Acid phosphatases of Escherichia coli: molecular cloning and analysis of agp, the structural gene for a periplasmic acid glucose phosphatase.
Several unknown Escherichia coli genes for different species of acid phosphatase were cloned in vivo with the plasmid Mu dII4042. When present in a multicopy state, each gene promoted hydrolysis of p-nitrophenyl-phosphate at acidic pH. Among seven recombinant clones that encoded periplasmic acid phosphatase activities, five different genes could be distinguished by the pH optimum and substrate preference for the enzyme and by the restriction enzyme pattern. A 1.7-kilobase recombinant DNA fragment, common to two clones, was inserted into plasmid pBR322 and shown to contain a new gene, agp, which leads to the overexpression of the periplasmic acid glucose-1-phosphatase, a dimer of a 44-kilodalton polypeptide. Fusions of agp to gene phoA deprived of its own signal sequence conferred an alkaline phosphatase-positive phenotype to bacteria, showing the presence of an export signal on agp. The resulting hybrid proteins were characterized by immunoprecipitation with an antiserum directed against purified acid phosphatase or against alkaline phosphatase, showing that agp is the structural gene of the acid phosphatase. The beginning, the orientation, and the end of gene agp on the cloned DNA fragment were determined by the characteristics of such hybrid proteins.
Pradel
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Département de Biologie, Centre d'Etudes Nucléaires de Saclay, Gif-sur Yvette, France.
Boquet
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PL
D000135:Acid Phosphatase / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005786:Gene Expression Regulation; D005796:Genes; D005798:Genes, Bacterial; D005952:Glucose-6-Phosphatase / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015183:Restriction Mapping; D013379:Substrate Specificity
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D000135:Acid Phosphatase; D005952:Glucose-6-Phosphatase