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標題: Xanthomonas albilineans 及 Xanthomonas campestris pv. glycines 細菌素之探討
Characterization of bacteriocins from Xanthomonas albilineans and Xanthomonas campestris pv. glycines
作者: 郭乃瑜
Guo, Nai-Yu
關鍵字: bacteriocin
出版社: 分子生物學研究所
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摘要: Xanthomonas albilineans (Xa) (BCRC13194), a phytopathogenic bacterium of sugarcane, was identified as a bacteriocin producing strain and used in this study. Maximal bacteriocin activity in culture filtrate was obtained in the early stationary phase of growth of the Xa strain in LB broth at 28℃. After 6 hours of mitomycin C (0.4 µg/ml) induction, approximately 62.5 folds increase in bacteriocin production was observed. The bacteriocin produced by Xa was purified from culture fluid by PEG precipitation and gel filtration chromatography (HiPrep 16/60 Sephacryl S-300 HR column). The inhibitory activity of the purified Xa bacteriocin was stable at temperature up to 55℃ for 10 minutes. The Xa bacteriocin was resistant to proteolytic enzymes such as trypsin, chymotrypsin, and proteinase K digestion. Ten protein bands, in the range of 10 kDa to 100 kDa, were identified by SDS-PAGE analysis. The two intense protein bands, 53 kDa and 12 kDa proteins, were chosen for LC/MS/MS analysis. By searching the MS/MS spectra against the proteome database, the 53 kDa protein is related to bacteriophage Mu tail sheath protein of Pseudomonas syringae pv. syringae B728a and the 12 kDa protein is related to a hypothetical protein PSPPH_0659 of P. syringae pv. phaseolicola 1448A. An 1.5-kb DNA fragment corresponding to the gene of the 53 kDa protein was amplified by PCR and used as probe. Results of Southern blot analysis identified a 2.5-kb DNA fragment and sequence analysis of the 2494 bp DNA fragment revealed four putative ORFs, ORF498, ORF115, ORF98, and ORF64. The ORF498 and ORF115 were the gene corresponding to the 53 kDa and 12 kDa protein, respectively. The morphology of the Xa bacteriocin was visualized by transmission electron microscopy and complete and contracted forms as well as empty sheaths were identified on the micrographs. Results from these studies indicated that the Xa bacteriocin is a high molecular weight phage-tail-like bacteriocin. The bacteriocin genes glyA and glyB from X. campestris pv. glycines YR32 (XcgYR32) were cloned and plasmid pMTc3-XgAB which produces MalE-fused GlyA and GlyB was constructed. The fused protein could be expressed as soluble form in E. coli Rosetta(DE3)(pLysS) and purified by passing through amylose affinity and Sephacryl S-300 HR columns. Unfortunately, some of the contaminant proteins were co-purified. The GlyA and GlyB of X. campestris pv. phaseoli 73 (Xcp73) has 24 and 6 amino acids difference with that of XcgYR32, respectively, and no antimicrobial activity could be detected. A set of gene fragments replacement in the glyA of XcgYR32 and Xcp73 was conducted to investigate the amino acid residues important for the bacteriocin activity. Results indicated that glyA and glyB may have equal importance for the bacteriocin activity.
本研究以實驗室過去篩選得到具有抑菌活性的甘蔗病原菌 Xanthomonas albilineans (Xa) 菌株為材料進行細菌素活性相關特性研究。Xa 分泌細菌素的最佳培養條件為以 LB broth 在 28℃ 之下培養至菌體生長接近 stationary phase 時期,且以 0.4 μg/ml mitomycin C 誘導 6 小時可使其表現量增加。純化策略為以終濃度 8% PEG6000、0.5 M NaCl 沉澱胞外蛋白後,再以分子篩管柱 HiPrep 16/60 Sephacryl S-300 HR 進行純化。純化的 Xa 細菌素蛋白其溫度最高耐受度為 55℃,10 分鐘。其對蛋白酶 proteinase K、trypsin、chymotrypsin 切割具有抵抗性。將純化所得蛋白以 SDS-PAGE 分析後,共觀察到將近十個蛋白條帶,取其中量較多的 53 kDa 與 12 kDa 蛋白以液相層析串聯質譜儀 (LC/MS/MS) 作分析比對,比對結果分別是 Pseudomonas syringae pv. syringae B728a 的 bacteriophage Mu tail sheath 與 P. syringae pv. phaseolicola 1448A 的 hypothetical protein PSPPH_0659。接著利用 PCR 增幅部份 53 kDa 的基因片段約 1.5 kb 為探針,以南方墨點法偵測並選殖出約 2.5-kb 的 DNA 片段,定序後確定其序列長度為 2494 bp,利用分析軟體推測其中含 4 個 ORF 序列,分別命名為 ORF498、ORF115、ORF98、ORF64,其中 ORF498 為 53 kDa 蛋白的基因序列,而 ORF115 為 12 kDa 蛋白的基因序列。純化之 Xa 細菌素蛋白以穿透式電子顯微鏡放大 10 萬倍觀察,可以看到類似噬菌體尾部之結構,綜合以上結果確認 Xa 細菌素可能為一種 phage-tail-like bacteriocin。 將具抑菌活性的 X. campestris pv. glycinesYR32 (XcgYR32) 菌株,其細菌素基因 glyA 與 glyB 選殖並構築至蛋白表現載體 pMTc3-XgAB,以 E. coli Rosetta(DE3)(pLysS) 進行大量表現及純化,結果發現以 MalE-fused GlyA 並與 GlyB 共同表現的構築可以得到可溶性的細菌素蛋白,但使用分子篩管柱 HiPrep 16/60 Sephacryl S-300 HR與 Amylose 管柱純化後,還是無法得到純度較高之 GlyAB 蛋白。菌株 X. campestris pv. phaseoli 73 (Xcp73) 與 XcgYR32 glyA 與 glyB基因 DNA 序列具有 92% 相同性,但其菌株並不具有抑菌活性。其中 Xcp73 與 XcgYR32 GlyA 有 24 個胺基酸差異,GlyB 具有 6 個胺基酸差異。為比較兩者之間的差異,將 Xcp73 與 XcgYR32 之 glyA 基因進行部份片段之交換與取代,實驗結果推測其抑菌功能表現並非只由 GlyA 單一個蛋白單元負責,必須由完整 GlyA 與 GlyB 互相結合,褶疊形成一個有活性的構造才能發揮作用。
其他識別: U0005-1112200715350200
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