The Helicobacter pylori UreI protein is not involved in urease activity but is essential for bacterial survival in vivo

Abstract

We produced defined isogenic Helicobacter pylori ureI mutants to investigate the function of UreI, the product of one of the genes of the urease cluster. The insertion of a cat cassette had a strong polar effect on the expression of the downstream urease genes, resulting in very weak urease activity. Urease activity, measured in vitro, was normal in a strain in which ureI was almost completely deleted and replaced with a nonpolar cassette. In contrast to previous reports, we thus found that the product of ureI was not necessary for the synthesis of active urease. Experiments with the mouse-adapted H. pylori SS1 strain carrying the nonpolar ureI deletion showed that UreI is essential for H. pylori survival in vivo and/or colonization of the mouse stomach. The replacement of ureI with the nonpolar cassette strongly reduced H. pylori survival in acidic conditions (1-h incubation in phosphate-buffered saline solution at pH 2.2) in the presence of 10 mM urea. UreI is predicted to be an integral membrane protein and may therefore be involved in a transport process essential for H. pylori survival in vivo.

FIG. 1

The urease gene cluster of H. pylori parental strains N6 and SS1 and of the derived mutants deficient in UreI, strains N6-823, N6-834, and SS1-834. The genes are indicated by boxes with arrows showing the direction of their transcription. The distances between the ure genes are given in base pairs. The sites hybridizing to the primers used to confirm correct allelic exchange in strains N6-823, N6-834, and SS1-834 are shown. Blank boxes represent the cassettes containing the genes conferring resistance to CM (cat) or to KM (aphA-3). The urease activities of these strains are given on the right side of the figure. Urease activity was measured at pH7 as the release of ammonia in crude extracts of bacteria grown for 48 h on blood agar plates as described previously (8). One unit corresponds to the amount of enzyme required to hydrolyze 1 μmol of urea/min/mg of total protein. The data are means ± standard deviations calculated from three to five determinations.

FIG. 2

Restriction map of pILL823, pILL824, pILL833, pILL834, and pILL845. Small boxes mark the vector of each plasmid; large boxes correspond to genes. Ori indicates the position of the ColE1 origin of replication. repA is the gene coding for the RepA protein, which is necessary for autonomous replication of pHel2 in H. pylori. Sp, Ap, and Cm indicate the genes conferring resistance to spectinomycin, ampicillin, and CM, respectively. The sequence of the DNA region comprising the ureI stop codon and the ureE start codon, including the BclI site where adapter H19 was inserted, is given below pILL824, which was obtained by the insertion of a 1.8-kb HpaI-HindIII fragment from pILL753 (8) into pBR322. Plasmid pILL825 was produced by the insertion of the H19 adapter (carrying an RBS and ATG in frame with ureE) (Table 1) into the BclI site of pILL824; the resulting ureI-ureE intergenic sequence is also shown. The stop codon of ureI and the start codon of ureE are boxed, and the RBS is underlined. In pILL833, the BamHI fragment of pILL825 was replaced by a 1.3-kb blunt-ended PvuII-BamHI fragment from pILL753. Plasmid pILL834 was obtained by replacement of the HpaI-BglII fragment of pILL833 with an 850-bp blunt-ended EcoRI-BamHI fragment of pUC18K2 containing the nonpolar KM cassette (18). Parentheses indicate the position of restriction sites removed by ligation.

FIG. 3

Alignment of the amino acid sequence of UreI from H. pylori with those of similar proteins and prediction of the two-dimensional structure of members of the protein family comprising UreI and AmiS. Residues identical at one position in at least four sequences are boxed; dashes indicate gaps inserted to optimize alignment. The organisms from which the sequences originated and the degree of identity of each with the H. pylori UreI protein are as follows: UreI-Hp, H. pylori (195 residues; accession no., M84338); UreI-Hf, H. felis; 74% identity over 196 residues (accession no., A41012); UreI-Lacto, L. fermentum; 55% identity over the 46-residue partial sequence (accession no., D10605); UreI-Strepto, S. salivarius; 54% identity over the 129-residue partial sequence (accession no., U35248); AmiS-Myco, M. smegmatis; 39% identity over 172 residues (accession no., X57175); AmiS-Rhod, Rhodococcus sp. strain R312; 37% identity over 172 residues (accession no., Z46523); and AmiS-Pseudo, P. aeruginosa; 37% identity over 171 residues (accession no., X77161). Predicted transmembrane α-helices are shown as shaded boxes. The regions separating these boxes are hydrophilic loops labeled IN when they are predicted to be intracellular and OUT when they are predicted to be extracellular.