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標題: Characterization of Mutants of Xanthomonas campestris Resistant to Phage phiL7
抗 phiL7 感染的Xanthomonas突變株之篩選與分析
作者: 劉柏延
Liu, Po-Yen
關鍵字: Xanthomonas;突變株之篩選與分析;Phage phiL7
出版社: 分子生物學研究所
Xanthomonas campestris pv. campestris 為十字花科黑腐病菌。噬菌體 L7則為專一感染 X. campestris pv. campestris的烈噬性噬菌體。為了了解噬菌體 L7感染寄主的機制,本研究利用mini-Tn5 誘變X. campestris pv. campestris P20H,得到一株能抗 L7 感染的突變株,命名為B236。分析顯示B236染色體上mini-Tn5插入致變的基因與X. campestris pv. campestris ATCC 33913 (Xcc33913) 的epimerase基因之胺基酸序列有98% 之同質性; 與Pseudomonas aeruginosa 的UDP-GlcNAc C6 dehydratase之胺基酸序列有36% 之同質性。將突變株B236染色體DNA上致變的基因命名為epiA。已知P. aeruginosa 的UDP-GlcNAc C6 dehydratase參與B-band lipopolysaccharide (LPS) 之合成,所以推測Xanthomonas的EpiA與LPS之合成有關。分析LPS含量的結果顯示P20H 的epiA突變株已失去合成LPS能力。Xcc33913的epiA 之上下游基因排列順序如下:orf864-orf863-epiA-galU-kefB。為了探討此基因串中各基因產物之功能,以及其與 L7 感染菌體之相關性,因此利用同質性DNA重組的原理,將P20H 染色體上的orf864、orf863、galU及kefB 基因分別進行插入突變。致變後於點測試(spot test)中發現 L7在含有orf863、epiA及galU突變株的培養皿上皆無法形成溶菌圈,但在含有orf864及kefB突變株的培養皿上則可形成溶菌圈。經LPS分析發現ORF863、EpiA及 GalU 皆與Xanthomonas 之LPS合成有關。另一方面,以噬菌體吸附方式證實,這些無法合成LPS的突變株對噬菌體 L7 的吸附能力比野生株降低約 1,500-4,300倍不等。利用分析型電子顯微鏡 (AEM) 觀察發現野生株與epiA回復株皆有被噬菌體吸附現象,但epiA突變株則無法觀察到噬菌體吸附在細胞外壁。以電孔法 (electroporation) 將 L7 基因體DNA送入epiA突變株後,仍然能釋放出具有感染性之噬菌體。由上述結果推測LPS是噬菌體 L7 感染X. campestris pv. campestris 的吸附座或吸附座組成之一,因此當突變株表面的LPS缺失時,會造成噬菌體無法吸附到寄主細胞進行感染。此外經由病原性測試與Etest 分析發現,當LPS發生缺失時也會造成菌株的致病力下降及對β-lactam類抗生素抗性減弱。曾義雄老師實驗室曾經利用mini-Tn5跳躍突變方式獲得一株EPS產量降低的突變株,命名為G76E。過去的實驗已證明mini-Tn5插入突變G76E的galU基因導致EPS產量降低。本實驗中,將完整的galU基因送入G76E時雖然EPS產量恢復,但卻無法回復LPS含量。若將完整epiA基因及galU基因送入該突變株中,則EPS及LPS含量回復正常。分析發現,G76E突變株中除了galU被mini-Tn5插壞突變,上游的epiA 基因也被轉位子IS1480 插入,造成二次突變。又分析galU單一基因突變株後,證明GalU 在X. campestris pv. campestris 中除了是合成EPS所需之外,也參與LPS合成。

The Gram-negative plant pathogenic Xanthomonas campestris pv. campestris is the causal agent of black rot in crucifers. Bacteriophage L7 is a lytic phage that specifically infects X. campestris pv. campestris ( Xc ). To understand phage-host interaction, a L7-resistant mutant was isolated from Xc strain P20H by mini-Tn5 transposition and designated B236. B236 could not plate L7 in double-layered assay and not form clear zones when the cell lawn was dropped with a high titer of L7. Sequence analysis showed that the mutated gene is epiA coding for an epimerase (EpiA). Homology searches of the encoded protein product indicated that EpiA is likely involved in lipopolysaccharide (LPS) biosynthesis. LPS samples isolated from epiA mutant exhibited different banding patterns from that of the wild-type LPS in silver-stained sodium dodecyl sulfate-polyacrylamide gels. These data showed that EpiA is important for the formation of LPS. In the phage adsorption assay, the adsorption efficiency of B236 decreased by 1,500-4,300 folds compared to that of the wild-type cells, suggesting that LPS is required for adsorption. B236 were able to release infective phage particles upon electroporation with the L7 genomic DNA, indicating that EpiA is not involved L7 replication. In addition, we found that mutant G76E, with a mutation in UDP-glucose pyrophosphorylase (galU) gene located downstream of epiA, also lacks LPS. This defect could not complemented by the cloned galU gene. However when the wild-type epiA gene was introduced into strain G76E containing the cloned galU gene, the LPS defects were corrected. These results suggested that the epiA of G76E also bears a mutation. Moreover, via LPS assay, we confirmed that both EpiA and GalU are essential for the biosynthesis of LPS in Xanthomonas campestris pv. campestris.
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