Extracellular secretion of Carocin S1 in Pectobacterium carotovorum subsp. carotovorum occurs via the type III secretion system integral to the bacterial flagellum

Background: Pectobacterium carotovorum subsp. carotovorum is a phytopathogenic enterobacterium responsible for soft rot, a disease characterized by extensive maceration of the affected plant tissue. This species also produces two or more antibacterial substances called bacteriocins, which enhance its competitiveness against related rival species. However, the secretion mechanism for low-molecular-weight bacteriocin is still unknown. Results: A mutant (flhC::Tn5) that did not secrete the low-molecular-weight bacteriocin (LMWB), Carocin S1, was generated by Tn5 insertional mutagenesis. Sequence analysis indicated that this insertion disrupted open reading frame 2 (ORF2) and ORF3 of this strain. Deletion and rescue experiments indicated that ORF2 and ORF3 were both required for extracellular LMWB secretion. The ORF2 and ORF3 sequences showed high homology with the flhD and flhC gene sequences of Pectobacterium carotovorum subsp. atroseptica, Serratia marcescens, Yersinia enterocolitica, and Escherichia coli, indicating that they likely encoded key regulatory components of the type III flagella secretion system. Conclusion: Thus, the extracellular export of Carocin S1 by Pectobacterium carotovorum subsp. carotovorum appears to utilize the type III secretion system integral to bacterial flagella

Tyrosine Aminotransferase Gene Negative Regulation Mechanism in Non-Hepatocyte Cell and Silencer Element Isolated

成體期肝臟在身體中主要工作是進行各種醣類﹑胺基酸﹑酯類等物質的新陳代謝以及儲存等工作，其在我們身體裡扮演一個重要的角色。本研究室在針對小鼠胎生期肝臟的分化過程中的研究裡，透過肝臟中的TAT 誘導調控機構的解析的瞭解，進而瞭解到肝前驅細胞如何受到胎生期肝臟環境的影響而分化成肝臟細胞的細胞內分子機構變化。雖然TAT、G6Pase 等基因並非是僅限於肝細胞中所表現的基因，傳統上也並不被當作是肝臟特有基因，但由於在胎生中期的肝臟中並不表現TAT、G6Pase 等基因，而是須等到出生後才會被表現出來，故傳統在做肝前驅細胞分化研究時，會將其作為肝臟的分化標誌基因，以瞭解肝細胞的分化狀態及細胞功能的轉變，由於老鼠胎兒在胎生中期(E14.5)時肝臟並不表現TAT、G6Pase 等基因，但是根據我們以前的研究中從老鼠懷孕中期(E14.5)胎兒中取出其胎生肝臟出來並從中分離出肝細胞初代培養系，然後將OSM、HGF 或其他因子加入初代培養系中直到肝前驅細胞被分化成肝細胞並使其表現G6Pase、TAT 等基因。在我們以前的研究結果中得知，在以轉錄起始點前方-230 至-270 區域有一個區域會對OSM 誘導反應的OSM response element(ORE)，然而這些element 除了在肝臟細胞中會被誘導活化外，也會在肝癌細胞來源的細胞株HepG2、非肝細胞的fibroblast 細胞株NIH3T 中及其他細胞中反應，此一結果無法說明為何只有在出生後的小鼠肝臟中能表現TAT基因的表現。我們在前三年的研究結果中確認了TAT DNA 結構中的intron A 區域中可能帶有有關辨識細胞分化與否的組織特異性element，此element 使非肝細胞的fibroblast 細胞株NIH3T3 或其他來源的細胞株在glucocorticoid 存在的狀態下，使其無論是在高密度細胞培養或是在OSM (或HGF) 存在狀態下低密度細胞培養中的TAT 基因誘導活性表現均被被壓抑；然而此現象則在肝臟前驅細胞中無法發現其抑制TAT 基因誘導活性表現的現象。因此我們確認其為對glucocorticoid 反應的組織特異性repressor。此一結果也證明了TAT DNA 結構中的intron A 區域其實也帶有調控基因表現與否的功用。我們逐段刪除intra A 的DNA 片段，並將起與Luciferase 結合成reporter vector後送入NIH3T3 及HepG2 細胞中測試其轉錄活性，在確認其最小有效活性抑制區域獲得後，我們發現該區域中有對AP-1 及NF-κb 反應的consensus sequence。在未來的2 年中，我們將以西方點墨法、EMSA、CO-IP 等方法近一步解析，以瞭解在非肝細胞中是何種轉錄因子在與我們所單離出的element 結合，已達到抑制TAT 基因的表現，而他們又是如何在肝細胞中被解除了其活性，使其TAT 能在肝細胞中表現

