Experiments involving fusion between the Staphylococcus aureus plasmid p1258-encoded mer operon and the reporter gene P-lactamase, mutational analysis, and trans-complementation studies have shown that the merR gene of p1258, which shows DNA sequence similarity with known merR genes from other bacteria, regulates the expression of the mer operon in vivo. The merR gene product is a trans-acting protein that activates mer operon transcription in the presence of the inducers Hg2' and Cd2+. A glutathione-S-transferase-MerR fusion protein specifically bound and protected a 27-nucleotide operator sequence from DNase I digestion. This operator sequence is highly homologous with mer operator sequences of other known systems.Mercury-resistant bacteria from environmental and clinical isolates enzymatically detoxify inorganic mercury salts and sometimes organomercurial compounds (for recent reviews, see references 3, 13, 20, 22, 23, 25, and 26). The mercury resistance (mer) genes are often found on plasmids and transposons and are usually clustered in an operon. The highly substrate-specific mercuric ion reductase catalyzes the reduction of Hg2f to chemically inert elemental Hg°, which is water insoluble and volatilizes because of its high vapor pressure. The organomercurial resistance operon encodes another enzyme, organomercurial lyase, that catalyzes the cleavage of the C-Hg bond from organomercurials such as phenylmercury to yield benzene and Hg2e.
Mammalian apolipoproteinB (apoB) RNA editing is a site-specific deamination reaction that mediates the C to U conversion responsible for apoB48 production in the mammalian small intestine. This process is not detected in chicken apoB RNA. Mammalian apoB RNA editing is mediated by a multicomponent enzyme complex that includes a single catalytic subunit, apobec-1. In order to examine the evolution of apobec-1, we have cloned and characterized an orthologous cytidine deaminase cDNA isolated from chicken small intestine. Northern blot analysis revealed expression restricted to the small intestine, colon and lung but not the liver or other tissues. The cDNA encodes a single 31 kDa protein with features reminiscent of other cytidine deaminases and with approximately 39% overall homology to rat apobec-1. The recombinant protein is a cytidine deaminase with activity on a monomeric substrate that was found to be zinc-dependent. However, no RNA editing activity was detectable towards cytidine nucleotides presented in the context of an optimally configured mammalian apoB RNA template. These studies provide information concerning the evolution of the apoB RNA editing machinery and indicate that a chicken small intestinal cytidine deaminase with homology to apobec-1 demonstrates no activity on an RNA substrate.
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 ...
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