嗜水性產氣單胞桿菌 Aeromonas hydrophila 質體 pAH163、pAH35 之性質分析

Abstract

嗜水性產氣單胞桿菌為革蘭氏陰性、兼性厭氧性菌，廣泛的存在於各類水域之中，一般被視為伺機性病原菌，對於人類及動物均有致病力，並為重要之水產食品病原菌。但目前為止本菌仍然缺乏適當的載體系統可研究其基因功能，所以本研究的目標是期望由 Aeromonas 屬分離內生質體，分析其質體相關之複製等性質，以建構成具有複製區域之選殖載體，提供本菌分子生物研究之利用。本研究中由實驗室所保存之 Aeromonas 環境及臨床株中測定其中存在之質體，其中有 6 株帶有質體，菌株分別為 A. hydrophila 25、A. hydrophila 35、A. hydrophila 132、A. hydrophila 163、A. hydrophila 191、A. hydrophila 1028，從中挑選A. hydrophila 35、A. hydrophila 163 株之質體 pAH35、pAH163 進行全基因組 DNA 定序，將所得序列以 WU-BlastN 與核酸資料庫進行比對，在 pAH35 上找到與存在 E. coli 之 ColE1 質體之 RNA II 及 RNA I 相似之序列，顯示其可能為 ColE1-type 之質體。另外 pAH35 上亦找到與 E. coli ColE2-type 質體類似之 RNA I 及 Rep 蛋白質，顯示其亦可能為 ColE2-type 之質體，但目前尚未明瞭 pAH35 以何種機制複製。pAH163之序列比對出與複製相關之 Rep 蛋白質之 coding region，因此有可能是以 rolling circle 的方式複製。以 SDS、EtBr 及提高培養溫度之方式進行質體剔除後，由 A. hydrophila 35及 A. hydrophila 163分別得到 1 株 (AH35c1) 及 4 株 (AH163c1~4) 質體剔除株，分析其生理特性大致與野生株相近。為探討複製起始區之確實位置及計算質體之備份數，分別再以野生株及質體剔除株計算質體之備份數，pAH163 之備份數約為 500 左右，屬於高備份數之質體，而 pAH35 之質體備份數大約在 50~70 之間。另以將 pAH35、pAH163 之不同片段接於不含複製起始區之 E. coli 載體，建構含質體片段之重組質體，並以抗生素之抗藥性基因作為篩選標記以尋找複製起始區，但尚無明確結果。

Aeromonas hydrophila, a gram negative, facultatively anaerobic freshwater bacterium, occurring widely in aquatic environment, is an opportunistic pathogen of a variety of aquatic and terrestrial animals, including humans. As there is no suitable cloning system to study the gene function in Aeromonas spp. The purpose of this work is to construct vectors able to replicate in Aeromonas which contain replication origin from indigenous plasmids of Aeromonas hydrophila. After examining several clinical and environmental strains, six strains, A. hydrophila 25, A. hydrophila 35, A. hydrophila 132, A. hydrophila 163, A. hydrophila 191 and A. hydrophila 1028 carry plasmids were found. Plasmids pAH35 and pAH163 were isolated from A. hydrophila 35 and A.hydrophila 163 respectively and then were determined using WU-BlastN in EMBL database. Sequences similar to those of RNAII and RNAI were found in pAH35, suggesting pAH35 might be a ColE1-type plasmids. However, sequences similar to those of the RNAI and Rep protein of ColE2-type plasmids were also found in pAH35, suggesting pAH35 also might be a ColE2-type plasmid. Right now, the replication mechanism of pAH35 is unknown. In pAH163, we found a region encluding a replication related protein, Rep, which is needed in rolling circle replication. We used SDS, EtBr ,and higher incubation temperature in order to cure the plasmids of A. hydrophila 35 and A. hydrophila 163. One AH35 curing stain (AH35c1) and four curing stains of AH163 (AH163c1~4) were obtained. The biological character of the curing strains almost the same with the wild-type. We determined the copy number of the plasmids with wild-type and plasmid curing strains. The copy number of pAH163 is about 500 copies, therefore pAH163 belongs to high copy number plasmid. In the other hand, the copy number of pAH35 is about 50 to 70 copies. Using different fragment of pAH35 and pAH163 fused with E. coli vector to find the replication origin is still proceeding.