Application of the Bacteriocin Gene Cloning from Erwinia carotovora and Soft-rot Disease Controlled

軟腐病菌可生產兩種不同型態的細菌素，一種是類似病毒結構的高分子量細菌素，另一種是以小段蛋白質形式存在的低分子量細菌素。我們利用transposon Tn5插入軟腐病菌菌株WF7的染色體中，並且個別分離出了約2000株插入突變株，分析了其低分子量細菌素表現活性，在經過了2次的以上的活性測試交叉分析後，我們確認了WF7相關的插入突變株15株完全喪失了低分子量細菌素表現能力。我們進一步的進行TAIL-PCR分析其插入位置周圍的基因序列，而插入突變株TW07的Tn-5是插入與可能是E. coli所生產的colicin E3有同質性DNA序列的位置，我們將此命名為Carocin S4。我們進一步的分析Carocin S4的基因，從親株WF7的染色體DNA的選殖與定序後，我們從所解出DNA序列中發現到2個完整的ORF，第1個ORF與E. coli所生產的colicin E3等有約24％的同質性，其有可能為DNA水解酵素低分子量細菌素，我們將第1個ORF命名為caroS4K gene；而第2個ORF與NCBI基因資料庫中無任何同質性蛋白資料可比對，但由於該基因是緊接於第一個ORF之後，有可能為其相對應的免疫蛋白，我們將其命名為caroS4I gene，這兩個基因我們稱之為carocin S4。我們這兩個基因導入pET32a載體中，製成pET32aS4-42表現載體中，並將其分別導入至宿主細胞大腸桿菌XL1-blue中，其結果顯示帶有pET32aS4-42質體的菌株均能表現出低分子量細菌素carocin S4活性，從此結果顯示我們成功的將carocin S4選質並表現於無致病性菌株中。 本年度計畫中我們將進一步的將Carocin S4蛋白大量純化出來，並確認其殺菌機制。除此之外，我們也將同時尋找能長期保存且且具有高度抗軟腐菌活性的方法，以利將來在田間實驗使用。此外，我們也計畫在今年度中大量蒐集中部地區軟腐菌株的種類，以期能獲得具有更多更廣泛的抗軟腐菌生長的細菌素高度表現能力菌株，以期本研究將來能有極高度的實用價值。 我們將在本年度內將carocin S4基因導入無病源性Erwinia carotovora及E. coli菌株中，以利用其基因轉錄、修飾、及分泌機制來將低分子量細菌素基因輸送至胞外，以期能做出高抗軟腐菌生產能力且無病源性的軟腐菌，使其能於生物防治上應用，以達成抑制軟腐病病害發生的可能性。We inserted the transposon Tn5 into the pectobacterium carotovorum subsp. carotovorum WF7 chromosomal, which can express the low-molecular-weight bacteriocins, then we obtained about 2000 insertional mutagensis strains. We test the Bacteriocin acticity, and there are 15 strains lose the production ability for Bacteriocin. We analyze the surrounding sequence from these strains by TAIL-PCR, and confirmed the sequence data by genebank database. The TW07 strain's data showed that the DNA sequence data is similar with colicin E3, which is a low-molecular-weight bacteriocin and produced by E. coli, and has 24％ homologous. We continue the sequence from the parent strain, WF7, and obtain about 2800 bp. After analyzed the DNA sequence, Two complete ORFs were obtained. The first ORF is similar with colicin E3 about 24％ homoglous, but the second ORF has bit any homologous with protein database. Both of two bacteriocins are nuclease activity. We named the first ORF gene is carocin S4K, and the second ORF gene is carocin S4I. After subclone the carocin S4 gene and transfer it into DH5 or BL21 cell, we found the carocin S4 gene can be expressed and Carocin activity can be detected in XL1-blue cell by bacteriocin activity assay. This year we will isolate the Carocin S4 protein, and confirm its antibiotic's activity. In addition, we also plan to collect a large number of Erwinia carotovora strains, and make a strain-bank of Erwinia carotovora for middle-Taiwan

