Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21439
標題: Functional analysis of small heat shock protein HspA in Xanthomonas capestris pv. campestris and characterization of plasmid pSM76 of Stenotrophomonas maltophilia
十字花科蔬菜黑腐病菌小熱休克蛋白質 HspA 之功能探討及 Stenotrophomonas maltophilia 質體 pSM76 之定序與分析
作者: Lin, Ching-Hsuan
林晉玄
關鍵字: 十字花科黑腐病菌;Xanthomonas campestris;熱休克蛋白質;HspA;蛋白質集結;Stenotrophomonas maltophila;質體 pSM76;heat shock protein;HspA;protein aggregation;Stenotrophomonas maltophila;plasmid pSM76
出版社: 分子生物學研究所
摘要: 
第一部份
本實驗室先前以二維膠體電泳分析發現 Xanthomonas campestris pv. campestris 17 (Xc17) 存在有一小熱休克蛋白質,命名為 Hsp20。 由轉錄起始點分析顯示 hsp20 的轉錄起始點位於轉譯起始點上游第 65 bp處。 在 X. campestris pv. campestris ATCC 33913序列解開後,依其命名方式將 hsp20 更名為hspA。 為了了解 hspA 在 Xc17 菌體內之功能,本研究由 DNA、 RNA、及蛋白質三個層面進行分析。 啟動子活性測試顯示, hspA 的啟動子受熱調控但不會受到 SDS、 H2O2、 pH 及高鹽滲透壓等逆境啟動。 從北方墨點分析結果亦証明受熱休克誘導後之 hspA 會大量表現。 以同源重組方法無法得到 hspA- 基因之突變株,推測其為一必要基因。 RT-PCR 結果顯示在一般培養條件之下, hspA 於 Xanthomonas 中持續低量表現。 為了了解此蛋白質之功能,以蛋白質表現載體 (pET30) 大量表現 HspAhis 及 HspAter 蛋白質, HspAhis 蛋白質 C 端帶有 6 個 histidines,而 HspAter 則否。 In vitro 活性測試顯示 HspAhis 及 HspAter 具有相似活性,可以有效降低熱逆境時蛋白質之集結 (protein aggregation) 或變性,所保護的目標蛋白質應屬廣泛性的。 當測試溫度達到 60℃ 時, HspAhis 與 HspAter 之活性逐漸降低,在 70℃ 時活性幾乎消失。 於 45℃ 培養環境中,可大量表現 HspA 蛋白質的 E. coli 比對照組之生長為佳。 上述結果顯示在 in vitro 及 in vivo 中 HspA 都有功能,可能扮演 chaperone 的角色。 利用 His-binding 管柱、硫酸銨沈澱法及陰離子交換法純化 HspAhis 及 HspAter 蛋白質後,再以gel filtration 分析得知 HspAhis 可形成分子量約為 820 (44 subunits) 及 410 (22 subunits) 兩種聚合體,此兩種聚合體並無明顯置換之情形;而 HspAter 只形成分子量約為 820 之聚合體。 HspAhis 會形成兩種不同之聚合體,可能是因為 C 端多了 6 個 histidines 所致,顯示 HspA 蛋白在形成聚合體時,C 端胺基酸可能參與作用。 當 luciferase 反應液中含有 HspA820his 或 HspA410his 蛋白質時,皆能改善此酵素遇熱變性的反應,顯示 HspA 功能為 chaperone,且兩蛋白質活性差異約為 2 倍。 由 EcoRI refolding assay 的結果顯示, X. campestris pv. campestris 17 之 HspA 具有協助變性蛋白質再摺疊的功能。
第二部份
Stenotrophomonas maltophilia 為一廣泛存在之革蘭氏陰性菌。 S. maltophilia T76 (SmT76)是從醫院內所分離到之一野生株,含有一迷你質體,命名為 pSM76。 定序結果顯示 pSM76 共計 2,927 bp, GC content 為 63.8%。 推測 pSM76 具有 4 個 open reading frames (ORFs)。 orf1 主導的產物含 194 個胺基酸,與質體 pPGZ500的 -integrase like protein 具有 50% 相似度。 orf2 主導的產物含 307 個胺基酸,與質體 pAsa11 的 RepA protein 具有 54% 相似度。 orf3 主導的產物含有 89 個胺基酸,與質體 pFAJ2600 的 RepB protein 具有 65% 相似度。 此外, ORF3 與 HTH_MERR (helix_turn_helix, mercury resistance) 胺基酸保守性序列也具有 67.7% 的相似度。 orf4 主導的產物含有 223 個胺基酸,與 X. campestris pv. campestris ATCC 33913 DNA repair protein 具有46% 的相似度。 將 Gmr 基因嵌入 pSM76 之 PstI 與 SalI 均不影響質體之複製,構築完成的質體分別命名為 pSM76PG 與 pSM76SG。 此衍生質體可穩定存活於 Xc17 與 S. maltophilia 10737 菌體中。 但無法得到 Gmr 插入於 XmnI 與 EcoRI 之 pSM76 衍生質體,顯示 ORF2 與ORF3與質體複製有關。 此外, pSM76無法在 Escherichia coli、 Enterobacter cloacae、 Klebsiella pneumonia、 Shigella sonnei、 Serratia marcessens 等菌體中存活,應屬窄寄主質體。 以南方墨點法分析,結果顯示 pSM76 與 S. maltophilia T29、 S. maltophilia T40、 S. maltophilia T76 及 S. maltophilia T103 等分離株中的小質體可能相同或相似,但未觀察到 S. maltophilia染色體 DNA 與質體 pSM76 有同質性序列存在。 以南方墨點分析,顯示 SmT76 菌體中未偵測到有 pSM76 之 ss-DNA,顯示質體可能是以 theta type 的方式進行複製。 從質體不相容性測定顯示,此質體無法與 pRK415 同時存在,顯示 pSM76 屬於 IncP 系列。 pSM76PG 複製套數在 Sm10737 中約為 70 套/cell,而在 Xc17 中則約為 40 套/cell。 經由不同質體之構築並採用 in trans 方法尋找質體 pSM76 之複製起始區 (ori),以 PstI site (CTGCAG) 之第一個 C 為1st base,推測 pSM76 之 ori 位於nt 787~1,200 區域內。 質體 pSM76 的限制酶切位不多,且穩定度高,應可改造為適用於研究 S. maltophilia 的質體。

Part 1
Previously we have identified a small heat shock protein, Hsp20, in Xanthomonas campestris pv. campestris 17 (Xc17) following a heat shock treatment. Primer extension result showed that the transcriptional start site of hsp20 is located 65 bp upstream of the translational start site. According to the gene nomenclature of Xanthomonas campestris pv. campestris ATCC 33913, hsp20 of Xc17 is renamed as hspA. In this study, the properties of Xc17 hspA was analyzed at DNA, RNA and protein levels, respectively. Promoter activity assay and Northern hybridization results revealed that the hspA could be induced and overexpressed by heat shock but not by SDS, H2O2, pH and osmosis. RT-PCR result indicated that hspA expressed at low level under normal condition. Since no hspA- mutant in Xc17 could be obtained, hspA could be an