土壤假单胞菌593使用Pcs途径合成磷酯酰胆碱

Soil bacterium Pseudomonas sp. 593 synthesizes phosphatidylcholine via phosphatidylcholine synthase pathway

【目的】原核生物有两条代谢途径N-甲基化途径(Pmt途径)和磷脂酰胆碱合酶途径(Pcs途径)合成磷脂酰胆碱(PC)。本文对土壤细菌Pseudomonas sp.593的磷脂酰胆碱合成进行了研究,试图弄清楚假单胞菌的磷脂酰胆碱的合成途径。【方法】通过氨基酸序列比较,获得已报道的磷脂酰胆碱合酶(Pcs)的氨基酸保守序列,并设计简并引物,从Pseudomonas sp.593总DNA中PCR扩增出磷脂酰胆碱合酶基因(pcs)的片段,然后用扩增的DNA片段作探针,对Pseudomonas sp.593基因组DNA亚克隆文库进行菌落原位杂交,获得pcs全基因序列;利用同源重组原理进行活体突变,获得Pseudomonas sp.593 pcs-突变体;采用薄层层析(TLC)法分析细菌总磷脂,检测PC含量以及pcs基因活性。【结果】TLC分析显示Pseudomonas sp.593细菌仅在添加外源胆碱的M9或LB培养基中生长时才合成PC;从Pseudomonas sp.593细菌中克隆出894 bp的DNA序列,编码的蛋白具有磷脂酰胆碱合酶活性;活体缺失pcs基因后,Pseudomonas sp.593 pcs-突变体在添加或不添加胆碱的条件下都不能合成PC。【结论】Pcs途径是土壤Pseudomonas sp.593乃至其它假单胞菌合成磷脂酰胆碱的唯一途径。

[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.