Figure 5 selleck Relative β-lactamase activity in PAO ampP and PAO ampG mutants.

Assays were performed on the parental PAO1, and the mutants, PAOampP and PAOampG in the presence of benzyl-penicillin at a concentration gradient of 0 to 125 μg/ml. Cultures at OD600 of 0.6-0.8 were induced for three hours before harvesting. Assays were performed on sonicated lysate using nitrocefin as a chromogenic substrate. The β-lactamase activity of PAO1 at 100 μg/ml of benzyl-penicillin was taken as 100%. Each value is the mean of at least three independent experiments. The asterisk refers to p-values of < 0.05 with respect to PAO1, which were calculated using the two-tailed Student's t-test. In PAOampG, the initial increase of β-lactamase activity was observed at 25 μg/ml, suggesting that this burst of β-lactamase production is ampG-independent (Figure 5). However, unlike Bortezomib PAO1, the induction level failed to increase after 25 μg/ml of benzyl-penicillin and even significantly decreased with addition of increased concentrations of benzyl-penicillin (Figure 5). Mutation of ampP also prevented maximum induction of β-lactamase, but the defect was

not quite as severe as in PAOampG. In PAOampP, the pattern of β-lactamase induction was very similar to PAO1 at concentrations of benzyl-penicillin up to 50 μg/ml (Figure 5). However, unlike PAO1, addition of benzyl-penicillin at concentrations greater than 50 μg/ml failed to further

induce production of β-lactamases (Figure 5). Thus, low induction is independent of ampG or ampP. The observation that PAOampP exhibited higher levels of β-lactamase expression at higher concentrations of benzyl-penicillin may suggest that ampG plays a greater role at higher concentrations of β-lactam. Most of the β-lactamase activity of P. aeruginosa can be attributed to AmpC, however, P. aeruginosa does contain another heptaminol chromosomally encoded β-lactamase, PoxB [9, 26]. To further analyze if the loss of β-lactamase induction in the PAOampG and PAOampP strains was due to loss of AmpC function, the ampC promoter (P ampC ) activity was measured in PAO1, PAOampG, and PAOampP. As expected, upon treatment with benzyl-penicillin, P ampC -lacZ activity increased approximately 15-fold (Figure 6). Benzyl-penicillin dependent induction of P ampC -lacZ was lost in PAOampG or PAOampP (Figure 6). Figure 6 Activity of the ampC promoter. Promoter activity of the ampC gene was analyzed using lacZ transcriptional fusions integrated at the att locus of PAO1, PAOampR, PAOampG and PAOampP (see Materials and Methods and text for details). Cells were grown to an OD600 of 0.6 – 0.8, at which time cultures were divided into two and one set treated with 100 μg/ml benzyl-penicillin. After three hours, cells were harvested and β-galactosidase activity assayed as described [10]. Each value is the mean of at least three independent experiments.