Studies the Protein Function and Secretory Mechanism of Low-Molecular-Weight Bacteriocins

Erwinia carotovora 是在十字花科作物上造成軟腐病的一種主要植物病源菌，該菌可以表現高分子量及低分子量兩種不同形式的細菌素，此類細菌素也因為只能殺死帶有相近基因序列的親緣性軟腐病菌，因此可提供在植物防治一種有效的選擇手段。本研究是在這5 年間的研究中，我們從Erwinia carotovora 89-H-4 菌株中選殖出carocin S1基因，並將carocin S1 基因其接入載體pACYC177 等載體中後，獲得pAYL4 表現載體。然後我們將此載體導入不生產細菌素的軟腐病菌宿主Ea1068 後可大量表現Carocin S1蛋白。確立軟腐病菌的低分子量細菌素基因carocin S1 基因， carocin S1 蛋白在藉由軟腐菌病源株Ea1068 當作指示菌株分析下，在大腸桿菌宿主及不生產細菌素的軟腐病菌宿主Ea1068 中所生產的Carocin S1 確實帶有其生物活性，同時也確保其在生物防治上的效用。Carocin S1 再經由DEAE Affi-gel 的液相層析純化分離並測試其生物活性後得知其具有nuclease 活性。此外，我們從Erwinia carotovora 3F-3 菌株中選殖出carocin S2 基因，並依同樣方法驗證得知其確為低分子量細菌素基因。我們在近兩年的研究中發現在同樣的生產Carocin S1 蛋白的H-rif-8-6及Carocin S2蛋白的F-rif-1-2菌株中，均可發現當以Tn5或以同質互換等方法破壞第三a型分泌蛋白系統中的胞內部份ysaT、yasU 等基因或第三b型分泌蛋白系統中的表現調控flhDC 基因或鞭毛部份的fliC 及flhA等基因時，carocin S1及carocin S2等基因雖然能被表現生產出有活性的蛋白，但卻無法將Carocin S1及Carocin S2 蛋白輸送至胞外。此一結果顯示Carocin S1及Carocin S2 蛋白的胞外輸送分泌機制同時與分屬T3aSS及T3bSS的第三型分泌系統的部分蛋白質有間接或直接的關係。本研究目的在於探討當輸送CarocinS1及Carocin S2 蛋白至胞外時，T3aSS與T3bSS分泌系統間的關係為何，其在Erwinia屬中是否混合成一個新型態的蛋白質分泌系統以專責分泌低分子量細菌素。Carocin S1及Carocin S2蛋白又是如何透過此一分泌系統被輸送至胞外，這三種不同系統的蛋白彼此間的關係及其作用機制是本研究的重點。Erwinia carotovora is a phytopathogenic enterobacterium, responsible for the softrot, blackleg, or stem rot of a number of economically important crops. Some bacterialspecies produce one or more antibacterial substances called bacteriocins, which enhancetheir competitiveness with other related bacterial species. Based on this knowledge,our laboratory tried to exploit the bacteriocins produced by E. carotovora to controlsoft-rot disease. Resounding success has been recorded with this system of control.From our the past 5 years studies, the low-molecular-weight bacteriocin carocin S1,genetic determinant comprised of two structural genes, caroS1K and caroS1I, whichencode the killing protein (CaroH1K) and the immunity protein (CaroH1I), was isolatedfrom Erwinia carotovora 89-H-4. The homology search result indicated that carocin S1gene is homologous to pyocin S3 and AP41 in P. aeruginosa from 1,078th bp to 1,704thbp. The Carocin S1 was incubated with Erwinia carotovora chromosomal DNA for 1 hourat 28℃, in order to confirmed the nuclease activity after isolated and purified byDEAE Affi-gel column. On the way, we also isolated the other LMW bacteriocin carocinS2 gene, which has low homologous with colicin E5, and confirmed the ribonucleaseactivity. From our other studies, the Carocin S1 protein also can not exported outocell when we destroyed the ysaT and ysaU gene, which is a members of the type Ⅲfactor-associated secretory system (T3aSS), or destroyed the flhD/C, fliC, or flhAgenes, is a members of the type Ⅲ bacterium-flagella secretory system (T3bSS) bytransposon Tn5 insertional mutagenesis or homologous recombination. These results aresuggested that Carocin S1 or Carocin S2 is secreted to extrocellular by the new typesecretory system, which recruit some components from T3aSS, T3bSS, or others. Thepurpose of this study is investigate the secretion mechanism, which is recruit thecomponents from T3aSS and T3bSS and develop the new secretion system, of Carocin S1and Carocin S2 to extracellular

