Molecular cloning and expression of the Escherichia coli dimethyl sulfoxide reductase operon.
The dimethyl sulfoxide (DMSO) reductase operon coding for a membrane-bound iron-sulfur, molybdoenzyme, which functions as a terminal reductase in Escherichia coli, has been isolated and cloned from an E. coli gene bank. Two clones, MV12(pLC19-36) and MV12(pLC43-43), overexpressed both DMSO and trimethylamine N-oxide (TMAO) reductase activities 13- to 15-fold compared with wild-type cells. Amplification was highest in cells grown anaerobically on fumarate, while cells grown on DMSO or TMAO displayed reduced levels of enzyme amplification. Growth on nitrate or aerobic growth repressed expression of the enzyme. A 6.5-kilobase-pair DNA restriction endonuclease fragment was subcloned from pLC19-36 into the vector pBR322, yielding a recombinant DMSO reductase plasmid, pDMS159. Two polypeptides were amplified and identified on sodium dodecyl sulfate-polyacrylamide gels of proteins from E. coli HB101 harboring pDMS159: a membrane-bound protein with molecular weight 82,600 and a soluble polypeptide with molecular weight 23,600. Three plasmid-encoded polypeptides with molecular weights of 87,500, 23,300, and 22,600 were detected by in vivo transcription/translation studies. The smallest subunit was poorly defined and not detectable by Coomassie blue staining. The DMSO reductase operon was localized to the 20.0-min position on the E. coli linkage map.
Bilous
P T
PT
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Weiner
J H
JH
D000693:Anaerobiosis; D001426:Bacterial Proteins / Q000032:analysis / Q000235:genetics; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004121:Dimethyl Sulfoxide / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005650:Fumarates / Q000378:metabolism; D005784:Gene Amplification; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D007506:Iron-Sulfur Proteins; D008744:Methylamines / Q000378:metabolism; D009154:Mutation; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D009876:Operon; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors; D010957:Plasmids; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005650:Fumarates; D007506:Iron-Sulfur Proteins; D008744:Methylamines; D009566:Nitrates; D010088:Oxidoreductases; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D009247:NADH, NADPH Oxidoreductases; C045664:dimethyl sulfoxide reductase; D004262:DNA Restriction Enzymes; C005855:trimethyloxamine; D004121:Dimethyl Sulfoxide