Femtomolar Sensitivity of Metalloregulatory Proteins Controlling Zinc Homeostasis

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E. coli strain BW25113 (37) was grown with shaking at 37°C in LB or Chelex-treated (Bio-Rad) A minimal medium (38) containing 0.2% glucose and 40 μg/ml of all 20 l-amino acids to an optical density at 600 nm of 0.5 to 0.6. The cells were harvested at midlog growth and washed three times with fresh media containing 1 mM EDTA and one time with Milli-Q water. The pelleted cells were dried overnight at 80°C in Teflon tubes, dissolved in concentrated nitric acid, and the metal content measured by ICP-MS (PQ ExCell, TJA Solutions, Franklin, MA). Parallel control experiments were run to determine the background metal contamination in the materials used. All glassware and plasticware was acid-washed before use.

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Cloning and purification of Zur will be reported elsewhere (C. Outten, D. Tobin, J. Penner-Hahn, T. O'Halloran, in preparation). For DNase I footprinting, a 340-base pair (bp) fragment including the znuACB promoter was amplified by the polymerase chain reaction from E. coli DH5α, subcloned into pUC19 (New England Biolabs), and confirmed by DNA sequencing. Footprinting procedures were as described (19). The Zur protein, as isolated, contains two zinc atoms per monomer and is denoted Zn₂Zur. It was treated with 1 mM TPEN overnight, and concentrated in a 10-ml Amicon ultrafiltration cell to 500 μl, then diluted up to 10 ml in buffer without TPEN. This process was repeated two more times to remove any weakly bound zinc. This form of the protein contained 0.7 to 0.8 Zn atoms per Zur molecule as determined by ICP-AES and is denoted Zn₁Zur.

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Supported by NIH training grant T32 GM08382 (C.E.O.) and NIH grants R01 GM38784 and DK52627 (T.V.O.). We thank F. W. Outten for znuC primer extension data, S. Shafaie for ICP-MS assistance, and Y. Hitomi, H. Godwin, and J. Widom for helpful discussions.