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. 2018 Nov 23;9:2752. doi: 10.3389/fmicb.2018.02752

FIGURE 5.

FIGURE 5

Impaired intracellular accumulation of anthranilate produced by an excessive kynurenine extrusion through MexCD-OprJ is not the cause for lower AQs production of the nfxB∗ mutant. Statistical significances were evaluated by using a Student’s two-tailed test and considered significant if P < 0.05, with a confidence interval of 95% (∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001). (A) The duplication time of PAO1, nfxB∗, and nfxB∗ΔmexD strains growing in minimal medium with succinate (control), tryptophan or kynurenine (both anthranilate precursors) as sole carbon sources was determined. As shown, the nfxB∗ mutant presents an impaired growth in both tryptophan and kynurenine, and deletion of mexD gene restored the growth rate in nfxB∗ mutant, suggesting these compounds might be substrates of MexCD-OprJ. (B) Anthranilate and kynurenine accumulation in cell-free supernatants was quantified by HPLC-MS from PAO1 and nfxB∗ cultures grown along 24 h in M63 minimal medium with succinate (10 mM) and tryptophan (10 mM) as sole carbon sources. Left panels anthranilate, right panels kynurenine. As shown, the supernatants from nfxB∗ cultures contained more kynurenine and less anthranilate than those from the wild-type PAO1 strain, indicating that kynurenine is a substrate of MexCD-OprJ and anthranilate is not extruded by this efflux pump. (C) The production of AQs in PAO1 and nfxB∗ strains growing in LB medium supplemented with anthranilate 1 mM was analyzed in early stationary phase (OD600 = 2.5) by TLC. (D) The extracellular vs. intracellular HHQ ratios were calculated measuring each one of the HHQ spots obtained in the TLC-assays by densitometry. (E) Real-time pqsABCDE expression was analyzed in both PAO1 and nfxB∗ strains growing in LB medium and LB supplemented with anthranilate 4 mM respectively, using a chromosomal insertion of the reporter construction PpqsA::luxCDABE. The results show that anthranilate supplementation of LB medium does not restore the AQs production in the nfxB∗ strain (C,E), reinforcing our hypothesis that HHQ extrusion (D) rather than kynurenine extrusion through MexCD-OprJ is the main cause for the QS-defective response of the nfxB∗ strain.