1887

Abstract

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 . To identify genes encoding proteins involved in protecting cells from acid stress, a screening method was developed using the electrogenic protonophore carbonyl cyanide -chlorophenylhydrazone (CCCP). CCCP was used to suppress intrinsic acid resistance of . 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 (Tn) insertion mutants screened, eight acid-sensitive 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 mc155. The DNA flanking the site of Tn was identified using marker rescue in , and DNA sequencing to identify the disrupted locus. Acid-sensitive mutants of 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 .

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2005-03-01
2024-03-29
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References

  1. Cole S. T., Brosch R., Parkhill J. & 39 other authors; 1998; Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544 [CrossRef]
    [Google Scholar]
  2. Cook G. M., Keis S., Morgan H. W., von Ballmoos C., Matthey U., Kaim G., Dimroth P. 2003; Purification and biochemical characterization of the F1F0-ATP synthase from thermoalkaliphilicBacillus sp. strain TA2.A1. J Bacteriol 185:4442–4449 [CrossRef]
    [Google Scholar]
  3. Dannenberg A. M Jr. 1993; Immunopathogenesis of pulmonary tuberculosis. Hosp Pract Off Ed 28:51–58
    [Google Scholar]
  4. Elferink M. G., de Wit J. G., Driessen A. J., Konings W. N. 1994; Stability and proton-permeability of liposomes composed of archaeal tetraether lipids. Biochim Biophys Acta 1193247–254 [CrossRef]
    [Google Scholar]
  5. Fisher M. A., Plikaytis B. B., Shinnick T. M. 2002; Microarray analysis of the Mycobacterium tuberculosis transcriptional response to the acidic conditions found in phagosomes. J Bacteriol 184:4025–4032 [CrossRef]
    [Google Scholar]
  6. Foster J. W., Bearson B. 1994; Acid-sensitive mutants of Salmonella typhimurium identified through a dinitrophenol lethal screening strategy. J Bacteriol 176:2596–2602
    [Google Scholar]
  7. Foster J. W., Hall H. K. 1990; Adaptive acidification tolerance response of Salmonella typhimurium . J Bacteriol 172:771–778
    [Google Scholar]
  8. Foster J. W., Hall H. K. 1991; Inducible pH homeostasis and the acid tolerance response of Salmonella typhimurium . J Bacteriol 173:5129–5135
    [Google Scholar]
  9. Gage D. J., Neidhardt F. C. 1993a; Adaptation of Escherichia coli to the uncoupler of oxidative phosphorylation 2,4-dinitrophenol. J Bacteriol 175:7105–7108
    [Google Scholar]
  10. Gage D. J., Neidhardt F. C. 1993b; Modulation of the heat shock response by one-carbon metabolism in Escherichia coli . J Bacteriol 175:1961–1970
    [Google Scholar]
  11. Guilhot C., Otal I., Van Rompaey I., Martin C., Gicquel B. 1994; Efficient transposition in mycobacteria: construction of Mycobacterium smegmatis insertional mutant libraries. J Bacteriol 176:535–539
    [Google Scholar]
  12. Iivanainen E., Martikainen P. J., Vaananen P., Katila M. L. 1999; Environmental factors affecting the occurrence of mycobacteria in brook sediments. J Appl Microbiol 86:673–681 [CrossRef]
    [Google Scholar]
  13. Krulwich T. A., Agus R., Schneier M., Guffanti A. A. 1985; Buffering capacity of bacilli that grow at different pH ranges. J Bacteriol 162:768–772
    [Google Scholar]
  14. Krulwich T. A., Quirk P. G., Guffanti A. A. 1990; Uncoupler-resistant mutants of bacteria. Microbiol Rev 54:52–65
    [Google Scholar]
  15. Liu Y., Zhou J., Omelchenko M. V. & 12 other authors; 2003; Transcriptome dynamics of Deinococcus radiodurans recovering from ionizing radiation. Proc Natl Acad Sci U S A 100:4191–4196 [CrossRef]
    [Google Scholar]
  16. Maloney P. C. 1979; Membrane H+ conductance of Streptococcus lactis. J Bacteriol 140:197–205
    [Google Scholar]
  17. Manganelli R., Provvedi R., Rodrigue S., Beaucher J., Gaudreau L., Smith I. 2004; Sigma factors and global gene regulation in Mycobacterium tuberculosis . J Bacteriol 186:895–902 [CrossRef]
    [Google Scholar]
  18. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. 1978; A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem 87:206–210 [CrossRef]
