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Current Microbiology

, Volume 50, Issue 5, pp 251–256 | Cite as

Identification, Cloning, and Expression of Pseudomonas aeruginosa Phosphorylcholine Phosphatase Gene

  • María J. Massimelli
  • Paola R. Beassoni
  • Marina A. Forrellad
  • José L. Barra
  • Mónica N. Garrido
  • Carlos E. Domenech
  • Angela T. LisaEmail author
Article

Abstract

Pseudomonas aeruginosa phosphorylcholine phosphatase (PChP) is a periplasmic enzyme produced simultaneously with the hemolytic phospholipase C (PLc-H) when the bacteria are grown in the presence of choline, betaine, dimethylglycine or carnitine. Molecular analysis of the P. aeruginosa mutant JUF8-00, after Tn5-751 mutagenesis, revealed that the PA5292 gene in the P. aeruginosa PAO1 genome was responsible for the synthesis of PChP. The enzyme expressed in E. coli, rPChP-Ec, purified by a chitin-binding column (IMPACT-CN system, New England BioLabs) was homogeneous after SDS-PAGE analysis. PChP was also expressed in P. aeruginosa PAO1-LAC, rPChP-Pa. Both recombinant enzymes exhibited a molecular mass of approximately 40 kDa, as expected for the size of the PA5292 gene, and catalyzed the hydrolysis of phosphorylcholine, phosphorylethanolamine, and p-nitrophenylphosphate. The saturation curve of rPChP-Ec and rPChP-Pa by phosphorylcholine revealed that these recombinant enzymes, like the purified native PChP, also contained the high- and low-affinity sites for phosphorylcholine and that the enzyme activity was inhibited by high substrate concentration.

Keywords

Recombinant Enzyme Phosphorylcholine Dimethylglycine Periplasmic Extract Phosphorylethanolamine 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Notes

Acknowledgments

We thank Dr. Fernando J. Irazoqui for advice and help with the preparation of mouse polyclonal anti-rPChP-Ec antibodies, the language consultants Iliana A. Martinez (UNRC) and Ann Lewis (USA) for assistance in the preparation of the manuscript, and Mariela Woelker for technical assistance. C.E.D., A.T.L., M.N.G., and J.L.B. are Career Members of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). M.J.M., P.R.B., and M.A.F. have a fellowship from CONICET. This work was supported by grants from CONICET, Agencia Córdoba Ciencia and SECYT-UNRC of Argentina.

