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Bioenergetics and Transport |
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
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–Guérin (BCG)] were used as examples of fast- and slow-growing 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 4·5 and 5·0, respectively, suggesting the ability to regulate internal pH. Both species of mycobacteria maintained their internal pH between pH 6·1 and 7·2 when exposed to decreasing external pH and the maximum 
pH observed was approximately 2·1 to 2·3 units for both bacteria. The pH of M. smegmatis at external pH 5·0 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 F1F0-ATPase. These results demonstrate that permeability of the cytoplasmic membrane to protons and proton extrusion by the F1F0-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 6·0. 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.
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; ZpH, transmembrane proton gradient expressed in mV
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