Analysis of F1F0-ATPase from Helicobacter pylori.
The adaptive mechanisms that permit Helicobacter species to survive within the gastric mucosa are not well understood. The proton-translocating F1F0-ATPase is an important enzyme for regulating intracellular pH or synthesizing ATP in many other enteric bacteria; therefore, we used degenerate primers derived from conserved bacterial F1F0-ATPase sequences to PCR amplify and clone the gene (atpD) encoding the H. pylori F1F0-ATPase beta subunit. The deduced amino acid sequences of the F1F0-ATPase beta subunits from H. pylori and Wolinella succinogenes were 85% identical (91% similar). To characterize a potential functional role of F1F0-ATPase in H. pylori, H. pylori or Escherichia coli cells were incubated for 60 min in buffered solutions at pH 7, 6, 5, or 4, with or without 100 microM N,N'-dicyclohexylcarbodiimide (DCCD), a specific inhibitor of F1F0-ATPase. At pH 5 and 4, there was no significant decrease in survival of H. pylori in the presence of DCCD compared to its absence, whereas incubation with DCCD at pH 7 and 6 significantly decreased H. pylori survival. E. coli survival was unaffected by DCCD at any pH value tested. We next disrupted the cloned beta-subunit sequence in E. coli by insertion of a kanamycin resistance cassette and sought to construct an isogenic F1F0-ATPase H. pylori mutant by natural transformation and allelic exchange. In multiple transformations of H. pylori cells grown at pH 6 or 7, no kanamycin-resistant F1F0 mutants were isolated, despite consistently successful mutagenesis of other H. pylori genes by using a similar approach and PCR experiments providing evidence for integration of the kanamycin resistance cassette into atpD. The sensitivity of H. pylori to DCCD at pH 7 and 6, and failure to recover F1F0 H. pylori mutants under similar conditions, suggests that the function of this enzyme is required for survival of H. pylori at these pHs.
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Department of Medicine, Vanderbilt University School of Medicine, and Department of Veterans Affairs Medical Center, Nashville, Tennessee 37232-2605, USA. mcgowacc@ctrvax.vanderbilt.edu
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D000251:Adenosine Triphosphatases / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002470:Cell Survival; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology; D016480:Helicobacter pylori / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007613:Kanamycin Resistance; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction
D004024:Dicyclohexylcarbodiimide; D000251:Adenosine Triphosphatases