    [Google Scholar]
  19. Martin C., Timm J., Rauzier J., Gomez-Lus R., Davies J., Gicquel B. 1990; Transposition of an antibiotic resistance element in mycobacteria. Nature 345:739–743 [CrossRef]
    [Google Scholar]
  20. O'Brien L. M., Gordon S. V., Roberts I. S., Andrew P. W. 1996; Response of Mycobacterium smegmatis to acid stress. FEMS Microbiol Lett 139:11–17 [CrossRef]
    [Google Scholar]
  21. Oh Y. K., Straubinger R. M. 1996; Intracellular fate of Mycobacterium avium: use of dual-label spectrofluorometry to investigate the influence of bacterial viability and opsonization on phagosomal pH and phagosome–lysosome interaction. Infect Immun 64:319–325
    [Google Scholar]
  22. Patterson J. H., McConville M. J., Haites R. E., Coppel R. L., Billman-Jacobe H. 2000; Identification of a methyltransferase from Mycobacterium smegmatis involved in glycopeptidolipid synthesis. J Biol Chem 275:24900–24906 [CrossRef]
    [Google Scholar]
  23. Piddington D. L., Kashkouli A., Buchmeier N. A. 2000; Growth of Mycobacterium tuberculosis in a defined medium is very restricted by acid pH and Mg2+ levels. Infect Immun 68:4518–4522 [CrossRef]
    [Google Scholar]
  24. Prasada Reddy T. L., Suryanarayana Murthy P., Venkitasubramanian T. A. 1975; Respiratory chains of Mycobacterium smegmatis. Indian J Biochem Biophys 12:255–259
    [Google Scholar]
  25. Quirk P. G., Guffanti A. A., Clejan S., Cheng J., Krulwich T. A. 1994; Isolation of Tn917 insertional mutants of Bacillus subtilis that are resistant to the protonophore carbonyl cyanide m-chlorophenylhydrazone. Biochim Biophys Acta 118627–34 [CrossRef]
    [Google Scholar]
  26. Rao M., Streur T. L., Aldwell F. E., Cook G. M. 2001; Intracellular pH regulation by Mycobacterium smegmatis and Mycobacterium bovis BCG. Microbiology 147:1017–1024
    [Google Scholar]
  27. Riebeling V., Thauer R. K., Jungermann K. 1975; The internal-alkaline pH gradient, sensitive to uncoupler and ATPase inhibitor, in growing Clostridium pasteurianum. Eur J Biochem 55:445–453 [CrossRef]
    [Google Scholar]
  28. Rius N., Loren J. G. 1998; Buffering capacity and membrane H+ conductance of neutrophilic and alkalophilic Gram-positive bacteria. Appl Environ Microbiol 64:1344–1349
    [Google Scholar]
  29. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  30. Saviola B., Woolwine S. C., Bishai W. R. 2003; Isolation of acid-inducible genes of Mycobacterium tuberculosis with the use of recombinase-based in vivo expression technology. Infect Immun 71:1379–1388 [CrossRef]
    [Google Scholar]
  31. Snapper S. B., Melton R. E., Mustafa S., Kieser T., Jacobs W. R. Jr 1990; Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol 4:1911–1919 [CrossRef]
    [Google Scholar]
  32. Stewart G. R., Wernisch L., Stabler R., Mangan J. A., Hinds J., Laing K. G., Young D. B., Butcher P. D. 2002; Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays. Microbiology 148:3129–3138
    [Google Scholar]
  33. Sturgill-Koszycki S., Schlesinger P. H., Chakraborty P. & 7 other authors; 1994; Lack of acidification in Mycobacterium phagosomes produced by exclusion of the vesicular proton-ATPase. Science 263:678–681 [CrossRef]
    [Google Scholar]
  34. Sung N., Collins M. T. 2003; Variation in resistance of Mycobacterium paratuberculosis to acid environments as a function of culture medium. Appl Environ Microbiol 69:6833–6840 [CrossRef]
    [Google Scholar]
  35. van de Vossenberg J. L., Ubbink-Kok T., Elferink M. G., Driessen A. J., Konings W. N. 1995; Ion permeability of the cytoplasmic membrane limits the maximum growth temperature of bacteria and archaea. Mol Microbiol 18:925–932 [CrossRef]
    [Google Scholar]
  36. van de Vossenberg J. L., Driessen A. J., da Costa M. S., Konings W. N. 1999; Homeostasis of the membrane proton permeability in Bacillus subtilis grown at different temperatures. Biochim Biophys Acta 141997–104 [CrossRef]
    [Google Scholar]
  37. Zhang Y., Scorpio A., Nikaido H., Sun Z. 1999; Role of acid pH and deficient efflux of pyrazinoic acid in unique susceptibility of Mycobacterium tuberculosis to pyrazinamide. J Bacteriol 181:2044–2049
    [Google Scholar]
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