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. 2018 Nov 9;9:2709. doi: 10.3389/fmicb.2018.02709

Figure 5.

Figure 5

Addition of OAA to SCFM results in increased CrcZ levels and in increased sensitivity toward gentamicin. (A) PA14 and PAO1 cultures were diluted to an initial OD600 of 0.5 and incubated for 20 h in SCFM with or without 40 mM OAA. The CrcZ levels were determined by Northern-blot analysis. 5S rRNA served as a loading control. The fold increase was calculated by dividing the signal intensity of CrcZ by the corresponding signal of the 5S rRNA and normalized to the signal without OAA that was set to 100%. Lanes 1 and 2, contain RNA extracted from PA14 in the presence and absence of OAA, respectively; lanes 3 and 4, contain RNA extracted PAO1 in the presence and absence OAA, respectively. (B,C) MIC determination for gentamicin of PA14 (B) and PAO1 (C). The cells were grown as described above in the absence (upper panel) and in the presence (lower panel) of OAA. The different concentrations of gentamicin added are indicated on top. Pictures were taken and the OD600 was measured 20 h after inoculation. The antibiotic concentrations in the presence of which the cells did not grow above OD600 of 0.5 (marked in red) were considered as MIC. All OD600 values above this gentamicin concentration are depicted in blue indicating toxicity. The experiments were performed in duplicate, revealing the same MICs. Only one representative experiment is shown.