Cloning of the Bacteriocin Gene from Erwinia Carotovora and Soft-Rot Disease Controlled

插入突變株的製出與細菌素生產能力喪失菌株的選出：本研究室從已擁有數十株可生產廣泛性殺菌能力細菌素菌種，選出對台灣來源軟腐病菌40菌株最有抗菌能力的菌株來進行低分子量細菌素基因的解析。由於本人以前在所做的相關研究中得知軟腐菌的低分子量細菌素基因是位於染色體中，而不是在質體DNA裡。因此我們以跳躍基因插入突變法選殖出低分子量細菌素基因。在本年度的目標中，我們計畫從HY21等菌株中選殖新規的低分子量細菌素基因。細菌素基因序列的在確認，我們將在本年度內確認軟腐病菌所生產低分子量細菌素基因的全長基因序列，以期在下一年度計畫中能被殖入其他宿主系統中表現確認，並且能被大量生產表現，以期能於生物防治上應用。The purpose of this study is to colne the new low-moleclar-weight bactericin from Erwin carotovora, in order to apply to bio-control agent for soft-rot disease. We use the E. coli 1830, which contains the tansposon Tn5 to mate with Erwinia crotovora HY21 strain, and make the Tn5 insertional mutanst, which defective the producing ability of low-molecular-weight bacteriocin. Using thermal asymmetric interlaced PCR, the DNA sequence from the Tn5 insertion site was analyzed and obtained

Studies the Hepatoblast Differentation Mechanism during Fetal Stage

成體期肝臟在身體中主要工作是進行各種醣類﹑胺基酸﹑酯類等物質的新陳代謝以及儲存等工作，然而胎生期的肝臟則不具有我們所熟知的肝臟功能；由於胎生期的肝臟中因為富含大量的造血幹細胞，而使肝臟成為胎生時期時身體中最主要的造血器官；肝臟本研究的目的，在針對小鼠胎生期肝臟的分化過程中，透過肝臟中的TAT 及G6Pase 誘導調控機構的解析的瞭解，進而瞭解到肝前驅細胞如何受到胎生期肝臟環境的影響而分化成肝臟細胞的細胞內分子機構變化。我們從前年度計畫的研究中得知G6Pase 基因轉錄區域對OSM 反應的element 有可能坐落在距離轉錄起始點上游由-13 Kb 到-0.6 Kb 的區域中。另外在TAT 基因的intron A 中可能帶有有關辨識細胞分化與否的組織特異性element，使其在高密度細胞中的誘導活性反到被壓抑。由於老鼠胎兒在胎生中期(E14.5)時肝臟並不表現TAT、G6Pase 等基因，同時因為我們所使用的肝細胞初代培養系(fetal liver primary culture)中含有肝前驅細胞，因此我們從老鼠懷孕中期(E14.5)胎兒中取出其胎生肝臟出來並從中分離出肝細胞初代培養系，然後將OSM、HGF 或其他因子加入初代培養系中直到肝前驅細胞被分化成肝細胞並使其表現G6Pase、TAT 等基因，將這些報告載體導入初代培養系細胞中觀察其轉錄活性，並從中分離出對OSM 刺激肝臟細胞分化過程中對OSM 反應的最小段轉錄區域的OSM response element，再將單離出來的element 更進一步的解析其相應之轉錄因子，及其轉錄調控機構，以瞭解肝前驅細胞在分化過程中的細胞內的分子變換，分化因子對胎生肝細胞的分化控制機構的瞭解，及各促進因子在胎生期肝臟中的階段性角色

