Fig. 3: UroM6 induces the expression of two regioselective dehydroxylases.
a Untargeted proteomics experimental design scheme. Treated isolates of E. asparagiformis (Ea), E. citroniae (Ec), and E. pacaense (Ep) were pooled by treatment group and lysed. Protein abundance was then determined by untargeted uHPLC-MS/MS. The scheme was generated using graphical elements from the NIAID NIH BIOART Source (bioart.niaid.nih.gov/bioart/42) and from BioRender. Castagner, B. (2025) https://BioRender.com/khbff31. b Aligned chromatograms (λ = 305 nm) of urolithins in DMSO or uroM6-treated (50 μM) mid-exponential phase isolates grown in mABB+H (4 h treatment with urolithins). Retention times of urolithin standards are shown with grey dotted lines. Data from n = 1 of 2 biological replicates are shown. c Venn diagram based on protein presence/absence in the untargeted proteomics dataset (from n = 2 biological replicates). d Heatmap of protein abundance (based on the average of total spectra from n = 2 biological replicates) for the 25 most abundant proteins detected in the uroM6 treatment group. Protein annotations from NCBI are shown on the right (along with the related taxon between brackets), and custom annotations are shown on the left. e Map of the pRG-Ea_uxdFOC plasmid for the heterologous expression of the E. asparagiformis (Ea) uxd operon in C. sporogenes. Plasmid map generated in Benchling. ori: origin of replication (E. coli); cat: chloramphenicol acetyltransferase; Pfdx-rbG: promoter of the C. sporogenes ferredoxin gene with riboswitch G. f Offset chromatograms (λ = 305 nm) of urolithins in uroM6-treated (100 μM) mid-exponential phase wild type (WT) and pRG-Ea_uxdFOC (uxd) C. sporogenes (Cs) cultures grown in BHI ± 30 μg/mL thiamphenicol (48 h treatment with uroM6). Retention times of urolithin standards are shown with grey dotted lines. Data from n = 1 biological replicate for each strain/transformant are shown. WT wild type; uxd #: Cs pRG-Ea_uxdFOC clone number. g Aligned chromatograms (λ = 305 nm) of urolithins in DMSO (black) or uroM7-treated (red, 100 μM) mid-exponential phase Cs WT, pRG-eGFP (eGFP), and uxd 1 cultures grown in BHI ± 30 μg/mL thiamphenicol (96 h treatment with DMSO or uroM7). Retention times of urolithin standards are shown with grey dotted lines. Data from n = 1 of 3 biological replicates for each strain/transformant are shown.
