Abstract:Abstract: [Objective] Prokaryotes synthesize phosphotidylcholine by using phospholipid N-methylation or phosphatidylcholine synthase pathway or both. To confirm which pathway the soil bacterium Pseudomonas sp. 593 utilizes, we tested its phosphotidylcholine synthesis, cloned the pcs gene encoding phosphatidylcholine synthase, examined Pcs activity, and constructed a pcs- mutant. [Methods] To clone the pcs gene from Pseudomonas sp. 593 genomic DNA, we firstly aligned amino acid sequences of phosphatidylcholine synthases in different pseudomonas strains reported in databases. Then we designed degenerate primers based on two amino acid segments conserved in sequences of phosphatidylcholine synthases. A partial fragment of the pcs gene was finally amplified from Pseudomonas sp. 593 genomic DNA. The amplified partial fragment was labeled with digoxigenin-dUTP (DIG) as a probe, sub-cloning library of Pseudomonas sp. 593 genomic DNA was prepared and then screened using DIG-labelled probe via in situ colony hybridization. DNA homologous recombination in vivo was preformed to delete pcs gene of Pseudomonas sp. 593. Thin-layer chromatography (TLC) assay was used to analyze total phospholipids, detect phosphotidylcholine content and determine pcs gene activity. [Results] TLC analysis revealed that Pseudomonas sp. 593 growing in the M9 or LB medium with choline was able to synthesize phosphotidylcholine, but wasn’t without addition of choline. A 894 bp DNA fragment coded a protein with phosphatidylcholine synthase activity was cloned from Pseudomonas sp. 593. The pcs- mutant obtained from in vivo mutagenesis was unable to form phosphotidylcholine, no matter choline was presented in the medium or not. [Conclusion] Phosphatidylcholine synthase pathway is a sole way for phosphotidylcholine synthesis in soil bacterium Pseudomonas sp. 593 or other Pseudomonas strains.
