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

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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.

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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.

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Authors and Affiliations

  1. Departamento de Biología Molecular, Facultad de Ciencias Exactas, Fisicoquímicas y Naturales, Universidad Nacional de Río Cuarto, 5800, Río Cuarto, Córdoba, Argentina

    María J. Massimelli, Paola R. Beassoni, Marina A. Forrellad, Mónica N. Garrido, Carlos E. Domenech & Angela T. Lisa

  2. CIQUIBIC, Departamento de Química Biológica, Universidad Nacional de Córdoba, Córdoba, Argentina

    José L. Barra

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  1. María J. Massimelli
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  2. Paola R. Beassoni
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  3. Marina A. Forrellad
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  4. José L. Barra
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  5. Mónica N. Garrido
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  6. Carlos E. Domenech
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  7. Angela T. Lisa
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Correspondence to Angela T. Lisa.

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Massimelli, M.J., Beassoni, P.R., Forrellad, M.A. et al. Identification, Cloning, and Expression of Pseudomonas aeruginosa Phosphorylcholine Phosphatase Gene. Curr Microbiol 50, 251–256 (2005). https://doi.org/10.1007/s00284-004-4499-9

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