G-Quadruplex Structures as a "Switch" Regulate ATF4 Expression in Ferroptotic HepG2 Cells.

G-quadruplex (G4) is a noncanonical structure folded in a widespread manner by guanine-rich tandem repeated sequences. As a key response factor, activating transcription factor 4 (ATF4) has dual functions in managing iron-dependent ferroptosis by regulating amino acid synthesis and antioxidant-related gene expression. In our study, the activity of ATF4 expression was elevated in HepG2 cells induced by erastin. Based on preliminary bioinformatics analyses, the G-tract region, named WT, had high potential to form G4, and it was found that PDS could markedly weaken the increase of ATF4 expression by reducing the sensitivity of HepG2 cells toward erastin. In circular dichroism spectra, WT oligonucleotides showed characteristic molar ellipticity at specific wavelengths of parallel G4 structures, while corresponding single-base mutants possessed a weaker ability to form G4, which were consistent with immunostaining results. In addition, endogenous G4 formed by the WT motif was significantly destroyed in HepG2 cells treated with erastin. After being transfected with WT oligonucleotides, the levels of ATF4 mRNA decreased significantly regardless of being treated with erastin or not. Meanwhile, mutations of G-tracts could advantageously impact the luciferase expression downstream of an ATF4 promoter in reporter assays, manifesting that the decrease of endogenous G4 in the ATF4 promoter was positively associated with the expression enhanced by erastin in HepG2 cells.

RpoS is a pleiotropic regulator of motility, biofilm formation, exoenzymes, siderophore and prodigiosin production, and trade-off during prolonged stationary phase in Serratia marcescens.

Prodigiosin is an important secondary metabolite produced by Serratia marcescens. It can help strains resist stresses from other microorganisms and environmental factors to achieve self-preservation. Prodigiosin is also a promising secondary metabolite due to its pharmacological characteristics. However, pigmentless S. marcescens mutants always emerge after prolonged starvation, which might be a way for the bacteria to adapt to starvation conditions, but it could be a major problem in the industrial application of S. marcescens. To identify the molecular mechanisms of loss of prodigiosin production, two mutants were isolated after 16 days of prolonged incubation of wild-type (WT) S. marcescens 1912768R; one mutant (named 1912768WR) exhibited reduced production of prodigiosin, and a second mutant (named 1912768W) was totally defective. Comparative genomic analysis revealed that the two mutants had either mutations or deletions in rpoS. Knockout of rpoS in S. marcescens 1912768R had pleiotropic effects. Complementation of rpoS in the ΔrpoS mutant further confirmed that RpoS was a positive regulator of prodigiosin production and that its regulatory role in prodigiosin biosynthesis was opposite that in Serratia sp. ATCC 39006, which had a different type of pig cluster; further, rpoS from Serratia sp. ATCC 39006 and other strains complemented the prodigiosin defect of the ΔrpoS mutant, suggesting that the pig promoters are more important than the genes in the regulation of prodigiosin production. Deletion of rpoS strongly impaired the resistance of S. marcescens to stresses but increased membrane permeability for nutritional competence; competition assays in rich and minimum media showed that the ΔrpoS mutant outcompeted its isogenic WT strain. All these data support the idea that RpoS is pleiotropic and that the loss of prodigiosin biosynthesis in S. marcescens 1912768R during prolonged incubation is due to a mutation in rpoS, which appears to be a self-preservation and nutritional competence (SPANC) trade-off.