腸炎弧菌磷脂醯絲胺酸脫羧酶之調控

The regulation of phosphatidylserine decarboxylase in Vibrio parahaemolyticus

張庭瑋(Ting-Wei Chang)

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(2015年)

https://doi.org/10.6342/NTU.2015.10942

摘要

磷脂醯絲胺酸脫羧酶(Psd)參與磷脂質代謝及內膜磷脂質比例的改變，會影響蛋白質正確折曡及Cpx膜壓力反應。Psd是磷脂醯乙醇胺(PE)生合成途徑最終端的重要酵素，將磷脂醯絲胺酸(PS)進行脫羧反應後形成PE。本研究以腸炎弧菌Vibrio parahaemolyticus no.93作為實驗菌株，建構腸炎弧菌cpxR刪除突變株及補償株，探討psd基因在腸炎弧菌中是否受CpxR調控。本研究發現腸炎弧菌psd啟動子區域具有與轉錄調控子CpxR保守性結合位相似之核苷酸序列。由即時定量聚合酶連鎖反應分析顯示，psd基因在cpxR刪除突變株的表現量高於野生株，而 cpxR補償株具有回補作用，證實psd基因受到CpxR負向調控。經由以psd啟動子觀察報導基因luxA及luxB的螢光表現發現，實驗菌株進入穩定生長期後，cpxR刪除突變株的螢光表現量高於野生株，推論腸炎弧菌CpxR透過與psd啟動子的作用以進行調控。此外，CpxR對於cpxP基因也具有抑制的現象。在野生株及cpxR刪除突變株之移動能力實驗發現，野生株的游走及泳動能力皆優於cpxR刪除突變株，顯示腸炎弧菌的游走及泳動能力受到CpxR正向影響。然而，腸炎弧菌之移動能力在cpxR補償株中並無回補現象，推測其移動能力可能也受其他因素影響。在psd過表現株的移動能力實驗中發現，psd過表現對腸炎弧菌的游走能力具有抑制的現象。本研究結果證實腸炎弧菌psd基因受CpxR負向調控，亦確認其啟動子位於轉譯起始點至上游第118個核苷酸間之區域。

關鍵字

腸炎弧菌 ； 磷脂醯絲胺酸脫羧?

並列摘要

Phosphatidylserine decarboxylases (Psd) is involved in phospholipid metabolism and alterations in the ratio of inner membrane phospholipids, which have been shown to affect both protein folding and the Cpx envelope stress response. Psd catalyzes the formation of phosphatidylethanolamine (PE) by decarboxylation of phosphatidylserine (PS). In this study, we construct V. parahaemolyticus cpxR-deletion mutant to investigate the relation between CpxR and psd. The promoter region of V. parahaemolyticus psd contains a conserved sequence, wich is similar with the conserved CpxR-binding site. The RLU/OD600 in cpxR-deletion mutant was higher than in wild type at the stationary phase. By qRT-PCR analysis, the transcript level of psd in cpxR-deletion mutant was higher than wild type, suggesting that CpxR may negatively regulate psd expression. The CpxR also negatively regulate cpxP expression. The swimming and swarming ability of wild type was better than cpxR-deletion mutant, suggesting that the swimming and swarming ability of V. parahaemolyticus is positively affected by CpxR. Conclude the results, V. parahaemolyticus psd is negatively regulated by CpxR, and the promoter of psd gene is located at upstream region.

並列關鍵字

vibrio parahaemolyticus ； phosphatidylserine decarboxylase

參考文獻

Acosta, N., Pukatzki, S., and Raivio, T.L. (2015). The Cpx system regulates virulence gene expression in Vibrio cholerae. Infection and Immunity 83, 2396-2408.

Batchelor, E., Walthers, D., Kenney, L.J., and Goulian, M. (2005). The Escherichia coli CpxA-CpxR envelope stress response system regulates expression of the porins ompF and ompC. Journal of Bacteriology 187, 5723-5731.

Bullock, W. (1987). XL-1 Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. BioTechniques 5, 376-379.

Cao, J., Woodhall, M.R., Alvarez, J., Cartron, M.L., and Andrews, S.C. (2007). EfeUOB (YcdNOB) is a tripartite, acid‐induced and CpxAR‐regulated, low‐pH Fe2+ transporter that is cryptic in Escherichia coli K‐12 but functional in E. coli O157: H7. Molecular Microbiology 65, 857-875.

Choi, J.Y., Duraisingh, M.T., Marti, M., Ben Mamoun, C., and Voelker, D.R. (2015). From protease to decarboxylase: the molecular metamorphosis of phosphatidylserine decarboxylase. The Journal of Biological Chemistry 290, 10972-10980.