Stenotrophomonas maltophilia的ampR-L2組裝之特性分析; Characterization of ampR-L2 module of Stenotrophomonas maltophilia

中國醫藥大學機構典藏 China Medical University Repository, Taiwan > 醫學院 > 醫學檢驗生物技術學系暨碩士班 > 博碩士論文 > Item 310903500/873

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题名:	Stenotrophomonas maltophilia的ampR-L2組裝之特性分析;Characterization of ampR-L2 module of Stenotrophomonas maltophilia
作者:	黃奕瑋;Yi-Wei Huang
贡献者:	中國醫藥大學：醫學檢驗生物技術學系碩士班
关键词:	嗜麥芽醣黃單胞菌;乙內醯胺酶;ampR-L2 module;Stenotrophomonas maltophilia
日期:	2009-01-06
上传时间:	2009-08-12 17:09:54 (UTC+8)
摘要:	Stenotrophomonas maltophilia可產生兩種可被誘發表現的b-lactamases蛋白，分別為L1和L2蛋白。而L1與L2基因的表現通常受LysR型轉錄調控因子AmpR蛋白所調控。在分類上，L2基因屬於class A型b-lactamase，而ampR基因位於L2基因的上游形成一ampR-L2組裝，且兩基因間含有一174（或175）-bp的intergenic（IG）區域，此組裝與腸內菌屬的ampR-ampC 組裝相似。 L1蛋白則屬於class B型b-lactamase，但其鄰近並無類似ampR之調控基因。本論文的第一部份是將S. maltophilia KH的ampR-L2組裝與其它微生物的ampR-b-lactamase組裝做進一步的分析，比較其AmpR蛋白、b-lactamase蛋白和ampR-b-lactamase IG片段。在所有被分析的菌株中，S. maltophilia與Xanthomonas campestris的演化關係最為相近。接著在評估AmpR蛋白對於L1與L2基因表現所扮演的調控角色方面發現，沒有誘發物的情況下，AmpR蛋白可活化L1基因的表現，抑制L2基因的表現；然而在有誘發物的情況下，AmpR蛋白對於L1、 L2兩基因表現皆扮演著活化者的角色。另外L1、 L2兩基因的誘發表現需要依靠AmpR蛋白的幫助（AmpR-dependent），而ampR基因的轉録作用是微弱且持續表現的，並無自我調控的現象。本論文的第二部份將16株S. maltophilia臨床分離菌株的ampR-L2組裝進行分析，並且將其AmpR蛋白、ampR-L2 IG區域和L2蛋白分組。結果發現，其L2蛋白和ampR-L2 IG區域之變異度高達32%，然而AmpR蛋白序列在這16株S. maltophilia分離菌株中卻被高度保留。接著利用縮短IG區域的DNA片段來鑑定足以誘發L2基因表現之區域和ampR基因之啟動子區域。結果證實S. maltophilia之ampR基因的啟動子與AmpR蛋白的結合區域並無重疊，說明AmpR蛋白對其ampR基因並無自我調控。然後將不同的IG-L2xylE transcriptional fusion質體送入S. maltophilia原始菌株中以評估AmpR蛋白對不同的ampR-L2 IG區域之誘發能力。另外，將一段突變之102-bp的IG片段送入S. maltophilia原始菌株中，即可同時減弱其染色體上L1和L2基因之表現，此oligo-nucleotides attenuator可發展為控制S. maltophilia菌株β-lactam抗藥性之新策略。 Stenotrophomonas maltophilia is known to produce at least two chromosomal-mediated inducible β-lactamases, L1 and L2. Expression of the L1 and L2 β-lactamase genes responsible for β-lactams resistance is generally regulated by a LysR type regulator of AmpR. Gene L2, which encodes a class A β-lactamase, and the ampR gene is located immediately upstream of L2 and is transcribed divergently, forming an ampR-L2 module with a 174 (or 175)-bp intergenic (IG) region. L1 belongs to the class B β-lactamase and has no neighbor ampR-like regulatory gene. Firstly, the ampR-L2 module from S. maltophilia KH was compared with ampR-β-lactamase modules from several microorganisms with respect to the AmpR and β-lactamase proteins and the intergenic (IG) region. S. maltophilia and Xanthomonas campestris showed the most closely phylogenetic relationship among the microorganisms considered. The regulatory role of AmpR towards L1 and L2 was further analysed. In the absence of inducer, AmpR acted as an activator for L1 expression and as a repressor for L2 expression, whereas AmpR was an activator for both genes in an induced state. In addition, inducibility of L1 and L2 genes depended on the presence of AmpR. The ampR transcript was weakly and constitutively expressed, but was not autoregulated. Secondly, the ampR-L2 modules of sixteen S. maltophilia isolates were analyzed in respect of AmpR protein, L2 protein, and the IG region. The results demonstrate that L2 proteins and IG regions show a significantly genetic diversity (up to 32% diversity) among S. maltophilia species, whereas AmpR proteins are highly conserved with identity of 98%. The DNA segments in the IG region, which are sufficient to support the L2 induction and ampR mutant complementation, were identified by deletion constructs assays. The AmpR binding domain and the ampR promoter were found to be not overlapped in the ampR-L2 IG region of S. maltophilia, signifying the fact that ampR gene is hardly autoregulated. Furthermore, the induction potential of AmpR toward the different ampR-L2 IG regions was evaluated by introducing the various IG-L2xylE transcriptional fusion constructs into a wild S. maltophilia strain. The induction levels achieved in the various AmpR-IG pairs display marked quantitative differences. In addition, introduction of a 102-bp mutated ampR-L2 IG segment into the wild strain attenuates the expression of chromosomal L1 and L2 genes. A concept of oligo-nucleotides attenuator for the development of antimicrobial agent is proposed.
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