Intracellular inducer Hg2+ concentration is rate determining for the expression of the mercury-resistance operon in cells

Abstract

Several mercury resistance (mer) operons isolated from different gram-negative bacteria have been cloned into Escherichia coli, and the regulation of expression of these operons has been studied (recently reviewed in references 5, 10, 16, 18, and 19). The mer operons encoded by transposons Tn21 and Tn501 and the plasmid pDU1358 have genes with common functions. The major regulatory gene merR is transcribed divergently from the other genes (Fig. 1). The MerR protein represses its own expression as well as the expression of other mer genes from a different promoter, P T . Genes encoding specific Hg 2ϩ transport proteins, mercuric ion reductase, organomercurial lyase (present in pDU1358), and an ancillary regulator protein, MerD, are expressed from the promoter P T . In the presence of Hg 2ϩ , MerR activates transcription from P T (3,6,8,13,15,20). Tn501-derived mer operon expression is activated at nanomolar concentrations of Hg 2ϩ in vitro (17), and this phenomenon was correlated with in vivo Tn21-derived mer operon expression by using mer operon-reporter gene fusions (1, 7). Induction of mer operon expression in the presence of unusually low concentrations of the inducer Hg 2ϩ is unique among all the known metal ion resistance operons in bacteria. In the present study, we found that the induction of the mer operon by nanomolar concentrations of Hg 2ϩ is not biologically relevant. mer operon expression is activated by the presence of nanomolar concentrations of Hg 2ϩ in liquid media only when the mercuric ion reductase function is artificially inactivated in cells, whereas cells with active mercuric ion reductase require micromolar concentrations of Hg 2ϩ for effective induction of the operon. Most importantly, the concentration of the inducer Hg 2ϩ determines the rate of transcription in resistant cells and the inducer concentration never reaches a saturation level in such cells.(H.Y. submitted this work in partial fulfillment of the requirements for a Ph.D. from the University of Illinois at Chicago.)Bacterial strain, medium, and plasmids. The bacterial strain used in this study is E. coli MC1061 (hsdR mcrB araD139⌬ (araABC-leu)⌬lacX74 galU galK rps-1 thi). Luria-Bertani (LB) medium supplemented with 100 g of carbenicillin or 10 g of tetracycline per ml was used to grow E. coli cells harboring different plasmids at 37ЊC. The plasmids used are listed in Table 1.Construction of merA-lacZ transcriptional fusion. In plasmid pDH1, sequences from the 79th codon of merB to the 3Ј end of the pDU1358-encoded broad-spectrum mer operon are deleted (11). A fragment from the EcoRV site (upstream of merR) to the EcoRI site (within merA) of pDH1 was blunt ended and cloned into the BamHI site (also blunted after BamHI digestion) of the promoterless lacZ transcriptional fusion vector pQF50 (2). Colonies expressing ␤-galactosidase activity were identified on 5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside (X-Gal) plates containing 100 g of carbenicillin per ml and 0.1 M HgCl 2 as described elsewhere (20). Plasmid DNA from one ...

Mercury Reduction and Methyl Mercury Degradation by the Soil Bacterium Xanthobacter autotrophicus Py2

“…One of the hallmarks of reduction of Hg(II) via merA is that previous exposure to low levels of Hg(II) initiates transcription of the operon, which increases the initial rate of Hg(II) reduction (38). This occurs because Hg(II) forms a complex with MerR, changing it from a transcriptional repressor to an activator.…”

Bacterial mercury resistance from atoms to ecosystems

“…Filtration [91] or autoclaving [92] inhibits Hg volatilization from natural waters. Although mer-lux transcriptional fusions can be induced at 6 pM of Hg(II) in oligotrophic conditions [97], induction is likely to be ine⁄cient [98]. However, Hg concentrations in most natural environments, including those where MeHg bioaccumulation occurs, are in the fM to pM range.…”

“…However, Hg concentrations in most natural environments, including those where MeHg bioaccumulation occurs, are in the fM to pM range. Although mer-lux transcriptional fusions can be induced at 6 pM of Hg(II) in oligotrophic conditions [97], induction is likely to be ine⁄cient [98]. In addition to the bacterial Hg(II) reductase, some algae reduce Hg(II) by both light-dependent [99] and light-independent [100] processes ; the latter transformation may be mediated by extracellular metabolites rather than by a speci¢c enzymatic activity.…”

Protein Method for Investigating Mercuric Reductase Gene Expression in Aquatic Environments

“…To collect released protein molecules and to reduce interference from particulate NADPH oxidase, the lysed cell suspensions were centrifuged at 80,000 ϫ g for 60 min. Because bacterial mercuric reductase is a cytoplasmic flavoprotein enzyme (11,14,26,29,32,33), the aqueous protein extracts were collected for enzyme assay and the particulate fractions were discarded. The concentration of proteins was determined by the Bradford dye assay (6) (U.S. Biochemicals, Cleveland, Ohio).…”