These results are consistent with what was previously shown for other mobile and integrative genetic elements as well as PAIs from E. coli, where excision occurs upon exposure to stress conditions such as sub-lethal UV-light irradiation [53, 55, 56]. Figure 2 Detection of VPI-2 excision by using real time quantitative PCR (QPCR) of attB levels in cell cultures grown under different conditions. The X-axis specifies culture conditions: 6 h, incubation time of 6 h; 24 h, incubation Tariquidar time of 24 h; 25C, incubation temperature of 25°C; 3%, the LB broth

contained 3% NaCl; M9+G, cell grown on AZD8931 minimal media supplemented with glucose; UV-light, bacterial cultures were UV-light irradiated. The Y-axis represent the ratio of the attB presence in the cultures tested compared with cultures grown on standard conditions 12 h at 37°C in LB. Unpaired t-test was used in order to infer statistical significance for the differences in VPI-2 excision. ***, p < 0.005. **, p < 0.05. Error bars indicate standard deviation. Each experiment was performed in triplicate a minimum of three times. VPI-2 encodes

two novel recombination directionality factors Both the high pathogenicity island HPI from Y. pestis and ICE SXT from V. cholerae encode small accessory proteins called recombination directionality factors (RDFs) or excisionases (Xis) that are required for efficient excision of these elements [29, 41]. In order to identify candidate RDFs within VPI-2 from V. cholerae N16961, we performed BLAST and PSI-BLAST searches GW3965 mw on the V. cholerae N16961 genome using RDFs, the V. cholerae Xis protein (ABA87014) from SXT, the Y. pestis Hef protein (NP_405464) from HPI and E. coli K12 AlpA protein (AAA18418) from λ phage as queries [57]. The most significant BLAST result in these searches

was ORF VC0497, which is annotated as a transcriptional regulator, and is encoded within Vibrio Seventh Pandemic island-II (VSP-II). VSP-II also encodes mafosfamide a tyrosine recombinase integrase at ORF VC0516 (IntV3) [58]. ORFs VC1785 and VC1809 encoded within VPI-2 were the second and third most significant hits retrieved from these BLAST searches, which we termed VefA (for Vibrio excision factor A) and VefB, respectively (Figure 3). The VefA and VefB proteins share 46% amino acid identity/72% similarity. VefA shares 37% amino acid identities with AlpA, 46% identity with Hef and 29% with Xis from the V. cholerae SXT element as was previously shown [53] (Figure 3). The vefB gene is located at the 3′ end of VPI-2 at ORF VC1809 marking the end of the island, and vefA (VC1785) is adjacent to neuraminidase gene, nanH (VC1784) in the middle of the island (Figure 1A). Figure 3 Alignments of VPI-2 RDFs VefA and VefB with other known RDFs: AlpA (AAA18418), Hef (NP_405464), Xis (ABA87014).