磷脂醯絲胺酸合成酶對腸炎弧菌運動性之影響

The phosphatidylserine synthase affects the motility of Vibrio parahaemolyticus

王婕(Chieh Wang)

指導教授 ： 李佳音

國立臺灣大學/生物資源暨農學院/農業化學研究所/碩士(2011年)

https://doi.org/10.6342/NTU.2011.10677

摘要

腸炎弧菌 (Vibrio parahaemolyticus) 屬於嗜鹽性的革蘭氏陰性菌，主要生長於海水中。如果食用了遭受污染的海鮮，會引發食物中毒造成腹瀉的症狀。腸炎弧菌與絕大部分細菌一樣，構成細胞膜主要的磷脂質成分為磷脂醯乙醇胺 phosphatidylethanolamine (PE)。在 cytosine diphosphate-diacylglycerol (CDP-DAG) pathway 中，上游基因磷脂醯絲氨酸合成酶 (phosphatidylserine synthase, PSS) 可合成出磷脂醯絲氨酸 phosphatidylserine (PS)，再經由去羧基化作用 (decarboxylation) 合成磷脂醯乙醇胺 phosphatidylethanolamine (PE)。 由此可知PS在磷脂代謝路徑中扮演了一個關鍵的中間角色。本研究以缺乏 tdh，trh 毒力基因的腸炎弧菌臨床菌株 V. parahaemolyticus No. 93 (VP93) 為研究材料，欲探討 pssA 基因對腸炎弧菌生長及運動性之影響。以同源基因置換方式建構出腸炎弧菌 pssA 缺陷株 (ΔpssA-VP93) 雖然已將 pssA 基因剔除，但以HPTLC進行分析仍含有少量的PE，但與野生株相比其 PE 產量已經大幅下降。20 mM 的 Mg2+ 有助於腸炎弧菌野生株與 ΔpssA-VP93生長，但即使含有 Mg2+， ΔpssA-VP93 菌體生長速率及菌體生成量皆比野生株低。ΔpssA- VP93的泳動及遊走能力較野生株差，隨著 Mg2+ 濃度的升高，ΔpssA-VP93 的泳動走能力會大幅減弱，遊走能力不會受到 Mg2+ 影響。進而以 QPCR 分析鞭毛相關基因的轉錄情形，當菌體生長於液態培養基中，ΔpssA-VP93 的 flaD 基因表現量比野生株降低 2.9 倍； fliS 基因表現量比腸炎弧菌野生株減少了 4.8 倍，且當菌體缺乏 pssA 基因時， Mg2+ 會抑制 lafK、cheY、motA 基因表相量。但當 ΔpssA-VP93 生長於固態培養基時，lafK、motA、motB 基因表現量比野生株高 0.8 – 2 倍，且 Mg2+ 會促進 ΔpssA-VP93 鞭毛相關基因的表現量。由此可知，pssA 基因會影響腸炎弧菌之生長，但 pssA 不是一個必須基因；膜脂質成分的改變與腸炎弧菌運動力有所相關，且Mg2+ 會抑制 ΔpssA-VP93 泳動能力。

關鍵字

腸炎弧菌 ； 磷脂醯絲胺酸合成酶運動性

並列摘要

Phosphatidylethanolamine (PE) is an abundant phospholipid in bacterial cell membrane. In bacteria, the biosynthesis of PE proceeds via CDP-DAG pathway. On the pathway, PS is generated by a reaction between CDP-diacylglycerol (CDP-DAG) and serine, which is catalyzed by the single identified PS synthase (PSS). The PS produced is decarboxylated to form the major membrane phospholipid PE. This shows that PS plays a central role in phospholipids biosynthesis pathway. The pssA mutants of E. coli were conditional lethal and temperature-sensitive. Since PS is a precursor of PE through the action of phosphatidylserine decarboxylase (PSD), the level of PE of these mutants declines dramatically. It is interesting that the pssA mutants of E. coli lack PE can be suppressed by the addition of sucrose and divalent cations, such as Mg2+, Ca2+. Apparently, the properties of cardiolipin change can substitute for PE in the presence of divalent cations. In this study, we constructed the pssA deletion mutant by using gene replacement method in a clinical strain V. parahaemolyticus No. 93, which lacking of tdh and trh. We examined the physiological role of PE biosynthesis gene pssA of Vibrio parahaemolyticus. The growth rate of pssA deletion mutant (ΔpssA) was severely impaired no matter in the medium of TSB3 and APSB3, but finding the addition of MgCl2 could help their growth. The ΔpssA was decreased in swimming motility when compared with that of wild-type in all test medium. The differential expression of the identified flagella genes in ΔpssA was further investigated by QPCR. The results showed that the transcription level of flaD and fliS were markedly lower by 2.9 and 4.8 folds in ΔpssA, respectively. It indicated that the pssA gene is required for the motility of V. parahaemolyticus.

並列關鍵字

Vibrio parahaemolyticus ； phosphatidylserine synthase ； pssA, motility

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