Membrane topology of CadA homologous P-type ATPase of Helicobacter pylori as determined by expression of phoA fusions in Escherichia coli and the positive inside rule

The only experimental data available on the membrane topology of transition metal ATPases are from in vitro studies on two distinct P-type ATPases (Ca dA and CopA) of a gastric bacterium, Helicobacter pylori, both postulated t o contain eight transmembrane domains (H1 to H8). In this study, H. pylori CadA ATPase was subjected to analysis of membrane topology in vivo by expre ssion of ATPase-alkaline phosphatase (AP) hybrid proteins in Escherichia co li using a novel vector, pBADphoA. This vector contains an inducible arabin ose promoter and unique restriction sites for fusion of DNA fragments to ph oA. The phoA gene lacking sequences encoding its N-terminal signal peptide was linked to the C-terminal regions of the postulated five cytoplasmic and four periplasmic segments of the H. pylori pump. The results obtained by h eterologous expression of ATPase-AP hybrid proteins showed consistence with a model of eight transmembrane domains. They also demonstrated that the H. pylori ATPase sequences are well assembled in the cytoplasmic membrane of E. coli, a neutralophilic bacterium. Cloning and amino acid sequence analys is of the homologous ATPase of Helicobacter felis further verified the topo logical model for the H. pylori pump analyzed here, although the degree of amino acid sequence identity varied between the corresponding transmembrane segments, from 25% for H1 up to 100% for H6. It was found that the topolog y of ATPase follows the 'positive inside rule'. With respect to the bioener getic capacities of H. pylori, we discuss here the membrane potential as a possible factor directing insertion of ATPases in the cytoplasmic membrane of gastric bacteria. (C) 1999 Editions scientifiques et medicales Elsevier SAS.