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The Structural Genomics of Pathogenic, Flagellar, and Other Proteins of Important Functions of Xanthomonas campestris
Xanthomonas campestris (Xcc) 為一引起十字花科植物黑腐病的革蘭氐陰性細菌， 它可產生許多的exopolysaccharide，xanthan gum及分泌一些胞外酵素，包括蛋白酶，pectinases及endoglucanases等。而這些胞外酵素一直都被認為是Xanthomonas campestris重要的致病因子。它是目前世界上許多結構基因体研究領域中，被研究的唯一植物病原菌。而它的一些分泌機制卻是細胞中不同分泌類別，被研究較廣泛的一株。由於這獨特性，Xcc是一株頗值得利用結構基因体學研究探討的細菌。但是它最特別的是並不俱cAMP及其接受蛋白CRP(或CAP)，而係利用另一類似蛋白Clp (cAMP-Receptor-Like-Protein)來執行所有訊息調控的功能。重要的是Clp並不須cAMP即可與操作子核酸結合，與CRP須cAMP結合成複合体才能與核酸結合完全不同。目前已有數據顯似Clp可能與c-di-GMP結合而失去活性。所以解出Clp蛋白結構及其與c-di-GMP複合体的結構是瞭解Xcc特殊信息調控網路的重要步驟。同時最近研究亦發現Clp蛋白可直接或間接調節約三百個與Xcc致病有關的基因表現。所以本計畫之一即為Xcc的致病結構基因体計畫，研究與Xcc致病有關的蛋白結構。本計畫的另一大項為Xcc的纖毛結構基因体研究。細菌的纖毛結構可說是生物界的一大奇妙奈米級結構，其對細菌的存活及致病性有很大的關聯。它一般由超過40個以上的基因控制，而這些數目極大的基因產物如何一步步的自行組裝成俱超級功能的纖毛實在是一頗值得探索的題目。同時另一值得關注的是，細菌纖毛的rod, hook,及filament等結構單体都必須在細胞質合成後再運送到胞外組裝。這些單体倒底如何避免在細胞質中產生聚合体，而只會在胞外產生聚合，必形成rod, hook,或filament的結構，實在另人好奇。目前已有證據證明這些單体的運送必須藉由特別chaperon與其結合，避免其在胞內形成聚合体。所以本計畫的另一項目為研究Xcc基因與纖毛結構形成有關的蛋白(當然暫時避免膜蛋白及已被發表的結構如hook及filament等)，並研究一些特殊chaperon及其與纖毛蛋白的複合体結構。本計畫的第三大項則為進行Xcc的俱重要功能且結構未曾報導的蛋白結構。如與核酸修補有關的RecX及與RecA複合体的結構及PilZ與di-c-GMP複合体的結構等等。相信以上三項有關Xcc結構基因体計畫將對Xcc如何致病、纖毛形成、及一些重要功能蛋白的結構等會有進一步的瞭解。
Xanthomonas campestris is a gram-negative bacterium causing black rot in crucifers. It is capable of producing great amounts of exopolysaccharide, xanthan gum, and secreting several extracellular enzymes, including proteases, pectinases, and endoglucanases. These extracullular products have long been considered important virulence factors of X. campestirs. Currently there are many structural genomics projects that are being carried out worldwide, but it is the only phytopathogen that is being studied using the structural genomics approach. Also, it is also the most studied bacterium regarding the various secretion systems existing in a cell. Due to these peculiar characteristics, Xcc is a bacterium that deserves detailed studies from structural genomics perspective.Furthermore, another important characteristic about Xcc is that it lacks cAMP and its receptor CRP (or CAP) protein. In other words, it uses an alternative secondary messenger system to transmit the various signals vital for its survival. Interestingly, CLP (cAMP-Receptor-Like-Protein) has been discovered as the missing link and can bind to the promoter region without cAMP. Recently, there is also unpublished data indicating that CLP may bind with c-di-GMP to lessen its binding with promoter. It is therefore extremely important to be able to determine the tertiary structure of CLP as a first step toward demystify CLP related network. Another important finding is that CLP is responsible for regulating approximately 300 pathogenic genes in Xcc. Therefore, in the first part of our project, we aim to work on the pathogenic structural genomics related to CLP factor.The second part in this project would be the Xcc flagellar structural genomics. The bacterial flagellar is one of the most amazing nano-structure in the living world. It is extremely important for a bacterium survival and pathogenicity. It is usually under control of more than 40 gene products, and it is really interesting to imagine how these 40 gene products are able to self-assemble it into a functional flagellum. Another point deserves consideration is that the rod, hook, and filament substructure are all polymeric and comprise of subunits that need to synthesized in the cytoplasm. However, somehow they are not allowed to polymerize in the cell, and have to be exported outside to start polymerize and assemble into a mature flagellum. Recently, there have been some proofs that some specific chaperons are required to bind with these monomer and escort them to the secretion apparatus to prevent their polymerization in the cell. So in the second part, we plan to determine all flagellar protein structures (membrane proteins are temporarily excluded, along with those that have PDB file already), and the chaperon/subunit complex structures in this flagellar structural genomics.The third part of this proposal is to study Xcc proteins that serve important functions, yet their structures are still not reported. For examples, the RecX protein and its complex with RecA that is involved in important DNA repair, and the PilZ protein and its association with di-c-GMP, which has been a very hot project regarding a novel messenger transduction.I believe that the above three projects, (pathogenic and flagellar structural genomics studies, and protein determination with important functions) would contribute significantly toward our understanding about the Xcc pathogenicity, flagellar formation, and complete structural annotation.
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