Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/14189
標題: 洛德乳酸桿菌內源性質體之抗藥基因與複製區的分子特性分析及其選殖載體之構築
Molecular Characterization of Antibiotic Resistance Genes and Replication Regions from Indigenous Plasmids of Lactobacillus reuteri and the Construction of Cloning Vectors for Lactobacillus reuteri
作者: 林春福
Lin, Chuen Fu
關鍵字: Lactobacillus reuteri
洛德乳酸桿菌
indigenous plasmid
antibiotic resistance gene
replication region
cloning vector
內源性質體
抗藥基因
複製區
選殖載體
出版社: 獸醫學系
摘要: 由於洛德乳酸桿菌 (Lactobacillus reuteri) 目前使用之選殖載體均源自外源性質體,不論轉形效率或穩定性均甚差;因此,本研究之目的為藉由對洛德乳酸桿菌內源性抗藥質體之分子特性分析,選擇出優良之抗藥基因與複製區,以建造可供本菌使用之良好選殖載體,俾作為本菌分子之研究、改良菌株、及開發為疫苗攜帶者 (vaccine carrier) 之基礎。 本研究共使用五個源自台灣雞隻消化道之洛德乳酸桿菌內源性抗藥質體,進行抗藥基因與複製區之選殖、定序及表現等分子特性分析,並已構築出預期之選殖載體。 試驗首先完成ㄧ個抗氯黴素質體(pTC82)及四個抗紅黴素質體(pTE31、pTE32、pTE15及pTE80)限制圖譜之建立,接著藉此完成各抗藥基因之定序及序列分子特性分析。有關各質體之結果,分述如下:pTC82之抗氯黴素基因及周圍區域 (flanking region) 共定序出1746-bp,經序列分析,預測含有一個714-bp之開讀框 (open reading frame; ORF),可轉譯出238個氨基酸,分子量約為26.2 kDa;此開讀框與其他已知之抗氯黴素基因比對,發現和Staphylococcus aureus pC194之氯黴素乙醯轉移 (chloramphenicol acetyltransferase; cat) 基因具有95%之相似性,故稱為cat-TC基因;在cat-TC基因上游區可發現 -35, -10啟動子 (promoter)、核醣體結合位 (ribosome binding site; RBS) 及引導序列 (leader sequence) 等基因表現調控子存在,經低劑量氯黴素誘導 (inducibility) 試驗,證實cat-TC基因之表現機制屬於誘導表現 (inducible expression) 型。經maxicell 法分析,結果得知cat-TC基因表現之蛋白產物大小與分子量預測值相近。 四個抗紅黴素基因及周圍區域則分別定序出1173-bp (pTE31)、1077-bp (pTE32)、1979-bp (pTE15) 及1076-bp (pTE80),彼此間序列相似性高,經序列分析,預測均含有一個開讀框,其中除pTE15之開讀框轉譯出248個氨基酸外,另三者均為250個氨基酸,分子量約為29.4 kDa,與其他已知之抗紅黴素基因比對,發現和E. coli pIP1527、Streptococcus faecalis Tn917、Streptococcus agalactiae pIP501及Lactobacillus fermentum pLEM3等之23S rRNA methylase gene具有93~99%不等之相似性,故分別稱為erm31、erm32、erm80及erm15基因,歸類為ermB class;在各個erm基因上游區可發現基因表現調控子存在。經maxicell 法分析,結果得知erm基因表現之蛋白產物大小與分子量預測值相符。本研究所定序出的抗氯黴素基因及抗紅黴素基因,均為首次在L. reuteri所發現。 為利於質體複製區 (replication region; RR) 之選殖,試驗以pTE80之erm80基因嵌入pUC18及pUC19之SspI site,完成質體複製區篩選載體pUE80 (+/-) 之構築,並應用此篩選載體選殖出該五個抗藥質體之複製區。複製區核酸片段大小及其重組質體分別為2.2-kb (pTE31-RO)、3.0-kb (pTE32-RO)、3.1-kb (pTE15-RO)、3.0-kb (pTE80-RO) 及3.3-kb (pTC82-RO),經分子特性分析結果顯示,pTE31-RO與L. fermentum pLEM3之複製區相似性高達99%,具有DSO (double strand origin), rep31 (replication initiation gene) 及SSO (single strand origin) 等結構,屬於pC194 family之一員,採用滾動環狀複製 (rolling-cirlce replication; RCR)。此外,pTE31經全長核酸定序,結果新發現二個開讀框,分別可轉譯出210及234個氨基酸,但功能未知。 pTE32-RO、pTE80-RO及pTC82-RO三者之複製區甚為相似,均具有DSO、rep及SSO等結構,屬於pC194 family之一員;各DSO雖然都具有典型之pC194 family切口 (nic) 保留序列 (CTTGATA),但周圍區域則相似性低。各rep基因預測可轉譯出749氨基酸,分子量約為86.7 kDa,Rep蛋白N端之286個氨基酸序列雖亦都保有pC194 family特有的三個motif結構,但與所有已知之Rep蛋白比對,相似性均低於23%,故屬於新發現之Rep蛋白。SSO則與Bacillus subtilis質體pBAA1之SSOT序列有66%相似性,此為乳酸菌RCR質體,首次發現SSO屬於SSOT type者。綜合以上之結果與RCR質體複製過程中,單股DNA中間產物之測定,證實三者以RCR方式複製;rep基因經E. coli pET表現系統進行表現,其蛋白產物大小與預測值相符,為目前最大的Rep蛋白。此外,pTC82經全長核酸定序,結果發現另外三個開讀框,分別可轉譯出419、221及72個氨基酸,其中轉譯出419個氨基酸者,發現靠近N端214個氨基酸序列與Lactococcus lactis pNZ4000及pCI528之mob基因具有40%相似性,而另二者則功能未知。 pTE15-RO複製區為新發現之核酸序列與RCR質體之複製區無相似性,其結構具有七個IR (inverted repeat) 、三個AT-rich之DR (direct repeat; iterons) 及二個ORF (RepA及RepB)。RepA及RepB氨基酸序列分別與Enterococcus faecalis質體pAD1之RepB及RepC具有57%及32%相似性,經限制刪除 (deletion)、質體複製數量 (copy number) 及質體穩定性 (stability) 等分析,證實二者與質體複製數量及穩定性有關之控制,但卻非質體複製所必須,故pTE15-RO複製無需Rep 蛋白,此種複製方式推測與q (theta) replication 型之ColE1-type質體或Bacillus subtilis pLS20相似,因此試驗以氯黴素處理含pTE15-RO之對數生長期洛德乳酸桿菌,結果pTE15-RO無法像ColE1-type質體呈現質體複製數量明顯增加之情形,故推測pTE15-RO之複製方式較類似pLS20。 質體複製區之性狀分析中,為求得各個質體之複製數量,試驗應用脈衝式電場膠體電泳 (pulse field gel electrophoresis; PFGE) 評估出L. reuteri DSM 20016 染色體之全長約為1.95-Mbp,藉此求得pTE31-RO、pTC82-RO及pTE15-RO之複製數量分別為21、13及2。