ANALYSIS OF F1F0-ATPASE FROM HELICOBACTER-PYLORI

The adaptive mechanisms that permit Helicobacter species to survive wi thin the gastric mucosa are not well understood. The proton-translocat ing F1F0-ATPase is an important enzyme for regulating intracellular pH or synthesizing ATP in many other enteric bacteria; therefore, we use d degenerate primers derived from conserved bacterial F1F0-ATPase sequ ences 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 8 5% identical (91% similar). To characterize a potential functional rol e of F,F,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 mu M N,N'-dicyclohexylcarbodiimide (DCCD), a specific i nhibitor of F1F0-ATPase. At pH 5 and 4, there was no significant decre ase in survival of H. pylori in the presence of DCCD compared to its a bsence, whereas incubation with DCCD at pH 7 and 6 significantly decre ased H. pylori survival. E. coli survival was unaffected by DCCD at an y pH value tested. We nest 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 trans formation and allelic exchange. In multiple transformations of H. pylo ri cells grown at pH 6 or 7, no kanamycin-resistant F1F0 mutants were isolated, despite consistently successful mutagenesis of other H. pylo ri genes by using a similar approach and PCR experiments providing evi dence for integration of the kanamycin resistance cassette into atpD. The sensitivity of H. pylori to DCCD at pH 7 and 6, and failure to rec over F1F0 H. pylori mutants under similar conditions, suggests that th e function of this enzyme is required for survival of H. pylori at the se pHs.