Fig. 2: Urolithin C treatment upregulates a putative dehydroxylase operon.
From: Diet-derived urolithin A is produced by a dehydroxylase encoded by human gut Enterocloster species
a Experimental design of uroC spike-in experiments during exponential growth. For each biological replicate in this design, (b) growth, (c, d) metabolism, (f, g) and RNA-seq results are matched. b Growth curve (optical density (OD) at 620 nm) of DMSO or uroC-spiked E. asparagiformis (Ea) and E. bolteae (Eb) cultures (n = 4 biological replicates). c Representative chromatograms (λ = 305 nm) of cultures sampled 2 h post-spike-in (from one representative biological replicate). The same scale was used for each chromatogram. IS, internal standard. d Quantification of urolithin concentrations over 4 h in uroC-spiked Ea and Eb type strain cultures (n = 4 biological replicates). e Quantification of urolithin A concentrations in DMSO- or uroC-treated Ea and Eb cell suspensions. The treated cells were washed and resuspended in PBS to halt the production of new enzymes, then treated with DMSO or 100 μM uroC (n = 3 biological replicates). f, g Manhattan plots of genes altered by uroC treatment in (f) Ea and (g) Eb based on DESeq2 (two-sided) analysis (n = 4 biological replicates for each bacterium). Data points are colored according to their adjusted P-value (based on the Benjamini-Hochberg-corrected Wald statistic). The genomic organization around the differentially expressed genes is depicted above the Manhattan plots. Genes are colored according to their log2fold-change (log2FC) values. h Primer (forward (F) and reverse (R)) design for RT-PCR (i) experiment targeting the Eb ucd metabolic gene cluster (MGC). i 1% agarose gel image of RT-PCR amplicons using primers (h) that span the full-length (~ 3.6 kb) Eb ucd MGC (from one biological replicate). NTC, no template control. j RT-qPCR expression of each gene in the Eb ucd operon following treatment with DMSO or uroC (100 μM) for 2 h (n = 3 biological replicates). Gene expression profiles of each target gene in the Eb ucd gene cluster displayed as log2FC with lines connecting paired biological replicates; repeated-measures one-way ANOVA with Tukey’s multiple comparisons test. Exact P-values are noted above the data points. Data are represented as mean ± SEM (behind symbols) in (b, d, e). Source data and statistical details are provided as a Source data file.
