Fig. 3: Impact of RpoN S-itaconation on transcriptional profile and anabolic glucose utilization in P. aeruginosa.
a Schematic of glucose metabolism in P. aeruginosa, highlighting RpoN S-itaconation prevents repression of genes (red) involved in the gluconate and Entner–Doudoroff (ED) pathways (blue), as well as enhanced glucose flux into anabolic product formation (red arrows) from primary metabolic pathways in WT rpoN versus C218/275A strains grown with itaconate. b Bulk RNA seq to determine differentially expressed genes in WT rpoN relative to C218/275A grown in LB media with itaconate. Grey dots represent all genes; colored dots represent genes of specific pathways. Y axis: logarithmic scale, cut off set on Y axis: −log10 (p value) ≥ 1.3; cut off set on X axis Log2FC (Fold change) > 1. c Volcano plot of differentially expressed genes specific to gluconate or ED pathway in presence of itaconate. Significantly expressed genes associated with gluconate and ED pathways as determined by d RNA seq (n = 2) and e RTqPCR (n = 6) in WTrpoN relative to C218/C275A with or without itaconate. Effect of itaconate on the 13C glucose carbon abundance in different isotopologues of metabolites in WTrpoN relative to C218/C275A (n = 3). Enrichment of 13C glucose into isotopologues of (f, g) cofactors: NAD (C7 p < 0.001 = 0.0003; C9 p < 0.0001 = 5.7988E-05) and NADP, (h) glycerol, (i–k) pyrimidines: UMP, CMP, dTMP, and (l, m) sugars (m, C13 p < 0.001 = 0.00037). Data are presented as mean ± s.e.m. Significance is determined by Wald t-test (b–d) and Unpaired two-tailed Student’s t-test (e, f–m). ns non-significant and * (significant) * (e) C218/275 A vs WTrpoN (p value for gcd = 0.042); C218/275A vs C218/275A itaconate (p values for kguT = 0.008, kguE = 0.0107); C218/275A vs WTrpoN itaconate (p values for gcd = 0.0004, eda = 0.0395); C218/275A itaconate vs WTrpoN itaconate (p values for gcd = 0.0164, kguD = 0.0164, kguT = 0.0275, kguD = 0.0015, eda = 0.015). Schematics in (a) is created in BioRender. Beg, A. (2025) https://BioRender.com/l3jqdlq. All statistical tests are two-sided. Source data are provided as a Source Data file 3.
