Transcriptomic response to the absence of IsrR sRNA and aconitase RNA-binding activity under iron-depleted growth conditions

IsrR is a regulatory RNA (sRNA) whose activity is decisive for optimum S. aureus fitness upon iron starvation and for full virulence. IsrR down-regulates several genes encoding iron-containing enzymes to spare iron for essential processes. Here we report that IsrR regulates the tricarboxylic acid cycle by controlling aconitase (CitB), an iron-sulfur cluster-containing enzyme, and its transcriptional regulator, CcpE. This IsrR-dependent dual-regulatory mechanism provides an RNA-driven feedforward loop. Overall design: 12 samples in total. 3 controls (HG003 wild-type strain, WT), 3 delta-IsrR mutant growths in iron-depleted media (DIP-DeIsrR), 3 CitB mutant (deficient in RNA-binding protein activity) and growth in iron-depleted media (DIP-RBPmin), 3 Delta-RsaE (Delta-rsaE). Total RNA were extracted, RNA was treated using Turbo DNase I (Ambion) and purified using the RNA Clean-up and concentration kit (Norgen Biotek), then the sequencing was performed with Illumina NextSeq 2000 using 100 bases run. Staphylococcus aureus is a major pathogen that adapts and thrives in iron-limited environments encountered during host infection. A key factor in adaptation is the reduction of iron requirements. IsrR is a regulatory RNA (sRNA) whose activity contributes to this reduction (Coronel-Tellez et al, 2022). It is decisive for optimum S. aureus fitness upon iron starvation and full virulence. We found that IsrR downregulates i) citB mRNA encoding the tricarboxylic acid cycle (TCA) aconitase (CitB), an iron-sulfur cluster-containing enzyme, and ii) the citB transcriptional regulator, CcpE. To uncover putative targets of isrR, we performed a transcriptomic study comparing strain HG003 and its ?isrR derivative in an iron-starved growth condition. Surprisingly, the transcriptomic profile was not much affected by the absence of IsrR suggestion that IsrR operates at the translational level. In iron-starved conditions, aconitase becomes an RNA binding protein (RBP) with regulatory activities. This moonlighting activity depends on conserved residues. We constructed an aconitase mutant (citB(rbp)) expected to have lost solely its RBP activity. We compared its transcriptomic profile to its parental strain in iron-starved growth conditions. We uncover that the staphylococcal aconitase RBP activity downregulates mRNAs encoding TCA cycle enzymes and upregulates an mRNA encoding an anaplerotic reaction. Overall design: 9 samples corresponding to 3 strains in triplicates: HG003 (parental strain, WT), ?isrR (deficient for isrR, DeIsrR), and citB(rbp, RBPmin) (citB mutant deficient for its RNA-binding protein activity). Strains were cultured overnight, followed by resuspension in fresh BHI medium supplemented with 2,2'-dipyridyl (DIP) at a concentration of 0.5 mM. Samples were collected at an OD600 of 1 and RNA was extracted, treated using Turbo DNase I (Ambion) and purified using the RNA Clean-up and concentration kit (Norgen Biotek). Sequencing was performed with Illumina NextSeq 2000 using 100 bases run.