D’Auria et al. BMC Genomics 2010, 11:181 http://www.biomedcentral.com/1471-2164/11/181 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems (C) CRISPR are bacteriophage resistance systems [33] that have been identified in the Alcoy and Lens (C1), and in Paris (C2) strains (Figure 5, pink track). Lpl also pos- sesses one almost identical CRISPR system on the plas- mid. Phylogenetic analyses reveal how Alcoy CAS (CRISPR-associated genes) genes are more closely related to the ones on the plasmidic CRISPR of the Lens strain than to the chromosomal one. The CAS genes identified were Cas1 (lpl2837, plpl0052, lpa01472), Cas3-helicase (plpl0051, lpl2838, lpa01473), and lpa01474 and lpa01475, which are two conserved pro- teins in bacteria, but with no relatives in other Legio- nella genomes. lpa01476, plpl0047 and lpl2842 belong to the CRISPR-associated Csy4 family proteins (see Additional file 2). Downstream of this cluster of proteins begins the repeats of the CRISPR system (see Additional file 3). The Alcoy and Lens strain repeats are almost identical, except for one base (adenine in the Alcoy strain and cytosine in the Lens, GTT(A/C)ACTGCCG- CACAGGCAGCTTAGAA). Fifty-seven direct repeats were found in Lpa, while 53 in the Lpl plasmid, and two clusters of 52 and 12 on the Lpl chromosome. None of the Alcoy spacers were found to be similar to other spacers from the CRISPR database [34]. The Paris strain genome hosts a CRISPR system, located at the beginning of the DT3 island. The first Page 8 of 13 CRISPR-related protein is coded by lpp0160 and shows a weak similarity to a putative CRISPR-associated large protein. The cluster is followed by Cas1 (lpp0161), Cas2 (lpp0162), Cas4 (lpp0163) and 34 direct repeats. Inter- estingly, the repeats and the CRISPR-associated genes are not related to the ones found in the Lpa and Lpl genomes. Phage related islands (PR) Up to seven different phage-related islands have been found. The Alcoy and Corby strains share PR1 and PR3 whereas all the other PR islands are strain-specific. PR1 in Lpa and Lpc are almost syntenic and could be consid- ered an ancient infection that took place before the split of the two lineages; a related region is also present in Lpg genome. This island contains several phage-related pro- teins, several transposases belonging to the IS4 family, an MviN virulence factor (lpa01685, LPC_2173, lpg1087), as well as other not well-defined proteins containing DNA cleavage or binding related domains. The MvinN-related protein is additional, but not equivalent to the constitu- tive one present on the chromosomes (see Additional file 4) of the five strains (lpa0385, LPC_0506, lpg2635, lpl2560, and lpp2688). MvinN has been described as an important factor of virulence in Salmonella typhimurium and Burkholderia pseudomallei [35]. Although its role in pathogenicity is still not clear, this protein is a homolog of the proposed lipid II flippase protein [36,37] that has no virulence activity. Figure 5 Islands distribution on L. pneumophila MLST tree topology from D’Auria et al. (2008) [9]. The left side (a) shows the islands appearance according to the tree topology obtained by the phylogenetic analysis. Each color is related to island type (see legend). On the right side (b) is reported the multiple genome alignment obtained by Mauve software applying default parameters [59]: homolog blocks are drawn with identical colors in forward and reverse strands; diagonal lines connect homologous blocks from each genome; colored blocks (according to island-type) on the light pink area between strands indicate the position of each island.