PA4496 is one of the genes responsible to encode acetylcholinesterase in Pseudomonas aeruginosa PA01

GONZÁLEZ, PJ; BEASSONI, PR; DOMENECH, CE; LISA, AT

Rosario, Argentina

Congreso; XLII Reunión Anual, Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2006

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

P. aeruginosa acetylcholinesterase (AChE) is produced when bacteria are grown with choline or betaine as nutritional source. Attempts to identify the gene encoding for AchE by random mutagenesis, were unsuccessful. The difficulty to found an AchE- phenotype led us to purify a protein with AchE activity, and use it to identify the encoding gene. Periplasmic extract obtained from betaine-grown cells, was precipitated with ammonium sulfate, and filtered in a Sephacryl S-300 column. An AChE activity 31,3 fold purified was obtained. The enzyme was found to be a monomer of 60,6 kDa with a pI of 8,5. After 2D electrophoresis AChE was detected with silver stain and by enzymatic histochemical assay. Peptide mass fingerprints (PMF) using MALDI-TOF mass spectrometry followed by in silico analysis showed that this purified protein corresponds to PA4496 gene from P. aeruginosa PAO1. Up to this moment, this gene is annotated in the data bank as a probable periplasmic component of the ABC transporter. PA4496 gene has a similarity of 41% with human AChE 1B41. PA4496 full-length gene was PCR amplified, cloned in pTYB12, expressed in E. coli ER2566 as an intein-fused protein, and purified by a chitin column. Even though we cannot discard the existence of other proteins with AchE activity, our present results indicate that PA4496 is at least one of the gene responsible to encode an AchE activity