Properties of the P-type ATPases encoded by the copAP operons of Helicobacter pylori and Helicobacter felis

Abstract

The cop operons of Helicobacter pylori and Helicobacter felis were cloned by gene library screening. Both operons contain open reading frames for a P-type ion pump (CopA) with homology to Cd2+ and Cu2+ ATPases and a putative ion binding protein (CopP), the latter representing a CopZ homolog of the copYZAB operon of Enterococcus hirae. The predicted CopA ATPases contained an N-terminal GMXCXXC ion binding motif and a membrane-associated CPC sequence. A synthetic N-terminal peptide of the H. pylori CopA ATPase bound to Cu2+ specifically, and gene disruption mutagenesis of CopA resulted in an enhanced growth sensitivity of H. pylori to Cu2+ but not to other divalent cations. As determined experimentally, H. pylori CopA contains four pairs of transmembrane segments (H1 to H8), with the ATP binding and phosphorylation domains lying between H6 and H7, as found for another putative transition metal pump of H. pylori (K. Melchers, T. Weitzenegger, A. Buhmann, W. Steinhilber, G. Sachs, and K. P. Schäfer, J. Biol. Chem. 271:446-457, 1996). The corresponding transmembrane segments of the H. felis CopA pump were identified by hydrophobicity analysis and via sequence similarity. To define functional domains, similarly oriented regions of the two enzymes were examined for sequence identity. Regions with high degrees of identity included the N-terminal Cu2+ binding domain, the regions of ATP binding and phosphorylation in the energy transduction domain, and a transport domain consisting of the last six transmembrane segments with conserved cysteines in H4, H6, and H7. The data suggest that H. pylori and H. felis employ conserved mechanisms of ATPase-dependent copper resistance.

FIG. 1

Fluorogram of a Southern blot of chromosomal H. pylori 69A DNA hybridized with various DNA oligonucleotides targeted to the phosphorylation signature sequences of P-type ATPases. The membranes containing HindIII-restricted DNA were hybridized with DIG-labeled primers I-405 (lane 1), I-406 (lane 2), I-407 (lane 3), I-408 (lane 4), and I-409 (lane 5), as described in Materials and Methods. Positive restriction fragments were detected by chemiluminescence. Molecular sizes are given in kilobase pairs.

FIG. 2

Organization of the copAP operons and flanking DNA cloned from H. pylori and H. felis. The figure shows the ORFs contained in plasmids pRH948 (H. pylori) and pHF8 (H. felis). The ORFs detected by translation of the DNA in the three possible frames (frames 1 to 3) are indicated by arrows. The ftsH gene and ORF5 of pRH948 are truncated (open arrows), as is ORF6 in pHF8. The locations of putative transcriptional termination sequences are indicated by open circles. Terminal EcoRI (pHF8) and XhoI-EcoRI (pRH948) restriction sites of plasmid insertional DNA as well as a unique DraIII site present in the DNA insertion of pRH948 are also displayed.

FIG. 3

Comparison of the amino acid sequences of CopZ from Enterococcus hirae and the CopP peptides from Helicobacter pylori (Hp) strains and Helicobacter felis (Hf). The CopP sequences of 66 amino acid residues encoded by plasmids pRH948 (H. pylori 69A) (this study) and pBHpC8 (H. pylori UA802) (13) or detected by H. pylori genome sequencing (H. pylori 26695) (60) have an overall identity of >95%. The CopP peptide predicted from the small ORF of the copAP DNA fragment of H. felis, also consisting of 66 amino acids, showed a lower degree of identity with the H. pylori peptides (about 60%), but a stretch of 10 identical amino acids is observed just after the CXXC motif. The degree of amino acid sequence identity of CopP peptides and E. hirae CopZ (69 amino acid residues) is still between 40 and 50%.

FIG. 4

Comparison of the CopA amino acid sequences of H. pylori (HP) and H. felis (HF). The CopA amino acid sequences are 741 (HP) or 732 (HF) amino acid residues in length. The overall level of sequence identity between the two pumps is about 55%. TM segments of H. pylori CopA, determined experimentally in this study, are boxed and lightly shaded (TM1 to TM8). Also in boxes are the putative membrane-spanning segments of the H. felis pump. More darkly highlighted are conserved sequence boxes: the putative N-terminal Gly-Met-Thr-Cys-Thr/Ser-Ala-Cys metal ion binding motif, the membrane-associated Cys-Pro-Cys sequence, the Asp-Lys-Thr-Gly-Thr-Leu-Thr phosphorylation sequence, and a Gly-Asp-Gly-Leu/Val-Asn-Asp-Ala-Pro motif for ATP binding.