質體宿主範圍分析顯示,pTE31能在L. reuteri DSM 20016及L. fermentum PO2中複製,而pTC82、pTE32-RO、pTE80-RO及pTE15-RO則僅能在L. reuteri DSM 20016中複製,屬於窄宿主範圍。質體穩定性分析顯示,除pTE15-RO在經歷144代不添加抗生素下僅剩4%之質體穩定性外,其餘在216代後均為100%。 本試驗接著選用erm80基因、四個複製區(pTE31-RO、pTE32-RO、pTE15-RO及pGK12)、及pBluescript II SK(-) 之ColE1、lacZ及MCS (multiple cloning site) 等零件,共建造出四個可供使用之E. coli-L. reuteri 穿梭選殖載體,分別稱為pSTE31 (4.9-kb)、pSTE32 (5.7-kb)、pSTE15 (4.0-kb) 及pSGK12-80 (4.2-kb)。其中pSGK12-80之複製區來自廣宿主範圍之外源性質體pWVO1,藉此可應用pSGK12-80攜帶L. reuteri之基因,轉形至其他細菌中,進行表現分析。四個選殖載體之特性分析顯示,MCS均至少具有13個常用之單一限制切割位;載體穩定性測試顯示,pSTE31及pSTE32在216代後均可達100%,而pSTE15及pSGK12-80則分別在144及180代後下降至4%及3%。其中pSTE31及pSGK12-80證實可分別承載抗氯黴素基因及Bacillus subtilis之aprA基因並能成功表現出產物。 為了構築L. reuteri專用之表現載體,以開發本菌成為疫苗攜帶者之目的,本研究已完成轉錄終止子 (transcriptional terminator) 及啟動子篩選載體之建構,分別稱為pSTE31-cat (5.6-kb) 及pSTE31-catT (5.9-kb),期能藉此選殖出功能強大的啟動子及轉錄終止子,作為日後構築表現載體所需之重要零件。
Most of cloning vectors used in Lactobacillus reuteri are adapted from systems already available in other microorganism and generally bear disadvantages of low efficiency and instability. Thus, the aim of this study was to search for some parts (i.e., antibiotic selection markers and replication regions) from indigenous plasmids of this microbe with good properties potentially used in constructing efficient cloning vectors for this bacteria. The final goal of these constructions is to provide tools for the molecular studies, the strain improvement, and the development as vaccine carrier of this bacteria species. This work began with the construction of physical maps from one chloramphenicol resistance (Cmr) plasmid (i. e., pTC82) and four erythromycin resistance (Emr) plasmids (i. e., pTE31, pTE32, pTE15, and pTE80) followed with sequencing determination and molecular analysis of their antibiotic resistance determinants. A total of 1746-bp, which include the Cmr determinant and its flanking region from the plasmid of pTC82, was sequenced. Further determination of the nucleotide sequence of the Cmr gene (cat-TC) on pTC82 revealed an open reading frame (ORF) for a 238-amino-acid Cm acetyltransferase (Cat) monomer. This structural cat-TC gene, 714-bp in length, was highly related (ca. 95% identity) to the cat gene from Staphylococcus aureus plasmid pC194. Immediately upstream to the Cat coding region, the likely genetic expression regulators including promoter, ribosome binding sites (RBS), and leader sequence were observed and were subsequently identified as a functional and inducible type of expression in this microbe. To determine the activity of this putative cat-TC gene, a maxicell analysis was conducted and was able to identify a synthesized protein with molecular weight in reasonable agreement with the prediction from the DNA sequence. To the four Emr determinants, totals of 1173-bp (pTE31), 1077-bp (pTE32), 1979-bp (pTE15), and 1076-bp (pTE80) were respectively sequenced. Determination of the nucleotide sequences of the genetic determinants encoding resistance to Em on the four plasmids revealed a very similar ORF for a 248 (or 250)-amino-acid rRNA methylase (Erm) existing in each of these plasmids. These putative structural erm genes, respectively called erm31, erm32, erm80 and erm15, were highly related (ca. 93-99 nucleotide identity) to the erm genes of E. coli pIP1527, Streptococcus faecalis Tn917, Streptococcus agalactiae pIP501 and Lactobacillus fermentum pLEM3 