1887

Abstract

Detailed molecular analysis of the ATP/ADP translocase, an obligate exchange transport system that is specific for ATP and ADP, has been extremely difficult due to limited quantities of material available from these obligate intracytoplasmic bacteria and by the toxicity and poor expression in recombinant expression systems. In this study, a stable and controllable system for the increased expression of the rickettsial ATP/ADP translocase was developed in where the expression of translocase from the bacteriophage T7 promoter in the pET11a vector led to a 26-fold increase in ATP transport activity and a 34-fold increase in translocase protein as compared to the expression with the native rickettsial promoter in When compared to ATP transport activity was increased sixfold and membrane translocase was increased threefold. Approximately 24% of the translocase protein produced was localized in an inclusion body fraction. This expression system was then used to determine whether the two cysteine residues in the ATP/ADP translocase were essential for activity or expression. The translocase was modified by oligonucleotide-directed site-specific mutagenesis such that the two cysteines were converted to alanines. The ATP transport properties and ATP/ADP translocase production kinetics, translocase protein concentration and subcellular localization were indistinguishable in the wild-type and mutant strains, proving that cysteines play no functional role in the ATP/ADP translocase and providing a system suitable for cysteine-scanning mutagenesis.

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/content/journal/micro/10.1099/00221287-143-11-3661
1997-11-01
2024-03-28
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References

  1. Atkinson W.H., Winkler H.H. 1985; Transport of AMP by Rickettsia prowazekii.. J Bacteriol 161:32–38
    [Google Scholar]
  2. Atkinson W.H., Winkler H.H. 1989; Permeability of Rickettsia prowazekii to NAD.. J Bacteriol 171:761–766
    [Google Scholar]
  3. Bachmann B.J. 1990; Linkage map of Escherichia coli K-12, edition 8.. Microbiol Rev 54:130–197
    [Google Scholar]
  4. Dasch G.A., Weiss E. 1977; Characterization of the Madrid E strain of Rickettsia prowazekii purified by renografin density gradient centrifugation.. Infect lmtnun 15:280–286
    [Google Scholar]
  5. Hanson B.A., Wisseman C.L. Jr Waddell A., Silverman D.J. 1981; Some characteristics of heavy and light bands of Rickettsia prowazekii on Renografin gradients.. Infect lmmun 34:596–604
    [Google Scholar]
  6. Henderson P.J.F., Baldwin S.A., Cairns M.T.C., Charalambous B., Dent H.C., Gunn F.G., Liang W.-J., Lucas V.A., Martin G.E.M., McDonald T.P. 1992; Sugar-cation symport systems in bacteria.. Int Rev Cytol 137:149–208
    [Google Scholar]
  7. Krause D.C., Winkler H.H., Wood D.O. 1985; Cloning and expression of the Rickettsia prowazekii ADP/ATP translocator in Escherichia coli.. Proc Natl Acad Sci USA 82:3015–3019
    [Google Scholar]
  8. Nishimura A., Morita M., Nishimura Y., Sugino Y. 1990; Arapid and highly efficient method for preparation of competent E. coli cells.. Nucleic Acids Res 18:6169
    [Google Scholar]
  9. Plano G.V., Winkler H.H. 1989; Solubilization and reconstitution of the Rickettsia prowazekii ATP/ADP translocase.. J Membr Biol 110:227–233
    [Google Scholar]
  10. Plano G.V., Winkler H.H. 1991; Identification and initial topological analysis of the Rickettsia prowazekii ATP/ADP translocase.. J Bacteriol 173:3389–3396
    [Google Scholar]
  11. 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]
  12. Sanger F., Nicklen S., Coulson A.R. 1977; DNA sequencing with chain-terminating inhibitors.. Proc Natl Acad Sci USA 74:5463–5467
    [Google Scholar]
  13. Smith D.K., Winkler H.H. 1977; Characterization of a lysine- specific active transport system in Rickettsia prowazekii.. J Bacteriol 129:1349–1355
    [Google Scholar]
  14. Williamson L.R., Plano G.V., Winkler H.H., Krause D.C., Wood D.O. 1989; Nucleotide sequence of the Rickettsia prowazekii ATP/ADP translocase-encoding gene.. Gene 80:269–278
    [Google Scholar]
  15. Winkler H.H. 1976; Rickettsial permeability: an ADP-ATP transport system.. J Biol Chem 251:389–396
    [Google Scholar]
  16. Winkler H.H. 1984; Potassium permeability of Rickettsia prowazekii.. J Bacteriol 157:197–201
    [Google Scholar]
  17. Winkler H.H., Daugherty R.M. 1984; Proline transport and metabolism in Rickettsia prowazekii.. J Bacteriol 158:460–463
    [Google Scholar]
  18. Winkler H.H., Daugherty R.M. 1986; Acquisition of glucose by Rickettsia prowazekii through the nucleotide intermediate uridine 5’-diphosphoglucose.. J Bacteriol 167:805–808
    [Google Scholar]
  19. Wu J., Kaback H.R. 1996; A general method for determining helix packing in membrane proteins in situ-, helices I and II are close to helix VII in the lactose permease of Escherichia coli.. Proc Natl Acad Sci USA 93:14498–14502
    [Google Scholar]
  20. Yanisch-Perron C, Vieira J., Messing J. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors.. Gene 33:103–119
    [Google Scholar]
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