Glyoxylate bypass operon of Escherichia coli: cloning and determination of the functional map.
In Escherichia coli, a single operon encodes the metabolic and regulatory enzymes of the glyoxylate bypass. The metabolic enzymes, isocitrate lyase and malate synthase, are expressed from aceA and aceB, and the regulatory enzyme, isocitrate dehydrogenase kinase/phosphatase, is expressed from aceK. We cloned this operon and determined its functional map by deletion analysis. The order of the genes in this operon is aceB-aceA-aceK, with aceB proximal to the promoter, consistent with the results of previous experiments using genetic techniques. The promoter was identified by S1 nuclease mapping, and its nucleotide sequence was determined. Isocitrate lyase and malate synthase were readily identified by autoradiography after the products of the operon clone were labeled by the maxicell procedure and then resolved by electrophoresis. In contrast, isocitrate dehydrogenase kinase/phosphatase, expressed from the same plasmid, was undetectable. This observation is consistent with a striking downshift in expression between aceA and aceK.
Chung
T
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Department of Biochemistry, University of Minnesota, Minneapolis 55455.
Klumpp
D J
DJ
LaPorte
D C
DC
D001483:Base Sequence; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004720:Endonucleases; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D006038:Glyoxylates / Q000378:metabolism; D007522:Isocitrate Lyase / Q000235:genetics*; D008292:Malate Synthase / Q000235:genetics*; D009876:Operon; D007652:Oxo-Acid-Lyases / Q000235:genetics*; D010749:Phosphoprotein Phosphatases / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000235:genetics*; D017346:Protein Serine-Threonine Kinases; D015719:Single-Strand Specific DNA and RNA Endonucleases
D006038:Glyoxylates; D008292:Malate Synthase; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; C042301:isocitrate dehydrogenase kinase; D004720:Endonucleases; D004262:DNA Restriction Enzymes; C042300:isocitrate dehydrogenase phosphatase; D010749:Phosphoprotein Phosphatases; D015719:Single-Strand Specific DNA and RNA Endonucleases; D007652:Oxo-Acid-Lyases; D007522:Isocitrate Lyase