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標題: 十字花科黑腐病菌XpsL蛋白在第二型胞外蛋白分泌過程中的角色探討
Significance of XpsL Protein in Type II Secretion Pathway of Xanthomonas campestris pv. campestris
作者: 鍾宜靜
Chung, Yi-Ching
關鍵字: Xanthomonas campestris
Type II Secretion Pathway
出版社: 農業生物科技學研究所
摘要: 摘 要 十字花科黑腐病菌屬於革蘭氏陰性菌,具有二層胞膜,其對於胞外蛋白的分泌是屬於第二型分泌模式,依賴二套分泌系統以二個步驟分別通過內膜與外膜。負責外膜分泌系統的成員有XpsE、F、G、H、I、J、K、L、M、N、D及O等共十二個蛋白,推測可能在內、外膜間形成蛋白複合體通道以輔助蛋白分泌,本研究針對位於內膜之XpsL蛋白的生化特性和功能作探討。首先分別構築XpsL N半端與C半端親水性區域蛋白的重組表現質體,於大腸桿菌中大量製造(His)6-tagged的融合蛋白,純化後作為抗原免疫兔子得到anti-XpsNL與anti-XpsCL抗體;同時利用同源性基因置換的方式,創造xpsL基因缺損的黑腐病菌菌株,利用上述二種材料以探討XpsL蛋白在外膜分泌系統中所扮演的角色。實驗結果發現XpsL為一個約41 kDa的蛋白,xpsL基因缺損菌株失去分泌澱粉酵素的能力,而當互補殖入含一段完整xpsL基因的質體時,便可恢復其分泌澱粉酵素的功能,顯示XpsL蛋白於外膜分泌系統中扮演不可或缺的角色。雖然所構築的xpsL基因缺損菌株可證實為一non-polar mutant,其下游 Xps蛋白的轉譯工作未受到影響,但菌株內XpsM、N及E三種蛋白的含量卻下降,並且產生一分子量較小的XpsN蛋白訊號,可能為XpsN蛋白因構形不同而導致降解的中間產物;另一方面,我們亦發現,於xpsM基因缺損菌株中, XpsL及N蛋白的含量下降,而於xpsN基因缺損菌株中,則XpsL及M蛋白的含量下降,這些都顯示XpsL、M及N蛋白間有相互穩定現象。利用分子篩管柱層析法探討XpsL、M及N蛋白是否有共分佈的現象,初步發現在野生株中XpsL及N蛋白確實同步出現,而XpsM蛋白則較晚出現,暗示XpsL、M及N蛋白並非緊密的複合體關係,此與遺傳上的結果不完全符合,因此更進一步,取經由Triton X-100萃取後之膜蛋白部分為材料進行免疫共沈澱分析,結果發現XpsL、M及N蛋白可同時被沈澱出,證明XpsL、M及N三個蛋白會形成一蛋白複合體以輔助胞外蛋白分泌。
Abstract In Xanthomonas campestris, a gram-negative bacteria, extracellular proteins are exported through the type II secretion pathway. At least 11 xps gene products are required for the secretory machinery, and are proposed to form a protein complex localized between the inner and outer membranes. In this study, we aim to elucidate roles of XpsL, a putative inner membrane protein, in the secretory machinery. The (His)6-fused truncated XpsL proteins were expressed and purified from E. coli. Specific antibodies against XpsL were raised by immunizing rabbits with purified recombinant proteins. Moreover, a mutant Xanthomonas strain with chromosomal in-frame deletion on xpsL was constructed by homologous recombination. Using the anti-XpsL antibodies and xpsL mutant, XpsL was identified as a 41 kDa protein mainly localized in membrane fractions. The xpsL mutant was deficient in extracellular secretion of α-amylase. Such defect could be complemented by introducing a plasmid carrying an intact xpsL gene, indicating that XpsL plays an essential role in formation of the secretory machinery. In the xpsL mutant, XpsM was not detected and its absence was correlated with a decreased stability of XpsN and XpsE. An extra band, detected only in xpsL mutant by anti-XpsN antibody, might represent the abnormal degraded form of XpsN protein. In addition, we observed that XpsL was not detected and the stability of XpsN was decreased in the xpsM mutant. Reciprocally, neither XpsL nor XpsM was detected in the xpsN mutant. Therefore, a relationship of mutual stabilization exist among XpsL,M and N proteins, strongly suggest that these three proteins constitute a protein complex. Further experiments demonstrated that XpsL, M and N proteins could be co-immunoprecipitated by any of the antibodies against them. These results argued for a critical role of protein-protein interaction among XpsL, M and N in assembly of the functional secretory machinery.
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