These results were validated using an approximate likelihood ratio test in PhyML . The phylogenetic tree of OMPLA conflicts with that
of AtpA, indicating multiple HGT events. The species found outside of their expected clusters might have adapted quickly to environmental changes as a result of HGT events, which accelerate the rate of adaption . This is illustrated in the epsilon INK1197 order cluster; three of the four non-epsilon bacteria in that clade colonize humans either as pathogenic bacteria or as part of the intestinal microbiota A-1155463 order (see Figure 3 and Additional file 2: Table S3 for details). Conclusions The pldA gene in Helicobacter pylori has high nucleotide sequence identity due to purifying selection at the vast majority of residues. The result is a conserved H. pylori protein that likely has an evolutionarily stable function, although some probable interaction sites are subject to positive selection. Although HGT was detected by codon bias, GC content, and phylogenetic analysis, the biogeography of the pldA sequences indicated that the transfer was ancient. The protein structure of H. pylori OMPLA will yield a better understanding
of the positively selected sites, which may be surface-exposed regions. Our analyses indicated that pldA may be a niche-adapted protein; it was horizontally acquired, is highly conserved, but positive selection occurs at sites needed for possible pathogenic interactions. Methods Helicobacter pylori sample collection and pldA sequencing The pldA gene of 227 H. pylori isolates was sequenced. The samples included 207 Norwegian and Sepantronium nmr 20 Korean isolates. The Norwegian samples consisted of a total of 155 isolates from the Farnesyltransferase Sørreisa study  and 52 isolates collected from four hospitals in the Oslo region. Among these isolates, 40 had been previously described . The Oslo isolates included samples with known foreign origins; four isolates with Indo-European
origins, two with Asian origins, and one with an African origin. DNA was isolated using BioRobot M48 and MagAttract DNA Mini M48 Kit (Qiagen Inc., Valencia, CA, USA). The pldA gene, including short parts of the up- and downstream genes, was amplified by polymerase chain reaction (PCR) with forward primer HP498/499-F (5’- ttatcgcgcctgtagtga -3’) and reverse primer HP499/500-R (5’- tatgatcgctggcatgga -3’) at an annealing temperature of 57°C. The 1068 base pair (bp) pldA-gene was sequenced using the ABI BigDye Terminators v 1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) with the PCR primers and the internal sequencing primers HP498/499-R (5’-ggttgatattggggtggta-3’), PLA-F (5’-tgtccaattcttggtatctc-3’), PLA-R (5’-atgcgataggtatagcctaag-3’) and HP499/500-F (5’-tatgatcgctggcatgga-3’). The sequencing products were analyzed with an ABI PRISM 3130 Genetic Analyzer (Applied Biosystems) and the sequences were aligned using Sequencher software (Gene Codes Corporation, Ann Arbor, MI, USA).