Results and Discussion VPI-2 excision

rates under different growth conditions It was previously shown that the four pathogenicity islands identified in V. cholerae N16961 can excise from chromosome 1 CHIR-99021 order and form circular intermediates (CI) [23, 28]. The excision of VPI-1 and VPI-2 occurs at very low OSI-027 nmr levels suggesting that excision is tightly controlled, although it may also suggest that the excision event is inefficient, possibly due to poor expression of the regulatory genes, an altered regulatory circuit, or mutations that might occur in these sequences as the region become evolutionarily integrated into the host chromosome [23, 28]. First, we quantified the excision levels of VPI-2 in cultures of V. cholerae N16961 grown for 12 hours in LB at 37°C (standard conditions) by measuring the presence of attB, the locus present on the chromosome after VPI-2 excises (Figure 1), and comparing it with the housekeeping gene mdh using QPCR. We used attB as a surrogate for VPI-2 excision Torin 2 cell line measurements since the copy number of attP in the CI is minuscule compared to attB, which replicates along with the rest of the chromosome unlike excised VPI-2. We compared the presence of attB

with mdh since all cells encode one functional copy of the latter. PCR products of attB and mdh were visually checked on an agarose gel and their melting temperature analyzed to ensure we had the correct PCR products. The reference gene was assayed both separately and in the same reaction. Both primer pairs used were tested by comparing the results obtained using previously quantified cloned copies of mdh and attB and gave comparable results. We found that attB was present in 1 in every 1.6 (±0.2) × 106 V. cholerae cells under optimal growth conditions. Next, we measured the presence of attB from V. cholerae cells cultured under different conditions compared with the Digestive enzyme presence of attB under our standard condition, growth for 12 hours at 37°C. We determined that incubation time does not affect the excision levels

of VPI-2 indicating that excision does not occur in a growth phase dependent manner (Figure 2). However, V. cholerae cultures grown at 25°C showed a 2-fold increase in the presence of the attB site compared to cells grown at the optimum temperature 37°C (Figure 2). In addition, we found that nutrient limitation affected the excision level showing over a 5-fold decrease in the presence of attB when compared to the growth on LB at the same temperature (Figure 2). Furthermore, we found that sub-lethal UV-light irradiation of cell cultures compared to untreated cells, resulted in a significant increase in the level of excision of VPI-2, over 4-fold compared to untreated cells grown under the same conditions (Figure 2).