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.
Mycobacterium smegmatis is able to grow and survive at acidic pH, and exhibits intracellular pH homeostasis under these conditions. In this study, the authors have identified low proton permeability of the cytoplasmic membrane, and high cytoplasmic buffering capacity, as determinants of intrinsic acid resistance of M. smegmatis. To identify genes encoding proteins involved in protecting cells from acid stress, a screening method was developed using the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). CCCP was used to suppress intrinsic acid resistance of M. smegmatis. The screen involved exposing cells to pH 5·0 in the presence of CCCP, and survivors were rescued at various time intervals on solid medium at pH 7·5. Cells capable of responding to intracellular acidification (due to CCCP-induced proton equilibration) will survive longer under these conditions than acid-sensitive cells. From a total pool of 5000 transposon (Tn611) insertion mutants screened, eight acid-sensitive M. smegmatis mutants were isolated. These acid-sensitive mutants were unable to grow at pH 5·0 in the presence of 1–5 μM CCCP, a concentration not lethal to the wild-type strain mc2155. The DNA flanking the site of Tn611 was identified using marker rescue in Escherichia coli, and DNA sequencing to identify the disrupted locus. Acid-sensitive mutants of M. smegmatis were disrupted in genes involved in phosphonate/phosphite assimilation, methionine biosynthesis, the PPE multigene family, xenobiotic-response regulation and lipid biosynthesis. Several of the acid-sensitive mutants were also defective in stationary-phase survival, suggesting that overlapping stress protection systems exist in M. smegmatis.
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