| Abstract: | 十字花科黑腐致病菌是屬革蘭氏陰性菌的一種，利用type II蛋白分泌機制分泌一些致病酵素，造成十字花科植物根部腐爛，萎縮而死亡。革蘭氏陰性菌之胞外蛋白分泌系統目前共被鑑定出六種，其中Type II是分佈最廣且最為常見的，又稱之一般分泌途徑(general secretory pathway; GSP)。該系統之外膜分泌機制主要由十二至十三個基因產物所構成，分別命名為GspCDEFGHIJKLMNO。在由這些基因所表現之蛋白中由於GspGHIJ的NH2-端具有與PilA纖毛的N-端有很當高的同源性，因此認為這些蛋白會形成pili-like的結構，但是截自目前GspGHIJ的纖毛結構一直未被鑑定分離出來。在本研究中，主要是利用生化與遺傳方法，對十字花科黑腐病菌蛋白質外膜分泌系統中XpsG的C端序列做結構與功能上的探討，於是構築了二個XpsG突變重組質體pFG-Y136及pFG-Y142。在互補實驗中，發現突變株喪失分泌澱粉酵素的功能；干擾實驗中突變株則不會影響野生株的分泌功能。以XpsG抗體偵測XpsG突變蛋白的分佈，結果突變與野生XpsG蛋白一樣具有水溶性及脂溶性兩種形式存在。以膠質篩濾層析法分析這兩種型式的突變與野生蛋白結構上的差異性，發現突變與野生XpsG蛋白一樣，在清潔劑0.5% DOC處理下，此兩種型式會由multimer form解離成雙體型dimer form。改以1% Triton X-100處理時，則複合體型式部分解離成較小分子的複合體。由於膠質篩濾層析法無法區別XpsG突變蛋白與XpsG蛋白在結構上的差異性，於是再以陰離子交換樹脂層析管柱分析，結果發現突變與野生XpsG蛋白之間有cofractionation的現象。為了進一步瞭解XpsG突變蛋白與XpsG蛋白之間是否有交互作用，於是再構築一突變株XC1701/pY142 (His)6-tag，利用Ni-NTA column親和性分析管柱分析。結果顯示， XpsG蛋白不會與XpsG-Y142(His)6-tag突變蛋白同時被高濃度的imidazole沖堤液沖堤出，由此可知，突變蛋白XpsG-Y142與XpsG蛋白之間沒有交互作用。以蛋白水解酵素切割的方法，探討兩者之間的結構是否有所不同。結果顯示，突變蛋白XpsG-Y136對蛋白proteinase K作用呈現敏感，XpsG蛋白則有抵抗性，暗示突變株的構形不同於野生株。總結，由本研究之結果推論XpsG的C端序列對於維持其構形是非常重要的。
Xanthomonas campestris, a Gram-negative bacterium, secretes toxic enzymes, through a type II protein secretion mechanism, to damage the roots of Xanthomonas plants. There are six different protein secretion mechanisms in Gram-negative bacteria were identified to date. Among these mechanisms, type II is the most commonly found and is called “the general secretory pathway” (GSP) for Gram-negative bacteria. Twelve to thirteen different gene products were discovered in GSP, and these proteins were named GspCDEFGHIJKLMNO. Amino acid analysis of GSP proteins have revealed that a significant sequence homology was found between the N-termini of GspGHIJ and PilA pili proteins, and therefore, it was speculated that the protein products of GspGHIJ might form a pili-like structure. However, the pili-like structures of GspGHIJ proteins have not been demonstrated yet. Using XpsG protein, a GspG protein from Xanthomonas campestris, as a model, we attempt to disclose the structure of the C-terminal region of XpsG. Two XpsG mutants, pFG-Y136 and pFG-Y142, were prepared and their enzymatic activities were measured. We found that both of these XpsG mutants lost their functions in amylase secretion in our complementary assays. However, wild-type amylase secretion remained normal even in the presence of the mutant XpsGs. Determined by immunoblotting analysis using an antibody against the XpsG protein, we found that either mutant or wild-type XpsG protein has both water-soluble and membrane-bound forms. No significant difference in structure was found between wild-type and mutant XpsG proteins, since their gel filtration chromatography profiles are similar even in the presence of 0.5% DOC or 1% Triton X-100. However, the mutant proteins and the wild-type XpsG proteins are found in the same fraction in an anion exchange chromatography. Suggesting that both wild-type and mutant XpsG protines might interact to each other. To determine the interaction between wild-type and mutant XpsG protein, another mutant called XC1701/pY142(His)6-tag was created. Using Ni-NTA affinity chromatography to purify this fusion protein, we found no wild-type protein was co-purified with the His-tagged mutant protein. Suggesting that no interaction between wild-type and mutant XpsG proteins. Using protease digestion of mutant XpsG protein, we found that the XpsG-Y136 was sensitive to proteinase K while the wild-type XpsG protein was not. Indicating that the protein structure of these proteins are different and the C-terminal region of XpsG protein might be important for its function. |
