In order to explore the occurrence of polymer degradation after metal addition, the effect of different Cu2+ concentrations on stationary

phase polyP levels was evaluated in MT + P cells (Figure 2A). A copper dependent decrease in polyP levels was observed in WT, pitA − pitB − , pitA − and pitB − after one-hour exposure to metal. PolyP degradation induced by copper was dependent on PPX, since metal addition did not affect the polymer levels in ppx mutant. PolyP degradation in WT cells took place IWR-1 immediately after copper addition (Figure 2B). Figure 2 PolyP levels of stationary phase cells exposed to copper. (A) Cells of the indicated strains grown in MT + P for 48 h were exposed to increasing copper concentrations for 1 h. After incubations, polyP was quantified as described in Methods

using DAPI fluorescence. (B) Time-course of polyP degradation in 48 h MT + P WT cells incubated with 0.25 mM Cu2+. Data are expressed as average ± SD of five independent experiments. DAPI emission was undetectable in cell free controls with or without copper addition. Ivacaftor ic50 Pi efflux in cells exposed to Cu2+ In view of the copper dependent polyP degradation and discarding the chelating effect of the polymer, Pi liberated from the reaction could form complexes with the metal which would be taken out from the cell by Pit system, contributing to detoxify the intracellular environment. Thus, Meloxicam we aimed to test if metal also induces Pi extrusion in stationary phase cells. Time-dependent Pi release was measured in cells exposed to 0.25 mM Cu2+. WT cells released around 40 nmoles Pi mL−1 at 30 min (Figure 3). For pitA and pitB single mutants, Pi exported at 30 min was 50% lower than that of WT cells. No Pi release was detected when pitA − pitB − was used (Figure 3). It is worth noting that Pi was not detected in supernatants of either WT cells incubated without copper or ppx − cells incubated with copper (data not shown).

Viability of all tested strains was maintained after 30 min-exposure to 0.25 mM copper in T buffer (data not shown). Taken together, Pi efflux would be associated to high polyP levels in stationary phase, its degradation in the presence of copper and to the functionality of the Pit system. Figure 3 Pi efflux from stationary phase cells exposed to copper. 48 h MT + P cells of the indicated strains were resuspended in T buffer and exposed to 0.25 mM Cu2+ for the indicated times. Pi was quantified in supernatants as described in Methods. Data are expressed as average ± SD of four independent experiments. Different letters indicate significant differences according to Tukey’s test with a p-value of 0.05.