Literature Cited

  1. 1.
    Beck, E, Ludwig, G, Auerswald, E, Reiss, B, Schaller, E 1982Nucleotide sequence and exact localization of the neomycin phosphotransferase gene from transposon Tn5Gene19327336CrossRefPubMedGoogle Scholar
  2. 2.
    Bradford, M 1976A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnal Biochem72248254PubMedGoogle Scholar
  3. 3.
    Collet, JF, Stroobant, V, Pirard, M, Delpierre, G, Schaftingen, E 1998A new class of phosphotransferases phosphorylated on an aspartate residue in an amino-terminal DXDX(T/V) motifJ Biol Chem2731410714112CrossRefPubMedGoogle Scholar
  4. 4.
    Cronin, CN, McIntire, WS 1999pUCP-Nco and pUCP-Nde: EscherichiaPseudomonas shuttle vectors for recombinant protein expression in PseudomonasAnal Biochem272112115CrossRefPubMedGoogle Scholar
  5. 5.
    Fiske, C, Subarrow, Y 1925The colorimetric determination of phosphorusJ Biol Chem66361375Google Scholar
  6. 6.
    Garcia, JL, Sanchez-Beato, AR, Medrano, FJ, Lopez, E 1998Versatility of choline binding domainMicrob Drug Resist42536PubMedGoogle Scholar
  7. 7.
    Garfin, D 1990One-dimensional gel electrophoresisMethods Enzymol182425441PubMedGoogle Scholar
  8. 8.
    Garrido, MN, Lisa, AT, Domenech, CE 1988Pseudomonas aeruginosa acid phosphatase contains an anionic site with a trimethyl subsite. Kinetic evidences obtained with alkylammonium ionsMol Cell Biochem844149CrossRefPubMedGoogle Scholar
  9. 9.
    Ho, C-L, Noji, M, Saito, K 1999Plastidic pathway of serine biosynthesis molecular cloning and expression of 3-phosphoserine phosphatase from Arabidopsis thalianaJ Biol Chem2741100711012CrossRefPubMedGoogle Scholar
  10. 10.
    Koonin, EV, Tatusov, R L 1994Computer analysis of bacterial haloacid dehalogenases defines a large superfamily of hydrolases with diverse specificity. Application of an iterative approach to database searchJ Mol Biol244125132CrossRefPubMedGoogle Scholar
  11. 11.
    Kovach, ME, Phillips, RW, Elzer, PH, Roop, RM, Peterson, KM 1994pBBR1MCS: a broad-host-range cloning vectorBiotechniques16800802PubMedGoogle Scholar
  12. 12.
    Lisa, TA, Casale, CH, Domenech, CE 1994Cholinesterase, acid phosphatase and phospholipase C of Pseudomonas aeruginosa under hyperosmotic conditions in a high-phosphate mediumCurr Microbiol287176CrossRefGoogle Scholar
  13. 13.
    Lisa, TA, Lucchesi, GI, Domenech, CE 1994Pathogenicity of Pseudomonas aeruginosa and its relationship to the choline metabolism through the action of cholinesterase, acid phosphatase and phospholipase CCurr Microbiol29193199CrossRefGoogle Scholar
  14. 14.
    Lucchesi, GI, Lisa, TA, Casale, CH, Domenech, CE 1995Carnitine resembles choline in the induction of cholinesterase, acid phosphatase, and phospholipase C and in its action as an osmoprotectant in Pseudomonas aeruginosaCurr Microbiol305560CrossRefPubMedGoogle Scholar
  15. 15.
    Lucchesi, GI, Lisa, AT, Domenech, CE 1989Pseudomonas aeruginosa phospholipase C activity in a high phosphate mediumFEMS Microbiol Lett57335338CrossRefGoogle Scholar
  16. 16.
    Nielsen, H, Brunak, S, Heijne, G 1999Machine learning approaches for the prediction of signal peptides and other protein sorting signalsProtein Eng1239PubMedGoogle Scholar
  17. 17.
    Ostroff, RM, Vasil, AI, Vasil, ML 1990Molecular comparison of a nonhemolytic and a hemolytic phospholipase C from Pseudomonas aeruginosaJ Bacteriol17259155923PubMedGoogle Scholar
  18. 18.
    Rella, M, Mercenier, A, Haas, D 1985Transposon insertion mutagenesis of P. aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene clusterGene33293303CrossRefPubMedGoogle Scholar
  19. 19.
    Salvano, MA, Domenech, CE 1999Kinetic properties of purified Pseudomonas aeruginosa phosphorylcholine phosphatase indicated that this enzyme may be utilized by bacteria to colonize in different environmentsCurr Microbiol3918PubMedGoogle Scholar
  20. 20.
    Sambrook, J, Russel, DW 2001Molecular cloning: a laboratory manual3Cold Spring Harbor Laboratory PressCold Spring Harbor, NYGoogle Scholar
  21. 21.
    Serra, AL, Mariscotti, JF, Barra, JL, Lucchesi, GI, Domenech, CE, Lisa, AT 2002Glycine betaine transmethylase mutant of Pseudomonas aeruginosaJ Bacteriol18443014303CrossRefPubMedGoogle Scholar
  22. 22.
    Simon, R, Priefer, U, Pühler, A 1983A broad host range mobilization system for in vitro genetic engineering: transposon mutagenesis in Gram negative bacteriaBio/Technology1784790CrossRefGoogle Scholar
  23. 23.
    Stonehouse, MJ, Cota-Gomez, A, Parker, SK, Martin, WE, Hankin, JA, Murphy, RC, Chen, W, Lim, KB, Hackett, M, Vasil, AI, Vasil, ML 2002A novel class of microbial phosphocholine-specific phospholipase CMol Microbiol46661676CrossRefPubMedGoogle Scholar
  24. 24.
    Stover, CK, Pham, XQ, Erwin, AL, Mizoguchi, SD, Warrener, P, Hickey, MJ,  et al. 2000Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogenNature406959964CrossRefPubMedGoogle Scholar
  25. 25.
    Vasil, ML, Prince, RW, Shortridge, VD 1993Exoproducts: Pseudomonas exotoxin A and phospholipase CFick, RB,Jr eds. Pseudomonas aeruginosa the opportunist: pathogenesis and diseaseCRC PressBoca Raton, FL6077Google Scholar
  26. 26.
    Vasil, ML, Vasil, AI, Shortridge, VD 1994Phosphate and osmoprotectants in the pathogenesis of Pseudomonas aeruginosaTorriani-Gorrini, AYagil, ESilver, S eds. Phosphate in microorganisms: cellular and molecular biologyASM PressWashington, DC126132Google Scholar

Copyright information

© Springer Science+Business Media, Inc. 2005

Authors and Affiliations

  • María J. Massimelli
    • 1
  • Paola R. Beassoni
    • 1
  • Marina A. Forrellad
    • 1
  • José L. Barra
    • 2
  • Mónica N. Garrido
    • 1
  • Carlos E. Domenech
    • 1
  • Angela T. Lisa
    • 1
    Email author
  1. 1.Departamento de Biología Molecular, Facultad de Ciencias Exactas, Fisicoquímicas y NaturalesUniversidad Nacional de Río CuartoCórdobaArgentina
  2. 2.CIQUIBIC, Departamento de Química BiológicaUniversidad Nacional de CórdobaCórdobaArgentina

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