essential gene in Xanthomonas. Two protein expression vectors that would overexpress HspAhis and HspAter were constructed. HspAhis possesses additional 6 histidines in C-terminal tail, but HspAter doesn't. In vitro protein activity assay revealed that HspAhis and HspAter had similar activities preventing crude extract proteins aggregated at high temperature. The activities reduced as the temperature achieved at 60℃ and disappeared at 70℃. The spectrum of protein substrates protected by HspA from aggregation at high temperature should be broad. Overexpression of HspA in E. coli has some effects on the growth of bacteria at high temperature. These results suggested that HspA acts as a chaperone in vitro and in vivo. We used His-binding column, ammonium sulfate and anion exchange column to purify HspAhis and HspAter, the purified proteins were subsequently assayed with gel filtration. It was found that HspAhis protein formed two different sizes of oligomers, 820 kDa and 410 kDa, and no changeover between them was observed. However, HspAter formed only one oligomeric complex with molecular weight of approximately 820. Therefore, it was suggested that the C-termini of HspA could be important for the formation of oligomeric structure. Luciferase protection assay result showed that HspA820his and HspA410his had similar activities on protecting luciferase from aggregation at high temperature, indicating that HspA is a kind of chaperone. In addition, based on the EcoRI refolding assay, it is believed that the small heat shcok protein, HspA, could possess ability to refold denatured proteins in X. campestris pv. campestris 17.
Part 2
A small plasmid of S. maltophilia T76, pSM76, was isolated and the complete nucleotide sequence was determined. No drug resistance properties were mediated by pSM76. The 2,927 bp pSM76 had a G-C residues content of 63.8%. Analysis of this sequence revealed the presence of 4 putative open reading frames (ORFs). orf1 encoded an ORF of 194 amino acids which showed 50% homology to -integrase like protein of plasmid pPZG500. orf2 encoded an ORF of 307 amino acids which showed 54% homology to RepA protein of plasmid pAsa11. orf3 encoded an ORF of 89 amino acids which showed 65% homology to RepB protein of plasmid pFAJ2600. ORF3 also showed 67.7% alignment with conserve domain of HTH_MERR. orf4 encoded an ORF of 223 amino acids which showed 46% homology to DNA repair protein of X. campestris pv. campestris ATCC 33913. Derivatives of pSM76 with a Gmr inserted into PstI or SalI were named pSM76PG and pSM76SG, respectively. Such derivatives can replicate stably in Xc17 and Sm10737. However, no derivative with Gmr inserted into XmnI or EcoRI of pSM76 was obtained, indicating that the ORF2 and ORF3 could be important for replication. pSM76 can only replicate in Xc17 and S. maltophilia 10737 (Sm10737) but not in Escherichia coli, Enterobacter cloacae, Klebsiella pneumonia, Shigella sonnei and Serratia marcessens, suggesting that this is a narrow host range plasmid. Southern hybridization results revealed that the small plasmids in S. maltophilia T29, S. maltophilia T40, S. maltophilia T76 and S. maltophilia T103 are homologus to pSM76, but no hybridized signal with genomic DNA of S. maltophilia were found. Moreover, no ss-DNA was detected in the cells containing pSM76, indicating that is a theta type replication plasmid. Incompatbility test indicated that pSM76 belongs to IncP group. The copy number of pSM76PG is about 70 copies/cell in Sm10737, and 40 copies/cell in Xc17. Plasmid pOM762xp can replicate in Xc17 only when the ORF2 and ORF3 of pSM76 were supplied in trans, suggesting the ori of pSM76 is located in the region between nt 787 to 1,200. Plasmid pSM76 has few restriction sites and possesses great stability, representing that it has the potential to be developed into vectors for gene cloning in S. maltophilia.
URI: http://hdl.handle.net/11455/21439
Appears in Collections:分子生物學研究所

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