Mutant of Escherichia coli K-12 with defective phosphorylation of two periplasmic transport proteins.
Bacterial periplasmic transport systems require the function of a specific substrate-binding protein, located in the periplasm, and several cytoplasmic membrane transport components. In Escherichia coli K-12, the arginine-ornithine transport system requires an arginine-ornithine-binding protein and the lysine-arginine-ornithine (LAO) transport system includes a LAO-binding protein. Both periplasmic proteins can be phosphorylated by a single kinase. The enzyme exhibits a kinase activity and an ATPase activity. A mutant, defective in the phosphorylation of the arginine-ornithine and the LAO periplasmic proteins, was isolated and characterized. The defective enzymatic activity was reflected in substantially reduced levels of transport activity of the periplasmic transport systems that include each of the binding proteins. The binding proteins, extracted from the mutant, showed no detectable alterations in terms of quantity, electrophoretic mobility, or affinity constants. An apparent Km value of 1.0 mM was calculated for the ATPase activity of the defective enzyme. The ATPase activity of the wild-type enzyme yielded an apparent Km value of 50 microM. The amount of inorganic phosphate incorporated in vivo and in vitro into the binding proteins by the activity of the defective kinase was reduced to very low levels. A structural gene for the phosphorylating enzyme was located near the serA marker on the linkage map of E. coli. These results indicate that phosphorylation of the periplasmic transport protein is obligatorily linked to the normal function of the periplasmic transport system.
Celis
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Department of Microbiology, New York University School of Medicine, New York 10016.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001120:Arginine / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002874:Chromosome Mapping; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005973:Glutamine / Q000378:metabolism; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D009952:Ornithine / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D010750:Phosphoproteins; D005973:Glutamine; D001120:Arginine; D009952:Ornithine; D011494:Protein Kinases; D000251:Adenosine Triphosphatases; D008239:Lysine