234 ± 0.014 0.223 ± 0.024 0.234 ± 0.048 0.241 ± 0.021 0.240 ± 0.015 0.278 ± 0.027 0.263 ± 0.054 0.215 ± 0.020 Ka 0.035 ± 0.003 0.028 ± 0.004 0.088

± 0.015 0.030 ± 0.005 0.034 ± 0.003 0.039 ± 0.005 0.062 ± 0.014 0.027 ± 0.004 Ka/Ks 0.150 ± 0.017† 0.125 ± 0.024 0.374 ± 0.100 0.125 ± 0.022 0.142 ± 0.016† 0.139 ± 0.023 0.234 ± 0.072 0.127 ± 0.024 * Out-of-frame sequences were excluded. Mol., molecular No., number nt, nucleotides Ks, Synonymous substitutions Ka, Non-synonymous substitutions Trichostatin A † PZ-Test <0.001 for purifying selection hypothesis (Ka/Ks <1). &Value ± Standard Error. Bold print highlights the higher molecular distance, Ka and Ka/Ks observed for segment 2, compared to the entire gene and to segments 1

and 3. Selonsertib analysis of the similarity plot of the 124 nucleotide sequences of homB and homA genes showed the existence of three distinct regions in both genes, named segments 1, 2 and 3, corresponding to the 5, middle and 3′ regions of the genes, respectively LCZ696 (Fig. 3). The analysis performed independently on the three segments of each gene showed that segment 2 displayed the highest molecular distance as well as the highest Ka, even when compared to the entire gene (Table 1). These results were confirmed by the analysis of the nucleotide substitution rate over a sliding window, which also showed a significant increase in the Ka in segment 2 of homB gene. In fact, the mean Ka for this region (0.191 ± 0.059) was five fold higher than for next the rest of the gene (0.037 ± 0.023). The same result was observed for homA gene (data not shown). These observations reveal a higher level of diversity of segment 2 in both genes. Figure 3 Similarity plot representation of homB (black lines) and homA (grey lines) genes of various Helicobacter pylori strains. The plot

was generated by using 16 strains representative of each gene, with the Jukes-Cantor correction (1-parameter), a 200-bp window, a 20-bp step, without Gap Strip and the jhp870 gene sequence as reference (GenBank accession number NC_000921). The arrow delineates the region which discriminates between homB and homA genotypes. bp, base pair. A phylogenetic analysis on each gene segment of 24 strains carrying one copy of each gene was also performed. The phylogenetic reconstruction of segment 1 showed that homB presented the highest similarity between orthologous genes, i.e., each homB was closely related to the homB in the other strains (Fig. 4A). A similar result was obtained for homA gene (Fig. 4A). In contrast, for segment 3, each homB was strongly correlated with the corresponding homA present in the same strain, indicating similarity between paralogous genes (Fig. 4B). The mean molecular distance and mean synonymous and non-synonymous substitution rates were calculated for all possible pairs of paralogous and orthologous genes, within the same strain and between strains.