Therefore, using qRT-PCR, we determined whether Vfr

regulates the expression of PA2782 and PA2783 in PAO1. We compared the expression of both genes in PAO1 and its vfr isogenic mutant PAO∆vfr at early (OD600 of 0.37 and 0.41) and mid (OD600 of 0.79 and 0.89) logarithmic phases of growth. As shown in Figure 2, at both time points and compared with PAO1, the expression of PA2782 and PA2783 was significantly reduced in PAO∆vfr. Figure 2 Vfr regulates the transcription of PA2782 and PA2783 at early and late stages of BGB324 clinical trial growth of PAO1. PAO1 and PAOΔvfr strains were grown in LB broth overnight and subcultured into fresh LB broth to a starting OD600 of 0.02. Cells were harvested at 4 h and 6 h, OD600 of 0.37 and 0.79 for PAO1 and 0.41 and 0.89 for PAOΔvfr, respectively, and total RNA was extracted. Levels of PA2782 and PA2783 mRNA in each sample were determined by qRT-PCR using specifically-designed primers. Values represent the means of three independent experiments PF-562271 ± SEM. ***P <0.001. Due to the presence of functional domains within the predicted protein encoded by PA2783 (see below), we decided to focus our effort on PA2783. We determined the regulation of PA2783 expression by Vfr

throughout the growth cycle of PAO1. This was done using the PAO1 mutant strain PW5661, which carries an in-frame PA2783::lacZ chromosomal fusion in which the first nine amino acids of the PA2783 protein are fused with the β-galactosidase protein (http://​www.​gs.​washington.​edu/​labs/​manoil/​two_​allele_​August2012.​xls)

and the vfr multicopy plasmid pKF917 (Table 1) [15, 28]. Cells were grown in LB broth for 12 h. Samples were obtained every 2 h and the levels of β-galactosidase activity was determined as previously described [29, 30]. Compared with PW5661 carrying a vector control (pUCP19), PW5661/pKF917 produced a significantly higher level of PA2783 expression from 2 h post-inoculation through 10 h, with a sharp peak of expression at 4 h post-inoculation (early to mid-log, OD600 0.15-1.24) (Figure 3). Following this peak, expression of PA2783 gradually declined towards the 12 h time point (late stationary phase, OD600 2.94-3.22) (Figure 3). This Dichloromethane dehalogenase pattern of expression did not result from the effect of pKF917 on the growth of PW5661 since its growth was comparable to that of PW5661 containing the cloning vector (Figure 3). Although in Figures 2 and 3, the time point at which the highest level of PA2783 expression was detected is different (6 h vs. 4 h post-inoculation), the growth of PAO1 at these two time points is close (OD600 of 0.89 for the 6-h time point in Figure 2 and OD600 of 1.2 for the 4-h time point in Figure 3). This variation in the growth is possibly due to the presence of a plasmid in PAO1 (pKF917 or pUCP19).