| Summary: | 博士 === 國立陽明大學 === 微生物暨免疫學研究所 === 84 === cutRS操作元為鏈黴菌中所發現之第一組雙配件訊號傳導系統，由cutR及下游cutS基因所組成。CutR與CutS蛋白分別屬於高保留性之反應調節蛋白族(response-regulator family)及組胺酸蛋白質激酉每族(histidine protein kinase family)。我以nuclease S1 mapping法定出cutRS操作元的轉錄起點，在cutR基因轉譯起點上游75至76核甘酸，而其表現在於過渡期及生長停滯期，顯示受到生長時期所調控。
對於研究質體上melCl基因轉譯起始突變(translational initiationmutation)的壓抑機制，我發現cutRS上游區域所內含之一個反向啟動子活性，可藉由促進下游melC基因的表現彌補其轉譯缺失。
在cutRS操作元生理功能的研究方面，我利用導向致變法(trgeted mutagenesis)分別構築CurR及cutS基因之失活突變株(nullmutnt)，並觀察其形性之改變。結果，發現此二種突變株均可提前產生至少二種抗生素素雅(actinorhodin及undecylprodigionsin)，且產量也較野生株多。富重新以質體送回完整的cutR基因時，這些突變株可以恢復為野生株的形性；但是當此段cutR成基因上負責接受磷酸基的ASp殘基發生改變時，則無法恢復。另外在cutS突變株中則可發現，抗生素actinorhodin而生合成所需之生產途徑專屬激活子基因actII-ORF4的表現亦多於野生株。這些結果顯示CutR-S訊號傳導系統扮演著多功能(pleiotropic)抑制的角色，透過抑制生產途徑專屬激活子基因的表現而調節抗生素的生合成。
本論文中也揭示CutR-S訊號傳導系統與另一正性調節系統AfsR-K在廣泛調節抗生素生合成上可能的交互關係。
The cutRS operon was the first two-component signal transduction system found in Streptomyces. The CutR and CutS proteins correspond to the response regulator and the histidine protein kinase, respectively. Transcription of the cutRS operon was detected during the transition and stationary phases of growth initiating at a single site upstream of cutR. This promoter region also possessed promoter activity directed away from cutRS, which appears to be responsible for the previously observed suppression of the'' translational deficiency of a melCl (that encoded a copper transfer protein necessary for the activation of apotyrosinase) mutation on plasmid.
To investigate the biological function(s) of the cutRS operon in Streptomyces, we disrupted the chromosomal cutR and cutS sequences by targeted gene replacement. The resulting mutants exhibited accelerated and increased production of actinorhodin (Act, a blue pigmented antibiotic) and undecylprodigiosin (Red, a red pigmented antibiotic) which could be reversed by introduction of cutR on a plasmid. However, when the mutagenized cutR on its phospho-acceptor site were introduced, the restoration ability was lost. Futhermore, the expression of a pathway specific activator (actII-ORF4) required for Act production was demonstrated to be repressed in cutS disrupted mutant. These results indicated that the functional CutR-CutS signal pathway played a pleiotropic role in the repression of antibiotic biosynthesis through their cognate pathway specific activators. In this study, we also uncovered a possible interaction between CutR-S signal transduction system and a positive regulatory system,AfsR-K in the global regulation of antibiotic biosynthesis.
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