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

張永麟(Yung-Lin Chang)

指導教授 ： 賴信志

國立臺灣大學/醫學院/醫學檢驗暨生物技術學研究所/碩士(2007年)

https://doi.org/10.6342/NTU.2007.00270

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。

關鍵字

靈菌 ； 表面移行

並列摘要

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.

並列關鍵字

serratia ； swarming

參考文獻

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