FIG. 5

Ion binding affinity of a synthetic ATPase peptide (P95-030) as determined by metal ion affinity chromatography. A 100-μg portion of peptide P95-030 was bound to a divalent-cation column as described in Materials and Methods. Bound peptide was eluted and separated by SDS-PAGE, using 15% acrylamide and Tricine buffer under nonreducing conditions, and stained with Serva Blue G. Lane 1, 5 μg of peptide P95-030 (control); lanes 3 to 8, peptide eluted from the matrix after binding to divalent ions and contained in 1 ml of the 3-ml elution volume. The column matrix was equilibrated with CuCl₂ (lane 3), NiCl₂ (lane 4), CoCl₂ (lane 5), CdCl₂ (lane 6), MgCl₂ (lane 7), or ZnCl₂ (lane 8). The peptide was able to form dimers, presumably due to the formation of intermolecular Cys-Cys bonds (lanes 1, 3, and 8).

FIG. 6

Electrospray mass spectra of synthetic ATPase peptide (amino acids 1 to 52) in the absence and presence of Cu²⁺ and Ni²⁺. (A) ESI spectra of unmodified Cop ATPase peptide. Three, four, and fivefold positively charged ions of the peptide were detected. (B) After preincubation with CuCl₂, 4.5-fold positively charged adducts of copper with the peptide were observed. (C) When the peptide was preincubated with NiCl₂, complexes of the peptide with nickel seemed to be detectable also, as indicated by the very faint peaks immediately following the [M + 4H]⁴⁺ signal. Binding to Ni²⁺, therefore, was much less significant than binding to Cu²⁺ (C). M, peptide molecule; H, proton; m, mass; z, charge of molecule.

FIG. 7

Kyte-Doolittle hydropathy profiles of H. pylori (A) and H. felis (B) CopA ATPases. (A) The putative TM segments determined by in vitro translation, H1 through H8, are highlighted. Also marked are hydrophobic regions HX and HY, which do not have membrane insertion activity. The localization of the conserved phosphorylation sequence is also shown (P site). (B) The membrane spanning segments of the H. felis pump are highlighted based on similarity to the CopA P-type ATPase sequence of H. pylori. As found for the H. pylori pump, the P site is between H6 and H7 in the CopA ATPase of H. felis.

FIG. 8

Autoradiograph of an SDS-PAGE gel with products of in vitro transcription-translation, in the absence and presence of microsomes (Mic), of the HK-M0 and HK-M1 vectors containing the first hydrophobic domain (lanes 1 to 4) and the second hydrophobic domain (lanes 5 to 8) of the H. pylori CopA ATPase.

FIG. 9

Autoradiograph of an SDS-PAGE gel with products of in vitro transcription-translation, in the absence and presence of microsomes (Mic), of the HK-M0 and HK-M1 vectors containing the third hydrophobic domain (lanes 1 to 4) and the fourth hydrophobic domain (lanes 5 to 8) of the H. pylori CopA ATPase.

FIG. 10

Autoradiograph of an SDS-PAGE gel with products of in vitro transcription-translation, in the absence and presence of microsomes (Mic), of the HK-M0 and HK-M1 vectors containing the HX hydrophobic domain (lanes 1 to 4), the fifth hydrophobic domain (lanes 5 to 8), and the sixth hydrophobic domain (lanes 9 to 12) of the H. pylori CopA ATPase.

FIG. 11

Autoradiograph of an SDS-PAGE gel with products of in vitro transcription-translation, in the absence and presence of microsomes (Mic), of the HK-M0 and HK-M1 vectors containing hydrophobic regions HY (lanes 1 to 4), H7 (lanes 5 to 8), and H8 (lanes 9 to 12) of the H. pylori CopA ATPase.

FIG. 12

Autoradiograph of an SDS-PAGE gel with products of in vitro transcription-translation, in the absence and presence of microsomes (Mic), of the HK-M0 vector containing regions encompassing HY to H7 (lanes 1 and 2) and HY to H8 (lanes 3 and 4) of the H. pylori CopA ATPase.