Identification of phosphorylcholine phosphatase gene in Pseudomonas aeruginosa PAO1 genome

MASSIMELLI, MJ; BEASSONI, PR; BARRA, JL; GARRIDO, MN; LISA, AT

Villa Carlos Paz, Córdoba

Congreso; XXXVIII Reunión Anual, Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2002

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

One of the challenges of the future is the functional analysis of putative ORFs identified in genome sequencing projects and their integration into metabolic pathways. The availability of the Pseudomonas aeruginosa genome sequence permits many strategies for elucidation of gene functions of this microorganism. By transposon mutagenesis, an acid phosphatase (AcP) deficient mutant strain was obtained (JUF8-00). AcP is an enzyme involved in choline metabolism and some kinetic studies allowed us to identify this enzyme as a phosphorylcholine phosphatase (PcP). Cloning and sequencing of Tn5::571 neighbouring DNA of JUF8-00 revealed that the knockout gene was present in P. aeruginosa PAO1 genome database. Sequence analysis of this locus revealed an open reading frame of 1,047-bp (349 aa) reported to encode a hypothetical protein. We named it pcP for phosphorylcholine phosphatase. The translated protein was compared with sequences in the Gene-Bank and it revealed homology with several phosphohydrolases, and phosphoserine phosphatase proteins. Complementation of JUF8-00 with wild type pcP gene on the pBBR1MCS-5 plasmid partially restored the PcP activity. Furthermore, the complemented strain was able to hydrolyze the artificial substrate (p-NPP) and also hydrolyzed phosphorylcholine. In conclusion, our results strongly suggest that the reported sequence is responsible for PcP activity in P.aeruginosa