Expression of functional Na⁺/H⁺ antiporters of Helicobacter pylori in antiporter‐deficient Echerichia coli mutants

Abstract

An open reading frame with a sequence homologous to Escherichia coli Na + /H + antiporter A (ENhaA) was found in the total genomic sequence of Helicobacter pylori, a pathogenic bacterium of gastric inflammation, and was named HNhaA. The primary sequences and the hydropathy profiles of ENhaA and HNhaA were very homologous except for one additional region found in HNhaA. This sequence has about 40 hydrophilic amino acid residues inserted at the position next to residue 235 of ENhaA which corresponds to residue 245 of HNhaA. HNhaA was expressed in E. coli mutants deficient in Na + /H + antiporters and complemented the salt-sensitive phenotype of the mutants. Membrane vesicles prepared from these transformants of HNhaA using mutants deficient in the antiporters had the antiporter activities. Surprisingly, the antiporter activity in the transformant membranes was high at acidic and neutral pH, while ENhaA did not function at these pHs. A hydrophilic region around residue 235 in ENhaA and the additional hydrophilic region of about 40 residues in the same region found in HNhaA might be responsible for this difference in activity by acting as putative pH sensors.z 1999 Federation of European Biochemical Societies.

The Monoclonal Antibody 1F6 Identifies a pH-dependent Conformational Change in the Hydrophilic NH2 Terminus of NhaA Na+/H+ Antiporter ofEscherichia coli

“…Various mutations in this loop change the pH profile of the antiporter toward acidic pH. Furthermore, recently an NhaA homolog was cloned from Helicobacter pylori (28). In contrast to the E. coli NhaA which is activated at alkaline pH, the H. pylori antiporter is active at acidic and neutral pH.…”

The Fourth Transmembrane Domain of the Helicobacter pylori Na+/H+ Antiporter NhaA Faces a Water-filled Channel Required for Ion Transport

“…We have previously shown that NhaA from Helicobacter pylori (HP NhaA) functions in E. coli where it exhibits a profile of pH sensing that is very different from that of EC NhaA (16). HP NhaA is constitutively active from weak acidic to alkaline pH (pH 6.0 -8.5) in contrast to EC NhaA, which functions at alkaline pH (pH 7.5-8.5), even though they have extensive homology at the primary sequence level.…”

“…Based on the hydrophilicity analyses of TM4 residues, we have proposed that residues between Ile-136 and Lys-154 are located within the hydrophobic intramembrane region (Fig. 1) (16). However, the precise location of TM4 residues within the membrane is not known.…”

“…Construction of Expression Plasmids-Construction of HP NhaA expression plasmids derived from pBR322 (pBR-HP) has been described previously (16). These plasmids express HP NhaA with a FLAG epitope tag at the 3Ј end of the coding region and under the control of the tet promoter.…”

“…The secondary structure model of HP NhaA predicted by hydropathy analysis is depicted (16). Putative transmembrane domains are indicated by numbered boxes.…”