D’Auria et al. BMC Genomics 2010, 11:181 http://www.biomedcentral.com/1471-2164/11/181 Several features relating to the virulence of L. pneumo- phila are well known. For example, the mechanisms responsible for entry into the macrophages [10,11], the intracellular (host) trafficking of effectors [12] and the membrane-associated protein involved in virulence [9,13]. The data available disclose an almost complete physiology of this organism and its relationships with protozoa and human macrophages. An interesting ques- tion relating to L. pneumophila is its high rate of DNA exchange, not only within species and other closely- related bacteria, but also with eukaryotic organisms [14]. Comparative genomics can give clues about the extent of this process. Nowadays, the genome sequencing of strains belonging to the same species offers the possibi- lity of defining their pangenome, which helps in under- standing the evolutionary dynamics of microbial species. The pangenome comprises the core-genome, made up of the genes shared by all strains, and the accessory or dispensable genome compartment, consisting of the genes that are strain-specific or shared by only some of the strains [15]. Pangenome studies can disclose charac- teristics that are not easily perceptible using standard annotation analysis [16]. For example, pangenome stu- dies have facilitated identification of virulence factors or anti-drugs systems in Escherichia coli and Streptococcus agalactiae [17,18]. The dispensable genome compart- ment can provide evidence of lateral gene transfer events that have occurred during the evolutionary his- tory of a strain, probably offering additional evolutionary potential to the organism. In this work, we report the main genomic features of L. pneumophila strain Alcoy 2300/99 and compare it with the four previously sequenced strains. A detailed description of the L. pneumophila pangenome is pro- vided, and strain-specific features are catalogued in terms of “islands”. Several islands containing virulence factors were identified and, where possible, their evolu- tionary origins were also hypothesized. Although the strains are phylogenetically closely related, the pange- nomic approach allowed identification of distinctive Table 1 Main features of L. pneumophila genomes Page 2 of 13 features, such as anti-drug related islands, strain-specific transport or secretion systems, DNA transfer-related islands, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems, and integrated phage insertions. Results and Discussion General features of the L. pneumophila Alcoy (Lpa) genome A total of 36,974 and 215,350 sequences were obtained by the Sanger method (with an average length of 781 nt) and 454-technology (with an average length of 242 nt), respectively. The contigs were finally assembled in one continuous strand, with average consensus cover- age of 22.1 ×. The Lpa chromosome length was of 3,516,335 base pairs, and 3,197 open reading frames were identified. The GC content was 38% and 1,175 pre- dicted proteins were found to have unknown functions, representing 36.7% of all CDSs. Similar to Lpc and Lpg genomes, no plasmids were found in Lpa. Table 1 sum- marizes the main features of this genome compared to the other four L. pneumophila sequenced strains. Pangenome of L. pneumophila Figure 1 summarizes the results obtained from compar- ing the five complete genomes of pathogenic L. pneumo- phila strains in relation to the orthologs/accessory gene distribution. The pangenome consists of 2,957 CDS with a core of 1,979 genes (66.9%) and a dispensable genome of 978 CDSs (33.1%). A total of 342 genes were found to be specific to the Lpa (53), Lpc (48), Lpg (88), Lpl (64) and Lpp (98) genomes. It is worth mentioning that 287 out of these 342 accessory genes (83.9%) were hypothetical proteins. Lpa and Lpc are the strains that share most genes; 2,560 out of 3,196 in Lpa (80%) and 3,207 in Lpc (79.8%). This result is in agreement with the phyloge- netic tree obtained using MLST [9]. Compared with the remaining genomes, Lpa and Lpc share 2,208 (69.1%) and 2,181 (68%) genes with Lpl; 2,271 (71.1%) and 2,284 Features Accession Number Serotype Genome length (bp) Plasmid GC content (%) Coding genes (%) Ribosomal operons Islands bp (%) Average GC of islands (%) Alcoy (Lpa) CP001828 I 3,516,335 0 38.38 86 3 157,442 (4.48) 37.75 Corby (Lpc) NC_009494 I 3,576,470 0 38.48 86 3 217,089 (6.07) 37.43 Philadelphia (Lpg) NC_002942 I 3,397,754 0 38.27 88 3 180,555 (6.13) 36.01 Paris (Lpp) NC_006368 I 3,503,610 1 38.37 87 3 163,637 (4.67) 38.17 Lens (Lpl) NC_006369 I 3,345,687 1 38.42 86 3 180,986 (5.40) 37.66