PMC:3091639 / 32625-35021 JSONTXT

{"project":"2_test","denotations":[{"id":"20646324-7984417-10032222","span":{"begin":1690,"end":1692},"obj":"7984417"},{"id":"20646324-17846036-10032223","span":{"begin":1986,"end":1988},"obj":"17846036"}],"text":"Methods\n\nChlamydia pneumoniae isolates\nThe complete genomic sequences of C. pneumoniae AR39 (GenBank accession number AE002161), CWL029 (GenBank accession number AE001363) TW183 (GenBank accession number AE009440), J138 (GenBank accession number BA000008) and LPCoLN (GenBank accession CP001713) were used in this study.\n\nStrategy for selection of genes used for comparisons\nThe selection strategy involved individual gene-by-gene comparisons of the complete koala LPCoLN genome. NCBI BLAST [61] searches using tblastx and tblastn were conducted in order to search for orthologs in the C. pneumoniae human genome and other organisms using an E-value cutoff of 1 × 10-4 with manual curation. This approach enabled us to identify regions of high SNP accumulation and to select target genes for comparison. These selected genes were grouped into five categories by their putative, predicted or hypothetical functions. The categories included: (1) C. pneumoniae-specific genes (with respect to the koala LPCoLN genome, n = 140); (2) genes with a prior demonstrated role in chlamydial biology and pathogenicity (n = 49); (3) genes encoding nucleotide salvage or amino acid biosynthesis proteins (n = 6); (4) extrachromosomal elements, including a plasmid (n = 9) and bacteriophage-related genes (n = 2) (Additional files 1 and 2). Selected genes were individually aligned in order to identify SNPs, indels and targets for strain-specific adaptations.\n\nAnalysis of nucleotide and amino acid sequences and phylogeny\nGene alignments were performed using the Clustal W program (MacVector 10.6 Genetics Computer Group, Madison, Wisconcin) to identify polymorphisms and indels (insertions/deletions) [62]. Conserved residues are outlined, similar amino acids are shaded in grey, mismatches are not shaded and dashes correspond to gaps in the sequence. A consensus line appears at the bottom of the alignment. Pmp sequences were subjected to multiple sequence alignment using ClustalW2 (EMBL-EBI) [63] and BioEdit Sequence Alignment Editor [64]. SNP position analysis for each group was performed using the Microsoft Excel program (Microsoft Corporation). Motif Scan [65] was used to identify motifs in a sequence.\nA phylogenetic tree was constructed by Neighbor-Joining, tie breaking = systematic, distance corrected by the Poisson method with gaps distributed proportionally and 1,000 bootstrap replicates."}