Unrooted tree based on the amino acid sequences of: <i>Helicobacter</i> spp. γGTs, <i>Campylobacter jejuni</i> γGTs and Bgh1 and Bgh2 homologues of <i>Helicobacter trogontum</i> and <i>Helicobacter bilis</i>.

<p>The evolutionary history was inferred using the Minimum Evolution method, and the evolutionary distances were computed using the Dayhoff matrix-based method. Bars indicate amino acid substitutions per position. Numbers at the nodes indicate support for the internal branches within the tree obtained by bootstrap analysis (≥70%; percentages of 500 bootstraps). (A) Phylogeny of almost complete pro-enzyme sequences (447 AA). (B) Phylogeny of almost complete N-terminal γGT sequences (Heavy chain; 333 AA). (C) Phylogeny of the almost complete C-terminal γGT sequences (Light chain; 190 AA).</p

Inhibitory effect on T-cell proliferation (Jurkats) by <i>Helicobacter bilis</i> γGT (Hb-γGT).

<p>Error bars in the graphs were calculated as SEM. The analysis was performed using Prism4 v4.03 (GraphPad Software). Different letters on the bars indicate significant differences at P<0.05. (A) Inhibitory effect with various amounts of recombinant Hb-γGT (Bgh2) protein; statistical analysis was performed using one-way ANOVA, followed by the Bonferroni test. (B) Inhibitory effect of the culture supernatant of <i>H. bilis</i> wild-type CCUG 23435 (WT) and <i>H. bilis</i> Δ<i>ggt</i> MR9 (Δ<i>ggt</i>) with or without pre-treatment with Acivicin (Sigma-Aldrich); statistical analysis was performed using unpaired <i>t</i>-test.</p

Unrooted tree based on complete amino acid sequences of different bacterial γGTs, <i>Helicobacter bilis</i> Bgh1 and Bgh2, and <i>Pseudomonas</i> Cephalosporin Acylase (CA).

<p>The evolutionary history was inferred using the Minimum Evolution method and the evolutionary distances were computed using the Dayhoff matrix-based method. Bar indicates amino acid substitutions per position. Numbers at the nodes indicate support for the internal branches within the tree obtained by bootstrap analysis (≥70%; percentages of 500 bootstraps).</p

Comparison of the kinetic constants for <i>Helicobacter bilis</i> γGT (Hb-γGT) at different pH.

<p>Error bars in the graphs were calculated as SEM. The analysis was performed using Prism4 v4.03 (GraphPad Software, San Diego, CA USA). At indicated pH values, kinetic constants (k_cat in black and KM in grey) for the hydrolysis of L-γ-glutamyl-p-nitroanilide (gGpNA) were determined.</p

Inhibitory effect on AGS proliferation by <i>Helicobacter bilis</i> γGT (Hb-γGT).

<p>Error bars in the graphs were calculated as SEM. The analysis was performed using Prism4 v4.03 (GraphPad Software). Statistical analysis was performed using one-way ANOVA, followed by the Bonferroni test. Different letters on the bars indicate significant differences at P<0.05.</p

Multi-alignment of amino acid sequences of different bacterial γGTs, <i>Helicobacter bilis</i> Bgh1 and Bgh2, and class IV Cephalosporin Acylase (CA).

<p>Multi-alignment between residues 379 and 477 (<i>Helicobacter pylori</i> γGT numeration) is shown. Sequences of γGTs of <i>H. pylori</i> (26695; HP1118), <i>H. acinonychis</i> (Sheeba; Hac_0598), <i>H. bizzozeronii</i> (CIII-1;HBZC1_08080), <i>H. salomonis</i> (O6A; EMBL FR821684), <i>H. suis</i> (HS1; HSUHS1_0265), <i>H. felis</i> (ATCC 49179; Hfelis_06880), <i>H. mustelae</i> (12198; HMU08020), <i>Esherichia coli</i> (K12; Swiss-Prot P18956), <i>Campylobacter jejuni</i> (81–176; CJJ81176_0067), <i>Pseudomonas</i> sp. (A14; Swiss-Prot P36267), <i>Bacillus subtilis</i> (168; Swiss-Prot P54422), <i>Arcobacter nitrofigilis</i> (DSM 7299; Arnit_0203), <i>Arcobacter butzleri</i> (RM4018; Abu_0961), <i>H. bilis</i> (Bgh1 = HRAG_01341; Bgh2 = HRAG_01828) as well as class IV CA of <i>Pseudomonas</i> sp. (SE82; Swiss-Prot P15557) are shown. Residues completely conserved among the sequences are indicated with a black background. The catalytic dyad is highlighted with a red box, and the conserved motif GXXGGXXI is enclosed in a black box. The Lid loop consists of the residues G428 to G438 of <i>H. pylori</i> γGT and is indicated in grey. Residues involved in the substrate recognition and the catalytic centre are highlighted in grey. Amino acid substitutions in Bgh1 potentially involved in functional change are indicated by arrows below the sequences.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

<i>Helicobacter</i> species used in this study.

<p><i>Helicobacter</i> species used in this study.</p

Purity and autoprocessing of recombinant Bgh1 and Bgh2.

<p>(A) SDS-page of the purified proteins after gel filtration: Bgh1 (Lane 1) and fully autoprocessed Bgh2 (Lane 2); (B) time-line for autoprocessing of Bgh1.</p

Comparison of the kinetic constants for <i>Helicobacter bilis</i> γGT (Hb-γGT) and <i>Helicobacter pylori</i> γGT (Hp-γGT).

<p>Comparison of the kinetic constants for <i>Helicobacter bilis</i> γGT (Hb-γGT) and <i>Helicobacter pylori</i> γGT (Hp-γGT).</p