and were thus categorized under the ermB class. The activities of these putative erm genes were also proved to be functional by a maxicell analysis in which synthesized proteins with a molecular weight of 29.4 kDa, being in agreement with the prediction from the nucleotide sequences, were observed. The nucleotide sequences of cat and erm genes revealed that in these works are the first report in L. reuteri. For facilitating the cloning of replication region (RR) from the five plasmids, a RR-probe vector, namely pUE80 (+/-) was constructed by the ligation of the erm80 gene of pTE80 into the SspI site of pUC18 and pUC19 respectively. Five DNA fragments each containing a RR from the five plasmids, being in the length of 2.2-kb, 3.0-kb, 3.1-kb, 3.0-kb and 3.3-kb respectively, were successfully cloned into the pUE80 (-) to generate the recombinant plasmids of pTE31-RO, pTE32-RO, pTE15-RO, pTE80-RO, and pTC82-RO respectively. Further sequence analysis of these RRs revealed that the RR from pTE31, with the presence of elements of DSO (double strand origin), rep31 (replication initiation gene), and SSO (single strand origin) typical of plasmids that replicate via a rolling-circle mechanism of replication (RCR) of pC194 family, was highly related (ca. 99% nucleotide identity) to the RR of pLEM3 from L. fermentum. Two more ORFs potentially encoding products, respectively, of 210- and 234-amino acid in length were also identifiable in the sequence of pTE31. Their functions remain to be determined. As to the nucleotide sequence of RRs from pTE32, pTE80, and pTC82, a highly sequence similarity was found among one another. Elements (i. e., DSO, rep, and SSO) with conservative characteristics of RCR plasmids of pC194 family were also detectable in these sequences. For examples, despite the low sequence similarities existing between these three RRs and other known RRs with respect to the flaking regions of the nic site of DSO, a conservative nucleotide sequence (CTTGATA) typical of the nic site of pC194 family was actually detectable in all three RRs. The putative rep genes from three RRs were predicted to encode proteins of a molecular weight of 86.7 kDa and 749 amino acids in length. Similar to the condition in nic site, despite little homologies (less than 23%) in amino sequence were found between these three Reps and other known Reps, a region containing three motifs typically found in Reps of pC194 family was also exactly identified in these three RRs. Analysis by employing the GCG software further indicated the deduced Reps as a novel protein belonging to the pC194 family. The SSOs from three RRs showed a strong homology of 66% to the typical sequence of SSOT type from pBAA1 of Bacillus subtilis. These are the first reported sequences of SSOT type in L. reuteri. Besides, three more ORFs, each with potential of encoding proteins of 419, 221, and 72 amino acids in length respectively, were also detected in the sequence of pTC82. To the deduced 419-amino acid product, the 214 amino acids located at the N-terminus was shown to share a homology of 40% in amino acid sequence with the mob gene of pNZ4000 and pCI528 from Lactococcus lactis. Functions of the other two deduced products are still cryptic. Nucleotide sequence of the RR from pTE15 revealed the presence of eight inverted repeats (IR), three AT-rich direct repeats (DR), and two ORFs (namely RepA and RepB). The amino