Fig. 2 | Communications Biology

Fig. 2

From: A TonB-dependent receptor constitutes the outer membrane transport system for a lignin-derived aromatic compound

Fig. 2

ddvT encodes the outer membrane transporter of DDVA. a The catabolic pathway of DDVA in SYK-6 cells. Enzymes: LigXa, oxygenase component of DDVA O-demethylase; LigXc, ferredoxin; LigXd, ferredoxin reductase; LigZ, OH-DDVA dioxygenase; LigY, meta-cleavage compound hydrolase; LigI, PDC hydrolase. Compounds: OH-DDVA, 2,2′,3-trihydroxy-3′-methoxy-5,5′-dicarboxybiphenyl; CHPD, 4-carboxy-2-hydroxypenta-2,4-dienoate; 5CVA, 5-carboxyvanillate; PDC, 2-pyrone-4,6-dicarboxylate. b Organization of the DDVA catabolic genes. Genes: SLG_07650 (ddvT), DDVA outer membrane transporter; ddvK, DDVA inner membrane transporter; ddvR, MarR-type transcriptional regulator. c Western blot analysis using anti-DdvT antibodies performed against total membrane fractions (10 μg of protein) obtained from cells of SYK-6, ∆ddvT and ∆ddvR incubated in LB supplemented with or without 1 mM DDVA. Ponceau S staining is shown as loading control. d Growth of ∆ddvT cells on DDVA. Cells of SYK-6(pSEVA338 [vector]), SYK-6(pS-ddvT), ∆ddvT(pSEVA338) and ∆ddvT(pS-ddvT) were cultured in Wx medium containing 5 mM DDVA. Cell growth was monitored by measuring the OD660. e DDVA conversion by resting cells of ∆ddvT. Cells (OD600 = 5.0) of SYK-6(pSEVA338), SYK-6(pS-ddvT), ∆ddvT(pSEVA338) and ∆ddvT(pS-ddvT) were incubated with 100 µM DDVA. Portions of the reaction mixtures were collected and the amount of DDVA was measured by HPLC. f DDVA uptake by ∆ddvT cells. The β-galactosidase activities of cells of SYK-6(pS-XR), ∆ddvT(pS-XR) and ∆ddvK(pS-XR) incubated in Wx-SEMP with or without DDVA (0.1, 1.0 and 5.0 mM) were measured. g DDVA uptake by ddvT-complemented ∆ddvT cells. The β-galactosidase activities of cells of SYK-6(pSEVA338 + pS-XR), SYK-6(pS-ddvT + pS-XR), ∆ddvT(pSEVA338 + pS-XR) and ∆ddvT(pS-ddvT + pS-XR) incubated in Wx-SEMP with or without DDVA (0.1, 1.0 and 5.0 mM) were measured. Each value is the average ± the standard deviation of n = 3 independent experiments. ns, P > 0.05, *P < 0.05, **P< 0.01, ****P < 0.0001 (one-way ANOVA with Dunnett’s multiple comparisons post-test). h Cellular localization of DdvT in SYK-6 cells. Western blot analysis using anti-DdvT and anti-His6 antibodies was performed against the total membrane fraction (10 μg of protein) and the outer membrane fraction (10 μg of protein) obtained from SYK-6 cells harbouring pJB-tonB2His grown in LB containing 1 mM m-toluate.

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