創傷弧菌YJ016中一套訊號傳遞系統vva0326~vva0329的特性分析探討

Abstract

在創傷弧菌YJ016的基因體中，我們發現了一組基因群和霍亂弧菌的三分子調控系統VieSAB序列相似。而分析這套基因組轉譯後的蛋白序列顯示VVA0329為感應蛋白，VVA0328 和VVA0327分別和VieA的N端、C端的序列相似，而VVA0326則為具有GGDEF功能區塊的蛋白。許多報導證實：含有GGDEF和EAL功能區塊的蛋白，分別具有雙鳥苷酸環化酶（Digunylate Cyclase）及磷酸雙脂鍵分解酶（Phosphodiesterase）的活性。在此論文研究中，我們發現將VVA0328中的EAL區域剔除，會降低創傷弧菌YJ016的泳動性，但會增加生物膜的生成。相對的，剔除VVA0326的GGDEF區塊，會使創傷弧菌的泳動力增加，而減少生物膜的生成。而量測VVA0326缺損的創傷弧菌細胞內環狀雙鳥苷酸（cyclic di-GMP）的濃度時，我們發現環狀雙鳥苷酸的含量在VVA0326缺損株中明顯降低，而在此缺損株轉形給予VVA0326的表現質體時，可以提高環狀雙鳥苷酸的濃度，這個結果暗示VVA0326蛋白具有雙鳥苷酸環化酶的活性。同時，我分別構築了攜帶VVA0326的GGDEF區域及VVA0328的EAL區域的表現質體，進一步在大腸桿菌中大量表現並純化這兩個重組蛋白。經酵素活性測試，我們發現這兩個僅帶有GGDEF及EAL區塊的重組蛋白分別具有微弱的雙鳥苷酸環化酶和磷酸雙脂鍵分解酶的活性。由此可推知，VVA0326和VVA0328蛋白可能藉著相對的酵素活性來調控細菌體內環狀雙鳥苷酸的濃度，進而影響細菌的表現型態。我們實驗室先前的實驗發現，分別將VVA0326和VVA0328和螢光表現蛋白作轉譯融合的重組蛋白，在大腸桿菌中會有極化的表現，顯示這兩個蛋白中可能帶有造成蛋白極化分佈的序列。我將這兩個蛋白分別截短後發現，這種極化分佈的現象便消失了。這項結果指出，VVA0326及VVA0328蛋白極化分佈的現象可能需要完整長度的蛋白；而另一個可能性是我們在作截短構築時破壞了特定接受極化信號的序列。最後，我以反轉錄核酸增幅反應（RT-PCR）確認vva0326~vva0329基因組同屬一操縱子（operon）。

In Vibrio vulnificus YJ016 genome, a gene cluster highly homologous to the three-component system VieSAB of V. cholerae was identified. This consists of VVA0329, encoding a VieS homolog, VVA0328 and VVA0327, exhibiting homology with the N- and C-terminal parts of VieA, respectively, and VVA0326, encoding a protein with a GGDEF domain. The GGDEF- and EAL-domain containing proteins have been reported to carry enzymatic activity of diguanylate cylase and phosphodisterase, respectively. Deletion of the EAL domain from VVA0328 reduced the swimming motility but enhanced the biofilm formation activity of V. vulnificus YJ016. On the other hand, deleting GGDEF domain from VVA0326 protein increased the bacterial swimming activity but diminished the biofilm formation. The reduced level of c-di-GMP could be complemented upon introducing a VVA0326 expression plasmid into the VVA0326 mutant, indicating that VVA0326 containing GGDEF domain likely possesses diguanylate cyclase activity involved in c-di-GMP synthesis. In addition, the GGDEF and EAL domains have been subcloned into expression vector, the recombinant proteins synthesized in Escherichia coli and purified. The subsequent enzyme activity analysis revealed that the recombinant proteins respectively carry low level of DGC and PDE activity. This indicates that VVA0326 and VVA0328 regulate intracellular c-di-GMP level inversely to influence the bacterial behaviors. We have previously reported that the recombinant E. coli JM109 carrying either VVA0326-GFP or VVA0328-GFP exhibited green fluorescence at the cell poles. To investigate if a specific sequence is present for the polar localization, several truncated forms of VVA0326 and VVA0328 were generated. The following fluorescent microscopy analysis revealed that all the truncation led to disperse distribution of the fusion proteins. This suggested that the entire protein of VVA0326 or VVA0328 is required for the polar localization. However, the possibility that the truncation interrupted the signature for the polar localization could not be ruled out. Finally, we have shown that the gene cluster vva0326~vva0329 is an operon using RT-PCR analysis.