A small RNA promotes siderophore production through transcriptional and metabolic remodeling

RyhB is essential for intracellular Fe homeostasis and Fe-sparing under low Fe conditions as determined by EPR. Cells were grown in the absence or presence of 1 μM of FeSO₄ until an OD₆₀₀ of 0.9, at which point they were assayed for free intracellular Fe (see Materials and Methods for details).

RyhB is required for normal expression of siderophore synthesis genes in Fe-restricted medium. (A) Quantitative RT-PCR (qRT-PCR) showing the ratio of transcript level (ΔryhB/WT) for several mRNAs involved in enterobactin synthesis (entCEBAH), enterobactin secretion (tolC and entS), enterobactin uptake (fepA), and transcriptional regulation (fur). The transcript levels were determined at OD₆₀₀ of 0.9 (white bars) and 1.2 (gray bars). (B) The addition of DHB to the culture medium restores siderophore production specifically in ΔryhB cells. Siderophore production as detected by TLC performed on E. coli WT and ΔryhB strains growing in M63 minimal medium in the absence or presence of 5 μM of DHB. The asterisk represents the loading spot on the TLC.

RyhB sRNA pairs with cysE mRNA to reduce translation initiation. (A) Potential pairing between the sRNA RyhB and cysE mRNA. The ribosome-binding site of cysE is underlined and the first AUG codon is in bold. (B) In vitro pairing between the sRNA RyhB and 5′-end radiolabeled cysE mRNA as determined by RNase TI, RNase TA, and PbAc probing. Lane C is the control 5′-end radiolabeled cysE mRNA alone, lane OH is treated with NaOH, and TI is treated with RNase TI in denaturing conditions. The 5′-end radiolabeled cysE mRNA was incubated either with RNase TI (lanes 4, without RyhB and 5, with RyhB), RNase TA (lanes 6, without RyhB and 7, with RyhB), or PbAc (lanes 8, without RyhB and 9, with RyhB). The observed pairing between cysE and RyhB correlates with the potential pairing as shown in A. (C) Toeprint assay indicating that RyhB prevents cysE translation initiation by blocking the binding of ribosomal 30S subunit on the cysE mRNA. The GcvB sRNA was used as a negative control.

RyhB directly reduces CysE expression in vivo. (A) β-Galactosidase assays of the translational cysE'–'lacZ reporter fusion in WT and ΔryhB cells grown in minimal M63 medium without Fe at different OD₆₀₀ (0.3, 0.6, and 0.9). (B) The effect of arabinose-induced WT RyhB and mutated RyhB6 on the translational WT cysE'–'lacZ and mutated cysE6'–'lacZ reporter fusions. See Fig. S5 for details. (C) The effect of arabinose-induced RyhB from pBAD-ryhB (as compared with the empty vector pNM12) on cysE mRNA, previously characterized target mRNAs (sodB and fumA), and a negative control mRNA (icd) as determined by qRT-PCR.