acid sequences of the deduced RepA and RepB from pTE15 showed homologies of 57% and 32% respectively to those of RepB and RepC from Enterococcus faecalis plasmid pAD1. Through a series of analysis including the restriction-deletion study, the copy number detection, and the segregational stability study, functions of the deduced RepA and RepB from pTE15, although dispensable for the plasmid replication, were determined as regulators for plasmid copy number and segregational stability. Judging from the organization mentioned above as well as the absence of Rep protein essential for plasmid replication in the nucleotide sequence, it is tempted to categorize the RR from pTE15 under the θ(theta) type of replication resembling that of pLS20 from Bacillus subtilis and ColE1-type plasmids. However, addition of Cm into the growing culture of L. reuteri, which contained the plasmid pTE15 failed to demonstrate the significant amplification of pTE15. It may thus suggest that the replication of pTE15 is more likely to be the same as that of pLS20. By employing the technique of pulse-field gel electrophoresis (PFGE), a molecular size of 1.95-Mbp for the chromosome of L. reuteri DSM 20016 was obtained. With the availability of this chromosome size, the copy numbers for the recombinant plasmids of pTE31-RO, pTE82-RO, and pTE15-RO were able to be determined as 21, 13, and 2 respectively. As to the host spectrum analysis, plasmids pTE15-RO, pTE32-RO, pTE80-RO, and pTC82 were capable of replicating in L. reuteri only, whereas plasmid pTE31 was found to have a wider host spectrum including L. reuteri and L. fermentum. For the last item of plasmid characteristics analysis, namely the segregational stability, all the recombinant plasmids tested in this study had a stability index of 100% after 216 generations except plasmid pTE15-RO which had an stability index of only 4% after 144 generations. By employing materials including the erm80 gene, four RRs (from pTE15, pTE31, pTE32, and pGK12), and parts (ColE1, lacZ, and multiple cloning site) from pBluescript II SK (-), which were analyzed in this study thus far as bearing good qualities for the construction of vectors, four E. coli-L.reuteri shuttle vectors, named as pSTE31 (4.9-kb), pSTE32 (5.7-kb), pSTE15 (4.0-kb) and pSGK12-80 (4.2-kb) respectively, were successfully constructed. Further evaluation of these vectors revealed that (1) at least 13 unique restriction sites were available for each of the vectors, (2) a high stability index of 100% after 216 generations was observed for the vectors of pSTE31 and pSTE32, while low stability indexes of 4% and 3% after 144 and 180 generations were respectively obtained for vectors of pSTE15 and pSGK12-80, (3) an aprA gene from Bacillus subtilis and a cat gene were cloned into pSTE31 and pSGK12-80 respectively and were successfully expressed and secreted from the recipient host of L. reuteri. Furthermore, a promoter-screening vector (pSTE31-cat, 5.6-kb) and a transcriptional terminator-screening vector (pSTE31-catT, 5.9-kb) had also been constructed in this study. Hopefully, more materials such as promoters and terminators from L. reuteri can be obtained by employing these vectors and the final goal of our research, i.e., construction of expression-secretion vectors, can be reached as early as possible.
URI: http://hdl.handle.net/11455/14189
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