Cloning and DNA sequence of the mercuric- and organomercurial-resistance determinants of plasmid pDU1358.

Abstract

The broad-spectrum mercurial-resistance plasmid pDU1358 was analyzed by cloning the resistance determinants and preparing a physical and genetic map of a 45-kilobase (kb) region of the plasmid that contains two separate mercurial-resistance operons that mapped about 20 kb apart. One encoded narrow-spectrum mercurial resistance to Hg2+ and a few organomercurials; the other specified broad-spectrum resistance to phenylmercury and additional organomercurials. Each determinant governed mercurial transport functions. Southern DNADNA hybridization experiments using gene-specific probes from the plasmid R100 mer operon indicated close homology with the R100 determinant. The 2153 base pairs of the promoter-distal part of the broadspectrum Hg2+-resistance operon of pDU1358 were sequenced. This region included the 3'-terminal part of the merA gene, merD, unidentified reading frame URF1, and a part of URF2 homologous to previously sequenced determinants of plasmid R100. Between the merA and merD genes, an open reading frame encoding a 212 amino acid polypeptide was identified as the merB gene that determines the enzyme organomercurial lyase that cleaves the C-Hg bond of phenylmercury.Resistance to mercurial compounds is widespread in prokaryotes and is usually specified by genes on plasmids or transposons (1-6). Two distinct mercurial-resistance phenotypes have been described: (i) resistance to mercuric ions (Hg2+) by enzymatic reduction to Hg°and to a limited number of organomercurials by an unknown mechanism (narrow spectrum) and (ii) resistance in addition to a wider range of organomercurials including phenylmercuric acetate (PhHgOAc) and thimerosal (broad spectrum). Both narrowand broad-spectrum determinants specify the enzyme mercuric reductase that converts Hg2+ to the less toxic Hg°, which is then volatilized. Broad-spectrum determinants encode, in addition, the enzyme organomercurial lyase, which cleaves C-Hg bonds to yield Hg2+ ions, which are then detoxified by mercuric reductase.The mercury-resistance determinants also encode transport functions, which carry mercurial compounds across the cell membrane and present the mercurials to the detoxifying enzymes. If the transport functions are expressed in the absence of reductase activity, the cells become hypersensitive to mercurial compounds (7).The narrow-spectrum mer resistance determinants from plasmid R100 (which contains transposon Tn2l encoding mercury resistance) and transposon TnSOI have been studied by genetic and DNA sequence analysis (8-13). The merT and merP gene products are involved in the uptake and transport of Hg2+; merA specifies the mercuric reductase enzyme; merC, merD, and the unidentified reading frames (URF1 and URF2) are of uncertain function. The merR gene of R100 is transcribed divergently from merT, merP, merC, merA, and merD and specifies a regulatory protein that normally represses the transcription of the mer operon (14). However, in the presence of subtoxic concentrations of Hg2+, the merR product acts as an inducer of the merc...

High diversity of bacterial mercuric reductase genes from surface and sub-surface floodplain soil (Oak Ridge, USA)

“…(Wang et al, 1987) and B. megaterium (Huang et al, 1999) confirmed that Firmicutes type merA primers, Fi-Fw/Fi-Rv1832 and Fi-Fw/Fi-Rv1892, resulted in amplicons of approximately 395 and 455 bp, respectively (data not shown). The b/g-type new primers worked with pDU1358 merA (Griffin et al, 1987) (data not shown). To test the applicability of the Actinobacteria primers, genomic DNA from the mercury-resistant Streptomyces isolate (Is-BDOE1) from soil B was used in PCR reactions.…”

“…The primer pairs were tested in the laboratory on available merA genes from g-Proteobacteria (merA from Serratia marcessens plasmid pDU1358, cloned into pHG103 (Griffin et al, 1987), kind gift of Dr Tamar Barkay, Rutgers University) and Firmicutes merA genes. Three different merA genes originating from Staphylococcus aureus pI258 (Laddaga et al, 1987), Bacillus sp.…”

A C⋅As lyase for degradation of environmental organoarsenical herbicides and animal husbandry growth promoters

“…There seems to be no specific ars gene associated with arsI, in contrast to arsD and arsA, which always go together because their functions are interrelated (32). MerB is a C·Hg lyase that confers resistance to organomercurials (33). Although superficially similar to ArsI, MerB is an unrelated enzyme with a different reaction mechanism (34).…”

A conservative region of the mercuric reductase gene (merA) as a molecular marker of bacterial mercury resistance

“…Bacterial mercury resistance has been found in a wide range of Gramnegative and Gram-positive bacteria. The operon varies in the number of genes present and is usually located on plasmids (3,5,8,19,25) and chromosomes (4,13,18,28); they are often components of transposons (5,11,15,20) and integrons (4,13,18) in a striking diversity of arrangements, often involving duplications and distributions of the enzymes, transporters or regulators among several replicons in one cell. Moreover, two major mer genes, the regulator merR and the merA, which codes the NADPH-dependent flavoenzyme mercuric reductase [E.C.…”

“…The BLASTn program was used with the initial searcher sequence of the partial sequence of the mercuric reductase enzyme, with length of 1141 base pairs (bp), for the Citrobacter freundii, access number AJ418049 (8). The alignment of the sequences, in which two regions with high similarity had been identified by visual approach in color key alignment score bigger or equal to 200, is shown in Fig.…”