eight mycobacterial species, M. tuberculosis was the most restricted for growth at pH 6.0 (Fig. 3 and 4). M. smegmatis and M. kansasii were also limited in growth in the pH 6.0 medium with low Mg²⁺ (10 µM) (Fig. 3). However, M. smegmatis and M. kansasii also failed to grow in the pH 7.0 medium with low Mg²⁺ (10 µM), indicating that they require higher concentrations of Mg²⁺ for maximum growth at neutral as well as acid pH.

View larger version (20K): [in this window] [in a new window]   FIG. 3.   M. tuberculosis, M. smegmatis, and M. kansasii require higher concentrations of Mg2+ for growth in acidic medium. Growth of mycobacterial species in modified Sauton media buffered to the indicated pH and containing 10 or 100 µM Mg2+ is shown. Data are expressed as the mean and standard error of the mean from three cultures.

View larger version (28K): [in this window] [in a new window]   FIG. 4.   Growth curves of mycobacteria which do not require higher concentrations of Mg2+ for growth in acidic medium. Growth of mycobacterial species in modified Sauton media buffered to the indicated pH and containing 10 or 100 µM Mg2+ is shown. Data are expressed as the mean and standard error of the mean from three cultures.

	DISCUSSION

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This study has demonstrated that in an environment which contains only simple nutrients, M. tuberculosis is very restricted in growth by acid pH and requires higher concentrations of Mg²⁺ for growth at a pH of 6.5 or lower. Using a defined medium (Sauton) containing moderate levels of Mg²⁺ (100 µM), the growth of M. tuberculosis was reduced by a pH of 6.25 and was almost completely absent at pH 6.0. The sensitivity of M. tuberculosis to a moderate acidic pH correlates with observations made with M. tuberculosis-infected macrophages. Mycobacteria containing phagosomes exhibit limited fusion with late endosomes and lysosomes. This results in the exclusion of the vacuolar ATPase from the phagosome membrane which limits acidification of the vacuole (8, 19). Nevertheless, the pH of mycobacteria containing vacuoles is mildly acidic and has been measured at pH values between 6.1 and 6.5 (14, 17, 19). Thus, the pH within the phagosome is slightly above the pH level (pH 6.0) which excluded growth of M. tuberculosis in the modified Sauton medium and is within the pH range that required higher levels of Mg²⁺ for growth. Gomes et al. recently suggested that the sensitivity of M. tuberculosis to acidic pH contributes to the control of infection (13). Using a coinfection with Coxiella burnetii, these authors demonstrated that M. tuberculosis could not grow in acidified vacuoles. On the other hand, M. avium grew in the acidified macrophage vacuoles, which coincides with our observation that M. avium grew well in the pH 6.0 defined medium. Thus, the limited growth of M. tuberculosis in defined medium with acid pH parallels the observation that M. tuberculosis fails to grow in acidified vacuoles of macrophages.

Growth of M. tuberculosis in acidic media was even more restricted when the level of Mg²⁺ was low. The requirement for Mg²⁺ during growth in acidic media was specific for Mg²⁺ and could not be replaced by the divalent cations Ca²⁺, Mn²⁺, or Zn²⁺. The need for Mg²⁺ was observed in the presence of millimolar levels of K⁺ and Na⁺ which are normal components of the modified Sauton medium. K⁺ and Na⁺ transport systems are associated with an electroneutral exchange for protons which contributes to pH homeostasis in bacteria (5). We have identified one of the M. tuberculosis genes required for growth under low-Mg²⁺, low-pH conditions. An M. tuberculosis mutants in the mgtC gene failed to grow in mildly acidic medium (pH 6.25) with limiting levels of Mg²⁺ (20 µM) (6). Furthermore, the mgtC mutant was attenuated for growth in human macrophages as well as for virulence in mice. These data suggest that the virulence of M. tuberculosis is dependent upon the growth of the bacteria in a mildly acidic low-Mg²⁺ environment. The concentration of Mg²⁺ in phagosomes has not been measured directly; however, it has been estimated to be in the low micromolar range (12). Thus, a limitation in Mg²⁺ within the phagosome would intensify the sensitivity of M. tuberculosis to moderately acidic pH.

Among diverse mycobacterial species, there was a spectrum of tolerance to acid pH and low levels of Mg²⁺. M. tuberculosis was the most restricted for growth at pH 6.0. Nontuberculosis mycobacterial species, which may grow in soil or aquatic environments, were much more acid tolerant and in fact M. kansasii, M. scrofulaceum, M. avium, and M. chelonae grew better at pH 6.0 than at pH 7.0. Previous studies investigating the sensitivity of mycobacteria to acid pH have reported growth at a broad range of pH values (7, 18). These studies reported that the optimum growth of M. tuberculosis in Dubos medium was between pH 5.8 and pH 6.5, with growth observed at as low as pH 5.4 (18). The disparity in results between these studies and those presented here may be explained by the influence of the different media used since the sensitivity to extremes in pH can be masked in complex media (5). Dubos medium contains caseinate extract, while Sauton medium contains no protein extract. Our results suggest that in a environment containing simple nutrients, mycobacteria are more sensitive to acid.

The role of Mg²⁺ in the adaptation of M. tuberculosis to growth under moderately acidic conditions is unknown. Mg²⁺ may be participating directly in the maintenance of a neutral internal pH. The Mg²⁺-dependent proton-translocating ATP synthase is reported to play a role in acid tolerance in bacteria (3). Homologues of the ATP synthase genes are present in the M. tuberculosis genome (9). Conversely, Mg²⁺ may not be directly involved in maintenance of a neutral internal pH. Instead, the Mg²⁺ transport systems of M. tuberculosis may function less efficiently under acidic conditions, and the bacteria may therefore require more Mg²⁺ in the surrounding environment for adequate uptake. Alternatively, under acidic conditions, greater amounts of Mg²⁺ may be required to perform the normal cellular functions of Mg²⁺ such as stabilization of membranes and acting as cofactors for enzymes. Ongoing studies to identify the function of the MgtC protein in M. tuberculosis should provide additional information on mechanisms involving Mg²⁺ which facilitate growth of M. tuberculosis under acidic conditions.