The numbers on the right indicate the number of amino acids of the predicted protein. As shown in Figure 2, UV-light irradiation increased excision of VPI-2 over 4-fold. In order to investigate this further, we determined the effect of UV-light irradiation on the expression of intV2, vefA and vefB in V. cholerae N16961 (Figure 4). We examined transcript levels of intV2, vefA and vefB in cells grown for 12 h in LB and in cells grown

for 12 h in LB followed UV-light irradiation treatment. We found that all three genes showed negligible levels of transcription under standard optimum growth conditions but after UV-light treatment both intV2 and vefA show a 10-fold and vefB a 5-fold increase in expression levels AZD2281 datasheet (Figure 4). These results indicate that UV-light induces expression of factors potentially involved in VPI-2 excision. Figure 4 Expression of intV2 (VC1758), vefA , and vefB from cultures grown in standard (black bars) or UV-light irradiated cultures (grey bars). The Y-axis represents the expression ratio of the genes relative to the expression of mdh. Unpaired t-test was used in order to infer statistical significance for the differences in gene expression between cultures of V. cholerae N16961 with or

without UV-light treatment. **, p < 0.05; ***, p < 0.005. Error bars indicate standard deviation. Each experiment was performed in triplicate a minimum of three times. IntV2 and VefA are essential for the excision of VPI-2 To determine MG-132 in more detail the role of intV2, vefA and vefB in VPI-2 excision, we created deletion https://www.selleckchem.com/products/AZD8931.html mutations in each gene and measured excision levels of VPI-2 by determining attB levels in cells. In V. cholerae RAM-1, an intV2 mutant, we did not detect any VPI-2 attB products, demonstrating that intV2 is essential for excision as was previously shown (Figure 5) [23]. We complemented RAM-1 with a functional copy of intV2 by transforming

V. cholerae RAM-1 with pIntV2 creating strain SAM-1. In our SAM-1 strain, we found that excision of VPI-2 was restored in addition, attB levels were approximately four-fold higher than wild-type levels which is represented by the dotted broken horizontal line in Figure 5. These data demonstrate that over expressing intV2 ectopically induces excision of VPI-2. In our control experiments, transformation of either wild-type N16961 or RAM-1 with pBAD33 alone (strains SAM-11 and SAM-12 respectively) did not affect attB levels (data not shown). Figure 5 Excision levels of VPI-2 in mutant strains and strains complemented with intV2 (VC1758), and vefA (VC1785). Excision levels of ΔintV2 mutant (RAM-1), ΔintV2 mutant complemented (SAM-1), ΔvefA mutant (SAM-3), ΔvefA mutant complemented (SAM-5), and ΔvefB mutant (SAM-4). Unpaired t-test was used in order to infer statistical significance for the differences in VPI-2 excision between V. cholerae N16961 and test strains. **, p < 0.05; ***, p < 0.005. Error bars indicate standard deviation.