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標題: | 靈菌多細胞表面移行行為之調控:rfaD與manA之角色 Regulation of swarming behavior in Serratia marcescens-Roles of rfaD and manA |
作者: | Yung-Lin Chang 張永麟 |
指導教授: | 賴信志 |
關鍵字: | 靈菌,表面移行, serratia,swarming, |
出版年 : | 2007 |
學位: | 碩士 |
摘要: | Serratia marcescens是一種會產生紅色色素,隸屬腸道桿菌科 (Enterobacteriaceae)中靈菌屬 (Serratia)的革蘭氏陰性菌。其為一伺機性致病菌,易造成院內感染,而導致免疫低下的病人致病。Swarming為其細菌群體在固體培養基表面移行的一種特殊型態。S. marcescens 野生株CH-1在30°C時,具有在0.8% 培養基上swarming的現象,但在37°C的環境下,卻無swarming的表現型態。為進一步瞭解其中的機制,利用轉位子突變(mini-Tn5-km1) 的方式,在37°C找到數株precocious-swarming mutant。對其中一株進行研究發現轉位子嵌入在sspA ORF(open reading frame)上,sspA的產物是lipoprotein,實驗室學長已經確定sspA缺乏是造成precocious swarming的原因之一。因此我選擇了sspA附近的ORF—rfaD作為研究主題,根據序列比對,推測此基因可能與細菌的LPS合成有關。rfaD的蛋白產物為nucleoside-diphosphate-sugar epimerases,主要是在LPS core合成的步驟將ADP-D-Glycero-D-manno-heptose轉為ADP-L-Glycero-D-manno-heptose。因此我針對野生株CH-1進行了rfaD基因的剔除得到了rfaD knock strain-YL121,可惜的是發現rfaD對於S. marcescens的swarming並無顯著影響;而觀察其LPS也發現到YL121與CH-1的LPS pattern並無差異,故推測在CH-1裡或許有其他的基因可以補償rfaD的功能。另有一株WW100,轉位子嵌入的位置,在manA ORF上游約211 nucleotides處,此位置位於預測promoter region的範圍內。推測可能是由於此基因的表現量改變,造成37°C時的表現型與野生株有所差異。而目前已知manA製造出的蛋白質mannose-6-phosphate isomerase,其功能為轉換fructose-6-P (Fru-6-P) 及 mannose-6-P (Man-6-P),是連接能量代謝及mannose代謝的橋樑。為了進一步研究37°C時precocious-swarming的原因是否導因於manA基因的過量表現或者降低表現,我們針對野生株CH-1進行了manA的基因剔除(WW101)以及overexpression(WW102)的實驗。取得突變株後,觀察其與CH-1在37°C時swarming的表現,推測manA的過量表現應為造成precocious-swarming的原因。我則著手確認manA的基因與蛋白表現是否是過量,並且針對mannose代謝與glucose代謝進行實驗。根據實驗發現葡萄糖能量代謝途徑對於S. marcescens的swarming比LPS合成途徑來的重要。另外也測量CH-1、WW100、WW101的ATP增加量與NADH/NAD ratio,最後推測WW100的manA過量表現,導致能量代謝途徑的活性增加,造成precocious swarming的行為。 Serratia marcescens is a red-pigmented gram-negative enteric organism. In addition, it is an opportunistic human pathogen and causes many nosocomial infections. Swarming is a specialized form of bacterial surface translocation when bacteria were cultured on surface of solid media. S. marcescens CH-1 swarms on 0.8% LB agar at 30°C, but not at 37°C. In order to understand the underlying mechanism of temperature-dependent regulation of swarming behavior, transposon mutagenesis had been performed to screen for the mutants that swarm at 37°C. One precocious-swarming mutant was isolated and further experiments showed that insertion site of transposon lay in the predicted open reading frame ‘sspA’. The sspA encoded a lipoprotein which was uncharacterized. This gene was characterized by others in our lab. I undertake another open reading frame, rfaD, which convers ADP-D-Glycero-D-manno-heptose to ADP-L-Glycero-D-manno-heptose in LPS synthesis pathway. In order to investigate whether the rfaD was involved in swarming, I use homologues recombination to knock out rfaD in CH-1, and performed series assay to identify the gene function. Furthermore I found that this potential rfaD mutant was not involved in swarming and LPS synthesis. The underlying mechanism is unknown. Another part of this thesis is manA, due to a previously screened precocious swarming strain WW100 which was inserted in manA promoter by transposon. The manA was predicted to encode a protein named mannose-6-phosphate isomerase whose function is interconverting fructose-6-phosphate and mannose-6-phosphate, and link the glucose and mannose metabolic pathway. A manA knock-out strain was constructed. Also, manA overexperssion in S. marcescens CH-1 was performed to test the WW100 precocious swarming phenotype. I concluded that this phenotype was due to manA overexpression. Subsequently, ATP assay, NADH/NAD ratio and LPS pattern were determined. The precocious swarming phenotype was due to increase in TCA cycle activity. Whether the LPS plays a role in this manA mutant phenotype remained to be determined. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/30334 |
全文授權: | 有償授權 |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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