D’Auria et al. BMC Genomics 2010, 11:181 http://www.biomedcentral.com/1471-2164/11/181 Philadelphia, Lens, Paris and Corby strains (see Table 1 for genome accession numbers). Ribosomal genes were identified by BLAST searches against “nt” databases. tRNAs were identified by the tRNAscanSE software [55]. tRNA genes with anticodon CAT (tRNAIle, tRNAMet and tRNAfMet) were identified by the method described by Silva and collaborators [56]. Comparative analyses CDSs from each genome were considered orthologous when reciprocal BLAST best hits gave at least 70% of overlap with a minimum of 80% similarity. A catalogue of orthologs was compiled. GenomeViz2 software was employed to draw genome plots [57]. Several Perl scripts were compiled in our laboratory for massive data handling (available upon request). To define the coverage of the L. pneumophila pangen- ome, rarefaction curves were calculated from pools of CDS from each genome. In ecology, rarefaction is a technique applied in order to standardize and compare species richness computed from samples of different size [58]. Here, it is applied to compare gene cluster richness among multiple genomes from the same spe- cies. The L. pneumophila pangenome was then com- pared with pangenomes from strains belonging to E. coli (8 genomes), Streptococcus pyogenes (8 genomes), Sta- phylococcus aureus (9 genomes), Streptococcus agalac- tiae (8 genomes)(accession numbers are reported in Additional file 6). For each genome BLASTCLUST soft- ware was used to define gene clusters (70% similarity and 70% overlap). Gene abundance within each cluster was used to calculate rarefaction curves by the Rarefact- Win.exe http://www.uga.edu/~strata/software/Software. html program. A comparative analysis among the five strains has been carried out using the Mauve, multiple genome alignment software [59]. Pangenome distribution All CDSs from each genome were pooled together and clustered by CD-HIT-EST software with at least 70% of overlapping and a minimum of 80% similarity [60]. One gene from each cluster was characterized by RPSBLAST best match (e-values lower than 10-15) against the COG database (Cluster of Orthologous Groups, [53]). Determination of specific islands Discontinuity of the homology (synteny) between CDSs from a given genome and its ortholog in every compari- son were considered to define an island. Generally, islands were defined when more than 5 consecutives CDSs were found to be specific for one strain. Syntenic Alcoy/Corby orthologous genes which did not match in Page 11 of 13 the other three genomes were also considered islands. Islands were named according to their proposed func- tion. A lowercase letter was added to the end of the name referring to the genome to which it belonged and letters were chosen according to the official locus tag definition (“a” for Alcoy, “c” for Corby, “g” for Philadel- phia, “l” for Lens and “p” for Paris; e.g. “TS1a": Trans- port/Secretion island number 1 from the Alcoy genome). Due to the fact that the original annotations of Lpc, Lpg, Lpl and Lpp genomes often report CDSs as “hypothetical protein”, similarity searches of genes within these islands were carried out against an updated Refseq (GenBank) database http://www.ncbi.nlm.nih. gov/RefSeq/. Additional file 1: Presence/absence of islands among the five considered genomes. The presence or absence of a given island (column headers) is reported for every genome (line). Orange island is common to all strain, blue islands are strain-specific islands, red islands are common to Lpa and Lpc genomes, the green island is present in Lpp, Lpg and Lpl but not in Lpa or Lpc genomes. For the other is not possible to make a clear hypothesis about their origin. Additional file 2: CRISPR associated gene trees. Rooted trees obtained by neighbor joining method applying Kimura distance. In bold the Legionella pneumophila str. Alcoy sequence. Relative sequences represent best hits from GenBank protein refseq database. Additional file 3: CRISPR repeats structure in Alcoy genome. Rooted trees obtained by neighbor joining method applying Kimura distance. In bold the Legionella pneumophila str. Alcoy sequence. Relative sequences represent best hits from GenBank protein Refseq database. Additional file 4: Mvin gene trees. Rooted trees obtained by neighbor joining method applying Kimura distance. In bold the Legionella pneumophila str. Alcoy sequence. Relative sequences represent best hits from GenBank protein Refseq database. Additional file 5: Bacilysin containing island from Alcoy genome. Bacilysin cluster begins with an ORF homolog of a phospho-2-dehydro-3- deoxyheptonate aldolase, which is an intermediate of the synthesis of chorismate (lpa03410); the next four ORFs are homologs of bacilysin biosynthesis BacA (lpa03409), BacB (lpa03408), BacC (lpa03407) and BacC (lpa03406); lpa03405 is a transporter of the multidrug/metabolite exporter family; lpa0304 is a purine metabolism-related protein. lpa03404 and lpa3403 are related to chorismate mutase and the subsequent amino-transferase could be related to the final steps in bacilysin biosynthesis [61]. Located further along the island, lpa03412 is a homolog of an S24-like peptidase, followed by two hypothetical proteins and a bacteriocin adenylyltransferase (lpa03416). The PR5 island ends with an IS10-related transposase, lpa03419, two hypothetical proteins and a phage related integrase (lpa03425). This island demonstrates evidence of cluster acting in bacilysin-like bacteriocin production that is specific to the Alcoy genome. Additional file 6: Genomes used for rarefaction analysis. For each organisms strain names and GenBank accession numbers are reported. Acknowledgements This work has been funded by contract with Consellería de Sanidad of Valencian Government to AL and AM, and by grants BFU2009-12895-CO2-01 and SAF2009-13032-CO2-01 from Ministerio de Ciencia e Innovación (MICINN) to AL and AM, respectively. NJH is recipient of a fellowship from Carlos III and GD has a research contract from CIBERESP. Sanger sequencing was carried out using facilities of the SCSIE from University of Valencia.