Cell-density-dependent sensitivity of a mer-lux bioassay.

The sensitivity of a previously described assay (O. Selifonova, R. Burlage, and T. Barkay, Appl. Environ. Microbiol. 59:3083-3090, 1993) for the detection of bioavailable inorganic mercury (Hg2+) by the activation of a mer-lux fusion was increased from nanomolar to picomolar concentrations by reducing biomass in the assays from 10(7) to 10(5) cells ml-1. The increase in sensitivity was due to a reduction in the number of cellular binding sites that may compete with the regulatory protein, MerR, for binding of the inducer, Hg2+. These results show that (i) the sensitivity of the mer-lux assay is sufficient for the detection of Hg2+ in most contaminated natural waters and (ii) mer-specified reactions, Hg2+ reduction and methylmercury degradation, can be induced in natural waters and may participate in the geochemical cycling of mercury.     

The merR regulatory gene in Thiobacillus ferrooxidans is spaced apart from the mer structural genes

Two distinct merR genes, which regulate expression of the mercuric ion resistance gene (mer), of Thiobacillus ferrooxidans strain E-15 have been cloned, sequenced and termed merR1 and merR2. As a result of gene walking around two merR genes, it was found that these two genes were quite close in distance. The nucleotide sequence of the region (5,001 base pairs; PstI-EcoRI fragment) containing the merR genes was determined. Between the two merR genes, there were five potential open reading frames (ORFs). Two of these were identified as merC genes, and the other three as ORFs 1 to 3. ORFs 1 to 3 show significant homology to merA, tnsA from transposon Tn7, and merA, respectively. Both merR genes consist of a 408 bp ORF coding for 135 amino acids. Their gene products, MerR1 and MerR2, differed at three amino acid positions, and shared 56-57% and 32-38% identity with the MerRs from other Gram-negative and Gram-positive bacteria, respectively. Competitive primer extension analysis revealed that both regulatory genes were expressed in the host cells. These merR genes were located more than 6 kb from either end of the mer structural genes (merC-merA). This is the first example of merR being separated from the mer structural genes. The two merC genes, each of which coded for a 140-amino-acid protein, appeared to be functionally active because Escherichia coli cells carrying these merC genes on plasmid vectors showed hypersensitivity to HgCl2. However, ORFs 1 and 3, which were homologous to merA, seemed to be inactive both structurally and enzymatically. The gene arrangement in this region took on a mirror image, with the truncated tnsA as the symmetrical centre. It is suggested that the Tn7-like factor may have participated in gene duplication events of the mer region, and in its chromosomal integration.