Hypersensitivity to Hg2+ and hyperbinding activity associated with cloned fragments of the mercurial resistance operon of plasmid NR1

Abstract

The region of plasmid NR1 concerned with resistance to Hg2' and organomercurials consists of sequences found on restriction endonuclease fragments EcoRI-H and EcoRI-I. When both fragments were cloned together into a derivative of plasmid ColEl, the hybrid plasmid conferred properties indistinguishable from those of the parental plasmid, NR1: resistance to Hg2' and to the organomercurials merbromin and fluoresceinmercuric acetate and the inducible synthesis of the enzyme mercuric reductase. When fragment EcoRI-I was cloned into plasmid ColEl, cells containing the plasmid were as sensitive to Hg2' and organomercurials as plasmidless strains. When fragment EcoRI-H was cloned into ColEl, cells with the hybrid plasmid were hypersensitive to Hg2' and organomercurials. This hypersensitivity was inducible by prior exposure to low, subtoxic Hg2+ or merbromin levels. It was associated with an inducible hyperbinding activity attributed to a gene governing Hg2+ uptake and found on fragment EcoRI-H (which contains the proximal portion of a mercuric resistance [mer] operon).

Molecular mechanisms of copper resistance and accumulation in bacteria

“…This effect was not observed with either copC or copD expressed alone. Similar observations led to the discovery of a mercury transport system encoded by merT and merP associated with mercury resistance [30,31]. Preliminary uptake studies indicated that cells containing copCD accumulated more copper than cells without these genes or with either copC or copD alone [23].…”

Bacterial mercury resistance from atoms to ecosystems

“…Work on the mechanism of HgR led to identi¢cation of the key detoxi¢cation enzyme, MerA, the mercuric ion reductase [12], and also of a second enzyme, MerB, which split the carbon^Hg bond in such compounds as the disinfectant phenylmercuric acetate (PMA) and the fungicide methylmercury chloride, a potent neurotoxic agent [13]. Membrane and periplasmic proteins involved in the seemingly paradoxical inward transport of ionic mercury [14,15] were also identi¢ed [16,17]. The ¢rst sequences of HgR loci revealed proteins corresponding to these biochemical and physiological functions as well as a candidate regulatory gene (merR).…”

“…The existence of an operon-speci¢c Hg(II) uptake system was ¢rst suggested on the basis of the Hg(II)-hypersensitive phenotype of merA mutants [14,15]; such variants were more sensitive to Hg(II) than cells lacking the operon altogether, consistent with there being some mechanism for bringing Hg(II) into the cell. In the absence of the mercuric reductase, the internalized Hg(II) is not de-toxi¢ed by reduction to Hg(q).…”

“…This 116-residue (12.4-kDa) inner membrane protein is strongly predicted to have three transmembrane helices, the ¢rst of which has a cysteine pair which would lie within the ¢rst hydrophobic helix and perhaps be accessible from the periplasmic side [157,162,178]. Mutations altering either of these cysteines decrease the Hg(II)-hypersensitive phenotype that is a surrogate for physical measurement of Hg(II) uptake [15]. As noted above, a Cys14Ser mutation in MerP blocks uptake of Hg(II) by wild-type MerT [169], perhaps by formation of a stable Hg(II)-bound trigonal structure involving the MerP Cys17 and MerT's N-terminus-proximal cysteine pair.…”

“…Thus, studies of Hg(II) uptake are only done in hypersensitive mutants lacking the reductase but capable of expressing the inducible transport genes. Not surprisingly, these strains are limited to taking up only as much 203 Hg(II) as can saturate their intracellular Hg(II) binding capacity without poisoning themselves [15,188]. However, initial rates of uptake by induced hypersensitive merA mutants are considerably faster than 203 Hg(II) uptake by cells lacking the mer operon and it is on such rates that work has been done to dissect the energetic basis of uptake.…”