Intracellular pH regulation by Mycobacterium smegmatis and Mycobacterium bovis BCG

Abstract

Mycobacteria are likely to encounter acidic pH in the environments they inhabit ; however intracellular pH homeostasis has not been investigated in these bacteria. In this study, Mycobacterium smegmatis and Mycobacterium bovis [Bacille Calmette-Gue! rin (BCG)] were used as examples of fast-and slowgrowing mycobacteria, respectively, to study biochemical and physiological responses to acidic pH. M. smegmatis and M. bovis BCG were able to grow at pH values of 45 and 50, respectively, suggesting the ability to regulate internal pH. Both species of mycobacteria maintained their internal pH between pH 61 and 72 when exposed to decreasing external pH and the maximum ∆pH observed was approximately 21 to 23 units for both bacteria. The ∆pH of M. smegmatis at external pH 50 was dissipated by protonophores (e.g. carbonyl cyanide m-chlorophenylhydrazone), ionophores (e.g. monensin and nigericin) and N,N'-dicyclohexylcarbodiimide (DCCD), an inhibitor of the proton-translocating F 1 F 0 -ATPase. These results demonstrate that permeability of the cytoplasmic membrane to protons and proton extrusion by the F 1 F 0 -ATPase plays a key role in maintaining internal pH near neutral. Correlations between measured internal pH and cell viability indicated that the lethal internal pH for both strains of mycobacteria was less than pH 60. Compounds that decreased internal pH caused a rapid decrease in cell survival at acidic pH, but not at neutral pH. These data indicate that both strains of mycobacteria exhibit intracellular pH homeostasis and this was crucial for the survival of these bacteria at acidic pH values.

The protonmotive force is required for maintaining ATP homeostasis and viability of hypoxic, nonreplicating Mycobacterium tuberculosis

“…3). To confirm this result, the ⌬⌿ was then measured with [ 3 H]tetraphenylphosphonium bromide (TPP ϩ ), as described (22)(23)(24). Results showed that ⌬⌿ in growing and nonreplicating hypoxic cells was identical ( Table 2), confirming that quiescent Mtb are able to maintain their membranes polarized to the same extent as growing cells.…”

“…The ⌬pH was measured by using [ 14 C]benzoic acid as a pH probe (23,24). We found a comparable ⌬pH in replicating and hypoxic nongrowing bacilli ( Table 2).…”

“…The ⌬⌿ was measured as described (22)(23)(24). Briefly, similar concentrations of aerobic and hypoxic Mtb cells (Ϸ1 ϫ 10 9 cfu/ml) were incubated with 10 M [ 3 H]TPP ϩ (380 mCi/mmol) for 30 min at 37°C.…”

“…The cells were centrifuged, and the radioactivity in pellet and supernatant was measured as described above. The internal pH was calculated as described (23,24 Genetic Deletion Mycobacteria. The nuoA-N gene was disrupted by allelic exchange method by using the pYUB854 plasmid (40).…”

Toxin-Antitoxin Systems of Mycobacterium smegmatis Are Essential for Cell Survival

“…The internal pH of cells was measured as described previously (36), with the following modifications. Samples of cultures were incubated with […”