D’Auria et al. BMC Genomics 2010, 11:181 http://www.biomedcentral.com/1471-2164/11/181 Conclusions The virulence and persistence of L. pneumophila are mainly due to specific mechanisms coded by part of its core genome that makes L. pneumophila able to infect, survive and replicate in macrophages [14,44-46]. Lpc has been described as one of the most virulent strains [47], while Lpp is responsible for sporadic cases but is frequently recognized worldwide [48]. Lpl was responsi- ble for important outbreaks in France during 2003-2004 with 86 registered cases resulting in 17 deaths [6]. Finally, although the Lpg strain was the first one isolated for which the genome sequence was defined, it turned out to be not so virulent as the other [49]. Comparative genomics of five strains isolated in dif- ferent parts of the world of L. pneumophila disclosed the presence of several HGT-related islands and an evident history of recombination events. Here, we reported a number of features connected to virulence that could have been exchanged, or acquired by the strains along with their evolution. The traces of these events are mainly part of the dispensable genome compartment. The islands encountered from the dispensable pangen- ome compartment of the five genomes revealed factors that can give additional virulence to each strain. Alcoy and Corby strains are those in which more islands have been found related to virulence and DNA transfer activ- ities. Multi-drug efflux systems have been found in Lpa, Lpc and Lpg, while stability systems have been found in Lpl and Lpp genomes (R1). Lpa and Lpc strains are probably potentially more resistant in the presence of heavy metals, due to an additional HelABC system in the TS1 island. Moreover, Lpa and Lpc seem to have acquired, before to their lineage split, the ability to be more successful in DNA transfer by the DT1ac and DT2ac systems. Interestingly, Alcoy strain also acquired a complete bacilysin system (PR5 island), probably by precedent phage contact after separation from the Corby lineage, which could represent an environmen- tally competitive advantage for this virulent strain. Lpa, Lpc the Lpg also carry an additional Mvin virulence fac- tor, although there is no experimental evidence of its activity (island PR1). Moreover Alcoy, as well as Lens and Paris, proved to carry phage resistance systems (CRISPR on C1al and C2p islands). Several additional specific features have also been reported, although their role could not be predicted. Finally, the data reported in this work show that the Alcoy strain possesses additional features, making it dif- ferent from other previously sequenced genomes, even with the most closely related Corby strain. This finding could be related to the recurrent and sometimes mortal outbreaks recorded in the Spanish town of Alcoy. Page 10 of 13 Methods Strains used in this work L. pneumophila strain Alcoy 2300/99 was isolated from sputum of a patient with Legionnaires’ disease (LD) and associated with the LD outbreak detected in Alcoy (Spain) in 1999. It belongs to the most predominant ser- ogroup 1 [8]. The same strain was further isolated in other successive LD outbreaks in 2000 and 2002. The publicly available genomes of L. pneumophila used for comparison were retrieved from GenBank database http://www.ncbi.nlm.nih.gov/Genbank/index.html. Abbreviations and accession numbers are reported in Table 1. DNA extraction, shotgun clone libraries and sequencing DNA from L. pneumophila Alcoy was extracted as described in D’Auria et al. (2008) [9]. Cloning and sequencing were carried out as follows: two libraries (inserts of 1-2 and 2-10 Kb) were generated by sonica- tion of genomic DNA, followed by cloning of the frag- ments using the TOPO XL PCR Cloning Kit (Invitrogen, #K4700-10). Plasmid DNA purification was done with a Montage Plasmid Miniprep96 kit (Millipore, #LSKP09624) on a MULTIPROBE II-Robot Liquid Handling System (Packard Bioscience). Sequencing reac- tions were mainly performed using the ABI PRISM Big- Dye Terminator v3.0 Ready Reactions Kit and resolved using the 3730 Xl Genetic Analyzer (Applied Biosys- tems). To complete the assembly we used 454 pyrose- quencing (Roche) performed on one half of a GS-FLX PicoTiter plate, obtaining a total of 52 Mb. The combi- nation of both sequencing methods allowed the genome to be defined in 4 contigs. Finally, inverse PCR was employed to fill the remaining gaps and close the genome. Genome assembly and annotation Base-calling of each Sanger read was carried out with the “Pregap4” interface from Staden Package [50]. All reads were then checked manually by the “Trev” pro- gram and the assembly of Sanger sequences was per- formed by Cap4 program, both from the Staden Package [50]. The 454 reads were assembled by the Newbler assembler http://www.roche-applied-science. com and then integrated with the previous Sanger assembly. Open reading frame predictions were carried out with the Glimmer3 program [51] assigning the “lpa” locus tag to each sequence. All CDSs were searched by BLAST [52] searches against the non-redundant Gen- Bank database, the Cluster of Orthologous Groups [53] and the Kyoto Encyclopedia of Genes and Genomes [54]. Annotation was then improved by homology searches against previously sequenced genomes of the