Effect of gene amplification on mercuric ion reduction activity of Escherichia coli.
The mercury resistance (mer) operon of plasmid R100 was cloned onto various plasmid vectors to study the effect of mer gene amplification on the rate of Hg2+ reduction by Escherichia coli cells. The plasmids were maintained at copy numbers ranging from 3 to 140 copies per cell. The overall Hg2+ reduction rate of intact cells increased only 2.4-fold for the 47-fold gene amplification. In contrast, the rate of the cytoplasmic reduction reaction, measured in permeabilized cells, increased linearly with increasing gene copy number, resulting in a 6.8-fold overall amplification. RNA hybridizations indicated that mRNA of the cytoplasmic mercuric reductase (merA gene product) increased 11-fold with the 47-fold gene amplification, while mRNA of the transport protein (merT gene product) increased only 5.4-fold. Radiolabeled proteins produced in maxicells were used to correlate the expression levels of the mer polypeptides with the measured reduction rates. The results indicated that, with increasing gene copy number, there was an approximately 5-fold increase in the merA gene product compared with a 2.5-fold increase in the merT gene product. These data demonstrate a parallel increase of Hg2+ reduction activity and transport protein expression in intact cells with plasmids with different copy numbers. In contrast, the expression level of the mercuric reductase gene underwent higher amplification than that of the transport genes at both the RNA and protein levels as plasmid copy number increased.
Philippidis
G P
GP
Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis 55455.
Malmberg
L H
LH
Hu
W S
WS
Schottel
J L
JL
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005784:Gene Amplification; D005798:Genes, Bacterial; D007700:Kinetics; D008628:Mercury / Q000378:metabolism*; D009876:Operon; D010084:Oxidation-Reduction; D012333:RNA, Messenger / Q000096:biosynthesis; D012044:Regression Analysis; D015183:Restriction Mapping
D001426:Bacterial Proteins; D012333:RNA, Messenger; D008628:Mercury