Summary
A strain of Escherichia coli exhibiting reduced activity of the periplasmic enzyme acid phosphoanhydride phosphohydrolase (pH 2.5 acid phosphatase) was isolated. The mutation designated appA1 was located at 22.5 min on the E. coli genetic map. Acid phosphatase purified from an appA −transductant showed less than ten percent of the specific activity of an isogenic appA +strain. The mutant enzyme was highly thermolabile and its Km for paranitrophenyl phosphate was increased about 20-fold. The mutant protein cross-reacted with antibody to the wild-type enzyme and had the same molecular weight and concentration in extracts as the wild-type enzyme. These findings strongly suggest that appA is the structural gene of the acid phosphatase.
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Abbreviations
- PNPP:
-
paranitrophenyl phosphate
- cAMP:
-
3′-5′-cyclic adenosine monophosphate
- Nitrosoguanidine:
-
N-methyl-N'-nitro-N-nitrosoguanidine
- TCY:
-
tetracycline
- KAN:
-
kanamycin
- STR:
-
streptomycin
References
Berg PE (1981) Cloning and characterization of the Escherichia coli gene coding for alkaline phosphatase. J Bacteriol 146:660–667
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Dassa E, Boquet PL (1981a) Is the acid phosphatase of Escherichia coli with pH optimum of 2.5 a polyphosphate depolymerase? FEBS Lett 135:148–150
Dassa E, Boquet PL (1981b) expA: A conditional mutation affecting the expression of a group of exported proteins in Escherichia coli K12. Mol Gen Genet 181:192–200
Dassa E, Tetu C, Boquet PL (1980) Identification of the acid phosphatase (optimum pH 2.5) of Escherichia coli. FEBS Lett 113:275–278
Dassa E, Cahu M, Desjoyaux-Cherel B, Boquet PL (1982) The acid phosphatase with optimum pH of 2.5 of Escherichia coli: physiological and biochemical study. J Biol Chem 257:6669–6676
Dvorak HF, Brockman RW, Heppel LA (1967) Purification and properties of two acid phosphatase fractions isolated from osmotic shock fluid of Escherichia coli. Biochemistry 6:1743–1751
Fangman WL, Nass G, Neidhardt PC (1965) Immunological and chemical studies of phenylalanine sRNA synthetase from Escherichia coli. J Mol Biol 13:202–219
Garen A, Levinthal C (1960) A fine structure genetic and chemical study of the enzyme alkaline phosphatase in E. coli. I) Purification and characterization of the alkaline phosphatase. Biochim Biophys Acta 38:470–479
Hafckenscheid JCM (1968) Properties of an acid phosphatase in Escherichia coli. Biochim Biophys Acta 167:582–589
Heppel LA (1971) The concept of periplasmic enzymes in Rothfield LI (ed) Structure and functions of biological membranes. Academic Press, New York, p 233
Hofsten B von, Porath J (1962) Purification and properties of an acid phosphatase from Escherichia coli. Biochim Biophys Acta 64:1–12
Inouye H, Pratt C, Beckwith J, Torriani A (1977) Alkaline phosphatase synthesis in a cell-free system using DNA and RNA templates. J Mol Biol 110:75–87
Inouye H, Michaelis S, Wright A, Beckwith J (1981) Cloning and restriction mapping of the alkaline phosphatase structural gene phoA of Escherichia coli and generation of deletion mutants in vitro. J Bacteriol 146:668–675
Low B (1973) Rapid mapping of conditional and auxotrophic mutations in Escherichia coli K12. J Bacteriol 113:798–812
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
Morris H, Schlesinger MJ, Bracha M, Yagil E (1974) Pleiotropic effects of mutations involved in the regulation of escherichia coli K12 alkaline phosphatase. J Bacteriol 119:583–592
Rogers D, Reithel FJ (1960) Acid phosphatases of Escherichia coli. Arch Biochem Biophys 89:97–104
Rothman F, Byrne R (1963) Fingerprint analysis of alkaline phosphatase of Escherichia coli K12. J Mol Biol 6:330–340
Schlesinger MJ (1965) The reversible dissociation of the alkaline phosphatase of Escherichia coli: II) Properties of the subunit. J Biol Chem 240:4293–4298
Schlesinger MJ, Barrett K (1965) The reversible dissociation of the alkaline phosphatase of Escherichia coli: I) Formation and reactivation of the subunits. J Biol Chem 240:4284–4292
Schlesinger MJ, Levinthal C (1963) Hybrid protein formation of E. coli alkaline phosphatase leading to in vitro complementation. J Mol Biol 7:1–12
Schlesinger MJ, Reynolds JA, Schlesinger S (1969) Formation and localization of the alkaline phosphatase of Escherichia coli. Ann NY Acad Sci 166:368–379
Tétu C, Dassa E, Boquet PL (1979) Unusual pattern of nucleoside polyphosphate hydrolysis by the acid phosphatase (optimum pH 2.5) of Escherichia coli. Biochem Biophys Res Commun 87:314–322
Wu TT (1966) A model for three-point analysis of random general transduction. Genetics 54:405–410
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Communicated by A. Böck
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Dassa, E., Boquet, P.L. Identification of the gene appA for the acid phosphatase (pH optimum 2.5) of Escherichia coli . Molec. Gen. Genet. 200, 68–73 (1985). https://doi.org/10.1007/BF00383314
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DOI: https://doi.org/10.1007/BF00383314