Increased and controlled expression of the Rickettsia prowazekii ATP/ADP translocase and analysis of cysteine-less mutant translocase.
Detailed molecular analysis of the Rickettsia prowazekii 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 Escherichia coli expression systems. In this study, a stable and controllable system for the increased expression of the rickettsial ATP/ADP translocase was developed in E. coli 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 E. coli. When compared to R. prowazekii, 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 R. prowazekii ATP/ADP translocase and providing a system suitable for cysteine-scanning mutagenesis.
Dunbar
Sherry A
SA
Winkler
Herbert H
HH
Laboratory of Molecular Biology, Department of Microbiology and Immunology, University of South Alabama College of Medicine, Mobile, AL 36688, USA.
D000255:Adenosine Triphosphate / Q000378:metabolism; D017123:Bacteriophage T7 / Q000235:genetics; D001692:Biological Transport; D003545:Cysteine / Q000502:physiology*; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D000226:Mitochondrial ADP, ATP Translocases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins; D019163:Reducing Agents / Q000494:pharmacology; D012283:Rickettsia prowazekii / Q000201:enzymology*
D011993:Recombinant Fusion Proteins; D019163:Reducing Agents; D008623:Mercaptoethanol; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases; D003545:Cysteine; D004229:Dithiothreitol