Related Experiment Videos
Biochemical characterization of HgCl2-inducible polypeptides encoded by the mer operon of plasmid R100
Abstract:
Minicells carrying the subcloned mer operon from plasmid R100 were pulse-labeled with [35S]methionine, and the labeled polypeptides were analyzed at various subsequent times by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The Hg(II) reductase monomer encoded by plasmid R100 occurred as two proteins of 69 and 66 kilodaltons (kd). The minor 66-kd protein is a modified form of the 69-kd protein. This modification occurs in vivo. Both of these mer proteins are found in the soluble fraction of the cell; however, the 66-kd protein appears to have a slight affinity for the cellular envelope. Both the 69- and 66-kd mer proteins have pI values greater (pI = 5.8) than that reported (pI = 5.3) for the analogous monomer encoded by plasmid R831. The 15.1- and 14-kd mer proteins are localized in the inner membrane and are probably elements of the mer-determined Hg(II) uptake system. These two mer membrane proteins, which are antigenically unrelated to the Hg(II) reductase monomer, are quite basic (pI values greater than 7.8). The 12-kd mer protein is also a basic polypeptide that is present in the soluble fraction of the cell. Unlike the two membrane-bound mer proteins, the 12-kd mer protein is processed from a 13-kd precursor.
Insights
Researchers identified mercury reductase proteins in minicells from plasmid R100. They found two forms of the mercury reductase monomer (69- and 66-kd) and other mer proteins involved in mercury uptake and transport.
Area of Science:
- Molecular Biology
- Environmental Microbiology
Background:
- The mer operon confers resistance to mercury.
- Understanding the proteins encoded by the mer operon is crucial for mercury detoxification mechanisms.
Purpose of the Study:
- To characterize the proteins encoded by the mer operon from plasmid R100.
- To investigate the localization and modification of these mer proteins.
Main Methods:
- Pulse-labeling of minicells with [35S]methionine.
- Analysis of labeled polypeptides using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
- Determination of isoelectric points (pI) and cellular fractionation.
Main Results:
- Identified two forms of the mercury reductase monomer (69-kd and 66-kd), with the 66-kd form being a modified version of the 69-kd protein, modified in vivo.
- Localized 15.1-kd and 14-kd mer proteins in the inner membrane, likely involved in Hg(II) uptake.
- Characterized a soluble 12-kd mer protein processed from a 13-kd precursor, distinct from membrane-bound proteins.
Conclusions:
- The mer operon encodes multiple proteins with distinct functions in mercury resistance.
- Post-translational modification of mercury reductase occurs in vivo.
- Specific mer proteins are involved in mercury transport and detoxification.