Figure 7.

RNA-seq shows (p)ppGpp regulates hundreds of genes in response to reactive oxygen species and pH stress, predominantly via derepression of the CodY regulon. RNA was extracted from bacterial cultures of WT, (p)ppGpp⁰, codY::Tn, or (p)ppGpp⁰  codY::Tn that had been exposed to either 100 mM H₂O₂ or 20 mM itaconic acid for 10 minutes. Four biological replicates were performed per experimental condition. A, PCA of RNA-seq count data collected under H₂O₂-stress conditions. PCA plot was generated by DESeq2 on the Galaxy web platform. B, Number of significant DEGs in CodY-dependent (WT vs [p]ppGpp⁰) and CodY-independent backgrounds (codY::Tn vs [p]ppGpp⁰  codY::Tn), following H₂O₂ or itaconic acid stress. Green indicates upregulated genes, red represents downregulated genes. C, Overlaps between H₂O₂ or itaconic acid-induced stress responses in CodY-dependent and CodY-independent backgrounds. D, Functional categorization of significant DEGs using the KEGG mapper search tool. Hypothetical proteins are excluded for clarity. Abbreviations: (p)ppGpp, guanosine tetraphosphate/guanosine pentaphosphate; (p)ppGpp⁰, (p)ppGpp null mutant; DEG, differentially expressed gene; Itac, itaconic acid; PCA, principal component analysis; RNA-seq, RNA sequencing; WT, wild type.