細菌D-glutamate生合成相關基因與青枯病菌gpsA基因之研究

Studies on bacterial D-glutamate biosynthesis related genes and Ralstonia solanacearum gpsA gene

饒益敏(E-Ming Rau)

指導教授 ： 鄭秋萍

國立臺灣大學/生命科學院/植物科學研究所/碩士(2012年)

https://doi.org/10.6342/NTU.2012.10652

摘要

細菌D-glutamate是細胞壁肽聚醣合成之關鍵成分，而glutamate racemase (MurI)與D-amino acid amidotransferase (Dat)為催化D-glutamate合成之酵素。先前在青枯病菌(Ralstonia solanacearum) murI的研究指出D-glutamate在其泳動力與致病力扮演重要角色，然而其具體機制尚不清楚，且murI與dat在D-glutamate生合成的相對重要性也有待釐清。藉由分析青枯病菌murI、ampG及pal跳躍子(Tn5)插入突變株，以及農桿菌(Agrobacterium tumefaciens)之murI基因剔除株，本研究指出細菌肽聚醣的缺陷皆會導致泳動力下降。此外，murI在不同細菌之分佈比dat廣泛，且農桿菌基因剔除株分析結果也指出murI在維持D-glutamate正常生合成與肽聚醣完整性的重要性高於dat1/2，而dat1之重要性則又略高於dat2。本研究為首次揭露革蘭式陰性菌之dat也具有維持肽聚醣完整性之重要性，且murI與dat(s)在細菌生理功能之重要性是有差異的報導。本研究第二部分希望了解可造成番茄青枯病最穩定抗病品系Hawaii7996 (H7996) 萎凋之高毒力青枯病菌株的致病機制。在進行Pss190菌株的毒力篩選後鑑定出一個在H7996致病力明顯下降但在感病品系L390則為延遲病程發展的突變株，其跳躍子插在RSc0356 (secB)中，經基因互補實驗證實其下游的RSc0357 (gpsA)表現缺失造成突變株需要glycerol做為碳源以維持其正常增殖與在茄番毒力下降的原因，且在另一高毒力菌株Pss1308 isogenic突變株中亦有一致的結果，但突變株之泳動力、生物膜生成及分泌細胞壁分解酵素等致病因子與野生菌株相比並無明顯差異。故推測在自根部入侵並系統性感染H7996的過程中，青枯病菌可能會遭遇glycerol匱乏的環境，故病菌必需有善用各式碳源的能力，方能成功地在植物不同組織中大量增殖並導致植物萎凋。

關鍵字

murI ； dat ； D-glutamate ； 肽聚醣 ； 青枯病菌 ； 農桿菌 ； gpsA

並列摘要

D-Glutamate is an essential meterial for the synthesis of bacterial peptidoglycan (PG). Glutamate racemase (MurI) and D-amino acid transaminase (Dat) catalyze independent reactions for D-glutamate biosynthesis. Our previous study on Ralstonia solanacearum (Rs) murI suggested that D-glutamate plays an important role in proper swimming activity and therefore pathogenicity on tomato; however, mechnism on how D-glutamate is involved in bacterial swimming was not known. In addition, contribution of MurI and Dat in D-glutamate biosynthesis and the related properties in Gram-negative bacteria remained to be determined. Characterization of murI, pal and ampG transposon (Tn5) insertional mutants of Rs as well as murI in-frame deletion mutant of Agrobacterium tumefaciens (At), suggested that defects in PG led to impaired in swimming activity. In addition, the fact that most bacteria contain murI rather than dat, along with phenotypes of various single/double mutants of At, revealed that murI plays a more important role in D-glutamate biosynthesis and PG integrity than dat1/2, and dat1 is more important than dat2. This is the first study not only evidences an important role of Gram-negative bacterial dat in PG integrity, but also elucidates the contribution between murI and dat. The second part of this study aimed, to decipher the mechanism involved in underlying the Rs high-virulence strains, which can overcome resistance of the most stable tolerant tomato cv. Hawaii 7996 (H7996). After virulence screening on H7996, a mutant of strain Pss190 displaying notably reduced virulence on H7996 but slightly delayed disease progress on the susceptible cv. L390 was identified. This mutant contains a transposon insertion in RSc0356 (secB). Isogenic mutants in another high-virulence strain Pss1308 displayed consistent defective phenotypes and complementation tests a polar mutation on the downstream gene RSc0357 (gpsA) was responsible for the mutant’s defects in virulence and bacterial proliferation under glycerol-limited conditions. However, the mutation did not cause significant changes in motility, biofilm formation and cell-wall-degradation enzyme activity. These results together suggested that, during the establishment of systemic colonization in H7996 initiated from the root, Rs may encounter glycerol-limited conditions. To achieve successful systemic infection, this bacterium must be capable of utilizing various carbon sources efficiently so that it can proliferate vigorously in different tissues of the plant.

並列關鍵字

murI ； dat ； D-glutamate ； peptidoglycan ； Ralstonia solanacearum ； Agrobacterium tumefaciens ； gpsA

參考文獻

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