Studies the Protein Function and Secretory Mechanism of Low-Molecular-Weight Bacteriocins

Erwinia carotovora 是在十字花科作物上造成軟腐病的一種主要植物病源菌，該菌可以表現高分子量及低分子量兩種不同形式的細菌素，此類細菌素也因為只能殺死帶有相近基因序列的親緣性軟腐病菌，因此可提供在植物防治一種有效的選擇手段。本研究是在這5 年間的研究中，我們從Erwinia carotovora 89-H-4 菌株中選殖出carocin S1基因，並將carocin S1 基因其接入載體pACYC177 等載體中後，獲得pAYL4 表現載體。然後我們將此載體導入不生產細菌素的軟腐病菌宿主Ea1068 後可大量表現Carocin S1蛋白。確立軟腐病菌的低分子量細菌素基因carocin S1 基因， carocin S1 蛋白在藉由軟腐菌病源株Ea1068 當作指示菌株分析下，在大腸桿菌宿主及不生產細菌素的軟腐病菌宿主Ea1068 中所生產的Carocin S1 確實帶有其生物活性，同時也確保其在生物防治上的效用。Carocin S1 再經由DEAE Affi-gel 的液相層析純化分離並測試其生物活性後得知其具有nuclease 活性。此外，我們從Erwinia carotovora 3F-3 菌株中選殖出carocin S2 基因，並依同樣方法驗證得知其確為低分子量細菌素基因。我們在近兩年的研究中發現在同樣的生產Carocin S1 蛋白的H-rif-8-6及Carocin S2蛋白的F-rif-1-2菌株中，均可發現當以Tn5或以同質互換等方法破壞第三a型分泌蛋白系統中的胞內部份ysaT、yasU 等基因或第三b型分泌蛋白系統中的表現調控flhDC 基因或鞭毛部份的fliC 及flhA等基因時，carocin S1及carocin S2等基因雖然能被表現生產出有活性的蛋白，但卻無法將Carocin S1及Carocin S2 蛋白輸送至胞外。此一結果顯示Carocin S1及Carocin S2 蛋白的胞外輸送分泌機制同時與分屬T3aSS及T3bSS的第三型分泌系統的部分蛋白質有間接或直接的關係。本研究目的在於探討當輸送CarocinS1及Carocin S2 蛋白至胞外時，T3aSS與T3bSS分泌系統間的關係為何，其在Erwinia屬中是否混合成一個新型態的蛋白質分泌系統以專責分泌低分子量細菌素。Carocin S1及Carocin S2蛋白又是如何透過此一分泌系統被輸送至胞外，這三種不同系統的蛋白彼此間的關係及其作用機制是本研究的重點。Erwinia carotovora is a phytopathogenic enterobacterium, responsible for the softrot, blackleg, or stem rot of a number of economically important crops. Some bacterialspecies produce one or more antibacterial substances called bacteriocins, which enhancetheir competitiveness with other related bacterial species. Based on this knowledge,our laboratory tried to exploit the bacteriocins produced by E. carotovora to controlsoft-rot disease. Resounding success has been recorded with this system of control.From our the past 5 years studies, the low-molecular-weight bacteriocin carocin S1,genetic determinant comprised of two structural genes, caroS1K and caroS1I, whichencode the killing protein (CaroH1K) and the immunity protein (CaroH1I), was isolatedfrom Erwinia carotovora 89-H-4. The homology search result indicated that carocin S1gene is homologous to pyocin S3 and AP41 in P. aeruginosa from 1,078th bp to 1,704thbp. The Carocin S1 was incubated with Erwinia carotovora chromosomal DNA for 1 hourat 28℃, in order to confirmed the nuclease activity after isolated and purified byDEAE Affi-gel column. On the way, we also isolated the other LMW bacteriocin carocinS2 gene, which has low homologous with colicin E5, and confirmed the ribonucleaseactivity. From our other studies, the Carocin S1 protein also can not exported outocell when we destroyed the ysaT and ysaU gene, which is a members of the type Ⅲfactor-associated secretory system (T3aSS), or destroyed the flhD/C, fliC, or flhAgenes, is a members of the type Ⅲ bacterium-flagella secretory system (T3bSS) bytransposon Tn5 insertional mutagenesis or homologous recombination. These results aresuggested that Carocin S1 or Carocin S2 is secreted to extrocellular by the new typesecretory system, which recruit some components from T3aSS, T3bSS, or others. Thepurpose of this study is investigate the secretion mechanism, which is recruit thecomponents from T3aSS and T3bSS and develop the new secretion system, of Carocin S1and Carocin S2 to extracellular

