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To compare the response of Zur, ZntR, RcnR, FrmR and FrmRE64H to Zn(II) and cobalt, Salmonella cells were cultured in minimal media (~4–5?h) supplemented with MNICs of each metal (giving ?10% inhibition of growth), ensuring multiple cell division cycles in the presence of metal (Supplementary Fig. 2). Transcript abundance was visualised by end point reverse-transcriptase PCR and enumerated by quantitative PCR (qPCR; Fig. 2a–e).
The DUF156 family of DNA-binding transcriptional regulators includes metal sensors that respond to cobalt and/or nickel (RcnR, InrS) or copper (CsoR) plus CstR, which responds to persulfide, and formaldehyde-responsive FrmR. Unexpectedly, the allosteric mechanism of FrmR from Salmonella enterica serovar Typhimurium is triggered by metals in vitro, and variant FrmRE64H gains responsiveness to Zn(II) and cobalt in vivo.
Author contributions: D.O. carried out the in vivo survival and gene expression experiments, bioinformatic analysis, and developed the fractional occupancy model for formaldehyde modification. C.P. did the in vitro metal-binding and fluorescence anisotropy experiments, and generated FrmRE64H crystals. E.P. and I.U. solved the X-ray crystal structure of FrmRE64H. J.C. and T.G.H performed the LC-MS/MS. L.N.S. and C.P. determined the intracellular glutathione concentration.
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