Femtomolar Sensitivity of Metalloregulatory Proteins Controlling Zinc Homeostasis
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Vallee B. L., Biofactors 1, 31 (1988).
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.
The total cellular concentration of each element was calculated assuming a maximal cellular volume of 1.8 μm 3 for cells grown in minimal medium and 3.5 μm 3 in LB medium (23 31). Metal concentrations in media were determined by inductively coupled plasma atomic emission spectroscopy (Thermo Jarrell Ash AtomScan 25 ICP-AES) for Mg Ca and K and ICP-MS for all other elements. Because transition metal concentrations in minimal medium were substantially lower than in LB ICP-MS measurements of minimal media samples employed the standard addition method. This reduced matrix effects.
H. Bremer P. P. Dennis in Escherichia coli and Salmonella : Cellular and Molecular Biology F. C. Neidhardt Ed. (ASM Press Washington DC 1996) vol. 2 pp. 1553–1569.
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 Zn 2 Zur. 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 Zn 1 Zur.
A. E. Martell R. M. Smith NIST Critical Stability Constants of Metal Complexes (NIST Standard Reference Database 46 v5.0 Plenum New York 1998).
Run-off transcription procedures were as described (19). The znu transcription template was prepared in the same manner as the zntA template in (19) and digested with Bsp MI and Bbv I to make a 233-bp fragment. Different size templates were tested for Zur transcription assays and a transcript was always observed for znuCB but not znuA. Both promoters are active in vivo (16 17). The znuCB promoter may be favored in in vitro transcription because it is stronger than the znuA promoter.
Y. Hitomi C. E. Outten T. V. O'Halloran in preparation.
F. C. Neidhardt J. L. Ingraham M. Schaechter Physiology of the Bacterial Cell: A Molecular Approach (Sinauer Associates Sunderland MA 1990).
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F. W. Outten unpublished data.
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.