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 Lisa

Research output: Contribution to journalArticleAcademicpeer-review

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.

Original languageEnglish
Pages (from-to)251-6
Number of pages6
JournalCurrent Microbiology
Volume50
Issue number5
DOIs
Publication statusPublished - May 2005
Externally publishedYes

Keywords

  • Base Sequence
  • Cloning, Molecular
  • Escherichia coli/metabolism
  • Ethanolamines/metabolism
  • Molecular Sequence Data
  • Nitrophenols/metabolism
  • Organophosphorus Compounds/metabolism
  • Phosphoric Monoester Hydrolases/biosynthesis
  • Phosphorylcholine/metabolism
  • Plasmids
  • Pseudomonas aeruginosa/enzymology

Cite this

Massimelli, M. J., Beassoni, P. R., Forrellad, M. A., Barra, J. L., Garrido, M. N., Domenech, C. E., & Lisa, A. T. (2005). Identification, cloning, and expression of Pseudomonas aeruginosa phosphorylcholine phosphatase gene. Current Microbiology, 50(5), 251-6. https://doi.org/10.1007/s00284-004-4499-9