靈菌二元系統RssAB 經由調控pig cluster影響靈菌紅素產生之研究

Regulation of prodigiosin production and pig cluster expression by RssAB signaling in Serratia marcescens

劉彥妮(Yen-Ni Liu)

指導教授 ： 蘇伯琦

元智大學/工程學院/生物科技與工程研究所/碩士(2009年)

https://doi.org/10.6838/YZU.2009.00010

摘要

靈菌(Serratia marcescens)CH-1 可生合成次級代謝產物靈菌紅
素，靈菌紅素具有抗微生物、抗瘧疾、抗癌症、抑制免疫等功能。由
文獻報導中已得知沙雷氏菌屬(ATCC 274、ATCC 39006)可經由pig
cluster 生合成靈菌紅素，pig cluster 的轉錄是屬polycistronic
mRNA，由pigA 的上游啟動子開始啟動。在本篇研究中，證實pig
cluster 的轉錄會經由靈菌CH-1 中RssA-RssB 二元訊號傳遞系統來控制，由macroscopy 及HPLC 的結果顯示rssA 或rssBA 的突變株
(CH-1ΔA、CH-1ΔBA)會產生較多的靈菌紅素；使用lacZ 當作pigA
的轉錄報導基因，結果顯示在CH-1ΔA 或CH-1ΔBA 的突變株中，pig
cluster (pigA–O)有較高的轉錄能力；另一方面，由膠體電泳位移分析法(electrophoretic mobility shift assay)顯示已磷酸化的RssB (RssB-P)是直接與pigA 的啟動子區域做結合，也經由DNaseⅠ足跡試驗分析法 (DNase I footprinting)證實RssB-P 的結合區域，RssB-P 結合在pigA基因起始的上游23 至46 bp 之間。利用軟體(ClustalW+, through SeqWeb v 3.1.2.)分析比對flhDC、rssB、pigA 的啟動子區域，發現在此三個啟動子的RssB 結合區域中約有18 bp 相似，利用單點突變建構一個全新的pigA_mut7 片段，實驗發現RssB-P 與pigA_mut7 不具有DNA binding shift 的情形，也證實這些突變的點可能就是影響RssB-P 結合到pigA 啟動子的重要區域。本篇研究指出RssA-RssB 的訊號傳遞所產生的活化態RssB 會直接抑制pigA 啟動子的活化，導致靈菌中其靈菌紅素的產生被抑制，另一方面藉由電腦的分析與實驗的證明確定了RssB-P 對於所結合DNA 的共有序列(consensus sequence)，ttaGCtaaaTTAaa·TtT，稱為RssB box。

關鍵字

靈菌 ； 靈菌紅素 ； 二元系統

並列摘要

Serratia marcescens CH-1 synthesize the red-pigmented
prodigiosin (2-methyl-3-pentyl-6-methoxy prodiginine) and display swarming behaviour on agar surface. The prodigiosin biosynthesis gene cluster (pig cluster) from two strains of Serratia (S. marcescens ATCC 274 and Serratia sp. ATCC 39006) has been reported. The pig cluster is transcribed as a polycistronic mRNA from a promoter upstream of pigA.
In this study, however, we demonstrate that Pig biosynthesis is also controlled by RssA-RssB two-component signal transduction system in S. marcescens CH-1. Mutation in rssA or rssBA resulted in more production of prodigiosin by macroscopy and using HPLC analysis. The transcription of the Pig biosynthetic operon (pigA–O) is also activating in
rssA or rssBA mutants using lacZ reporter fusions to pigA.
Characterization of phosphorylated RssB (RssB-P) binding site by electrophoretic mobility shift assay showed direct interaction of RssB-P with the pigA promoter region. Alignment of RssB-P binding region on upstreams of rssB, flhDC, and pigA revealed that the conserved core sequence, ttaGCtaaaTTAaa·TtT, which is proposed as RssB box.
Site-specific mutations of the conserved nucleotide in the RssB box resulted in unbinding of RssB-P onto pigA upstream region. Collectively, the present results indicate that activated RssA-RssB signaling directly inhibits the pigA promoter activity leading to repress the prodigiosin
production in Serratia marcescens CH-1.

並列關鍵字

Serratia marcescens ； prodigiosin ； two-component system

參考文獻

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