Cloning and Sequence Analysis of the Mercury Resistance Operon of Streptomyces sp. Strain CHR28 Reveals a Novel Putative Second Regulatory Gene

Abstract

A DNA library of pRJ28, a large linear plasmid encoding mercury resistance, was constructed, and the mercury resistance genes were cloned. The 5,921-bp sequence was analyzed and showed a high degree of similarity to the Streptomyces lividans 1326 mercury resistance operon. Genes merR, merT, merP, and orfIV were found in a similar order and in a single transcription unit. merA and merB were found to be transcribed in the opposite direction to genes merR, merT, merP, and orfIV, as in S. lividans 1326. A novel putative regulatory gene, orfX, was found 22 bp downstream of merA. orfX encodes a 137-amino acid protein with a potential helix-turnhelix motif in the N-terminal domain, characteristic of the MerR family of transcriptional regulators. Transcriptional studies showed that orfX is cotranscribed with merA and merB. It is hypothesized that orfX plays a role in the regulation of the mercury resistance operon, probably by binding at the MerR operator site.Mercury resistance is widespread among prokaryotes, and resistance genes are often found on plasmids or transposons (11). The major mechanism of resistance is reductive detoxification of Hg(II) to elemental mercury Hg(0), which is extremely volatile and leaves the cell by diffusing through the cell membrane. The process is mediated intracellularly by a mercuric reductase (MerA). Mercuric ions are transported from outside the cell by a series of transporter proteins. MerP is an extracellular mercuric ion binding protein, and MerT is a membrane-anchored protein responsible for transporting Hg(II) into the cell. All gram-positive and some gram-negative systems are resistant to a broad range of mercuric compounds, including organomercurials like phenylmercuric acetate (PMA) (7). This ability is due to the presence of an organomercurial lyase (MerB) which cleaves the carbon-mercury bonds and releases Hg(II). Narrow-spectrum resistance is observed when the merB gene is missing (17). The systems are regulated by transcriptional regulator MerR. In all cases studied, with the exception of in Streptomyces lividans where MerR is a repressor (14, 16), MerR is an activator/repressor transcriptional regulator. In the presence of Hg(II), MerR binds Hg(II) and activates its own transcription as well as that of the other mer genes. In the absence of Hg(II), MerR binds tightly to an operator and represses the system (7). In a few mercury resistance operons, a second regulator gene, merD, is present and binds weakly to the MerR operator site. MerD has been shown to down-regulate the system (9, 10).We have previously described mercury-resistant Streptomyces strain CHR28, in which mercury resistance genes are encoded by the large linear plasmid pRJ28 (330 kb) (13). CHR28 is an environmental strain isolated from a heavily polluted site in the Baltimore Harbor and might have developed resistance and/or regulation mechanisms adapted to its environment which differ from those of S. lividans 1326. Mercury resistance genes of the laboratory strain S. lividans 1326 have previously been clone...

Signature proteins that are distinctive characteristics of Actinobacteria and their subgroups

“…MerR is a transcriptional regulator of the mercury resistance genes (Rother et al 1999). All gram-positive and some gram-negative bacteria are resistant to a broad range of mercuric compounds, and other bacteria besides actinobacteria possess proteins that are annotated as MerR family proteins (Ravel et al 2000). However, they share very little similarity with actinobacterial MerR sequences as seen by the results of BLAST analyses.…”

Genomics ofActinobacteria: Tracing the Evolutionary History of an Ancient Phylum

“…(We note that the inclusion in this set of four strains of M. leprae has resulted in the exclusion of a significant number of genes found in all free-living actinobacteria.) Most of these proteins are of unknown function, except for WhiB, which has roles in development in Streptomyces and cell division in Mycobacterium smegmatis (400), and the transcriptional regulator of mercuric compounds MerR (360). Genes related to whiB have also been found in some actinophages, which may have acquired them from Actinobacteria (336).…”

Heavy metal resistance in Arthrobacter ramosus strain G2 isolated from mercuric salt-contaminated soil

“…strain CHR28 (GenBank accession numbers CP000454, CP000455, CP000456, CP000457) [12]. However, the merA gene of Streptomyces strain CHR28 itself was identified based on sequence similarity to merA from Streptomyces lividans [13], which in turn, was identified by its similarity to merA from other bacteria [14]. Thus, in this long series of similarity-based predictions, there is no direct experimental evidence for the presence of merA gene or enzyme.…”