The MerE protein encoded by transposon Tn21 is a broad mercury transporter in Escherichia coli

Abstract

a b s t r a c tIn order to clarify the physiological role of the merE gene of transposon Tn21, a pE4 plasmid that contained the merR gene of plasmid pMR26 from Pseudomonas strain K-62, and the merE gene of Tn21 from the Shigella flexneri plasmid NR1 (R100) was constructed. Bacteria with plasmid pE4 (merR-o/p-merE) were more hypersensitive to CH 3 Hg(I) and Hg(II), and took up significantly more CH 3 Hg(I) and Hg(II), than the isogenic strain. The MerE protein encoded by pE4 was localized in the membrane cell fraction, but not in the soluble fraction. Based on these experimental results, we suggest for the first time that the merE gene is a broad mercury transporter mediating the transport of both CH 3 Hg(I) and Hg(II) across the bacterial membrane.

Role of MerC, MerE, MerF, MerT, and/or MerP in Resistance to Mercurials and the Transport of Mercurials in <i>Escherichia coli</i>

“…19) The primers UKpnmerF (5′-GGG GTA CCA TGA AAG ACC CGA AGA CAC T-3′) and LEcomerF (5′-CGG AAT TCT CAT TTT TTT ACT CCA TTG A-3′) were used to amplify the merF region (0.25 kb) of Tn5053 (accession no. L40585) in the plasmid pUC18F.…”

“…11) After digesting the polymerase chain reaction (PCR) product with KpnI and EcoRI, the fragment was cloned into the KpnI-EcoRI site of pR2, 19) which contained the merR-o/p gene of plasmid pMR26 (accession no. D83080) from pKF19k.…”

“…19) Subcellular Fractionation, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), and Western Blot Analysis E. coli XL1-Blue cells that contained the plasmids pE4, pF17, pT5, pC7, pPE62, pPF8, pTP4, or pPC20 were grown to an optical density of 0.80 at 600 nm with 0.5 µM HgCl 2 . The cells were centrifuged, washed with buffer (50 mM Tris-HCL, pH 8.0, 0.15 M NaCl, 1 mM EDTA, and 10% glycerol), and suspended in 1.2 mL of the same buffer.…”

“…The proteins were separated by SDS-PAGE and transferred to an Immobilon-P membrane (Millipore, Billerica, U.S.A.). After blocking with de-fatted milk, the membrane filter was incubated with anti-MerE, 19) anti-MerF, anti-MerT, anti-MerC, 19) or anti-MerP antibodies to detect the proteins of interest. The membranes were washed and reacted with peroxidase-conjugated anti-rabbit immunoglobulin G (IgG) (Sigma-Aldrich, MO, U.S.A.).…”

“…Mer protein expression in the transformants was measured by immunoblot analysis using polyclonal anti-MerE, 19) antiMerF, anti-MerT, anti-MerC, 19) and anti-MerP antibodies with SDS-PAGE. The following novel protein bands were identified in the crude cell extracts and membrane fractions from cells that contained pE4, pF17, pT5, pC7, pPE62, pPF8, pTP4, and pPC20 (Fig.…”