Examining the influence of frontline service employees' job cognition and behavior on customer satisfaction and customer-company identification

[[abstract]]現今服務產業在台灣社會和經濟中的地位日益提高，並且講求顧客導向行為，員工主動且積極滿足顧客的需求。研究指出，服務不良的公司將會影響競爭力，並嚴重影響經營績效。本研究以服務利潤鏈為基礎，藉由員工工作認知的三個變數-自我效能、工作滿意度、員工對公司認同，以及員工跨界服務行為之外部表現、內部影響及服務傳遞，了解對於顧客滿意度及認同度的影響。 本研究以上海曼都髮型十家分店的設計師及顧客為探討對象，利用問卷方式進行調查，問卷預試部份，有效問卷員工為10份、顧客為30份；實測部份，有效問卷為員工41份、顧客204份，回收率98 8%。並透過SPSS 12 0、AMOS 6 0、HLM 6 06統計軟體進行分析，研究結果顯示： (1) 自我效能正向顯著影響顧客滿意度、顧客對公司認同；工作滿意度負向顯著影響顧客滿意度、負向不顯著影響顧客對公司認同；員工對公司認同對顧客滿意度、顧客對公司認同呈現負向不顯著影響。 (2) 外部表現與服務傳遞分別正向顯著影響顧客滿意度、顧客對公司認同；內部影響對顧客滿意度、顧客對公司認同呈現正向不顯著影響。 本研究導入員工自我效能、員工跨界服務行為等過去未曾出現在服務利潤鏈架構的變數，並且採用社會認同理論 (員工對公司認同、顧客對公司認同)在此架構裡，冀望可使服務利潤鏈架構更加完善。本研究樣本針對美髮服務業作發放，顧客對於員工的技能，或是所提供之服務才有非常明顯的感受，故員工工作滿意度、員工對公司認同屬於較心理層面之工作認知，且內部影響較無法直接使顧客感受到。[[abstract]]Nowadays the service industry in Taiwan is increasingly important and focuses on customer-oriented behavior which means employees actively to meet customers’ need This research is based on service-profit chain model uses three variables of employee job cognition: self-efficacy job satisfaction employee-company identification and three variables of customer-oriented boundary-spanning behavior: external representation internal influence service delivery to understand the influence of customer satisfaction and customer-company identification This study investigates hair designer and customer of Mentor Hair Stylist in Shanghai with questionnaire survey There are 10 valid questionnaires for employees 30 valid questionnaires for customers of the pre-test questionnaires In formal questionnaires there are 41 valid questionnaires for employee 204 valid questionnaires for customers Through SPSS 12 0 AMOS 6 0 HLM 6 06 statistical analysis software to conduct hierarchical linear model The results show that: (1) Self-efficacy has positively significant effect on customer satisfaction and customer-company identification Job satisfaction has negatively significant effect on customer satisfaction but insignificant on customer-company identification Employee-company identification has negatively insignificant effect on customer satisfaction and customer-company identification (2) External representation and service delivery have positively significant effect on customer satisfaction and customer-company identification Internal influence has positively insignificant effect on customer satisfaction and customer-company identification This research incorporates employee self-efficacy social identity and customer-oriented boundary-spanning behaviors to build a more comprehensive service-profit chain model to fill previous theory’s gap The research samples are hair stylist sevice industry Customers toward employee’s skills and services have very clear fealing Therefore employee job satisfaction and employee-company identification belong more psychological level of job cognitive and internal influence can not let customer that have directly feelin