大腸桿菌多套數質體的分離機制

多套數質體不具有類似低套數質體的主動分離機制，其分離機制目前仍不明確。多套數質體會在細菌細胞內特定位置形成質體群聚。細胞分裂過程質體群聚裂成兩個小群聚，分裂的小群聚移動遠離原有群聚的位置，將質體傳遞至子代細胞。對於這樣的多套數質體群聚與分離過程，我們採取兩種實驗觀察方式：細胞分裂多套數質體數量變化與多套數質體群聚內單一質體運動。首先，細胞多套數質體數量觀察，我們設計測量兩種具有相同複製原點、不同螢光抑制操作系統(fluorescent repressor-operator systems, FROS)標記之 ColE1衍生質體，轉形於同一菌株，統計細胞分裂親代細胞與子代細胞質體數量。發現ColE1衍生質體之間的質體不相容，以及證實細胞分裂過程質體分配具有子代分配傾向。質體群聚內的單一質體追蹤，我們突變ColE1複製原點，利用抑制子-啟動子配對(repressor-promoter pair) PhlF與PphlF ，調控RNAII的表達，控制質體複製數量。藉此限定質體群聚當中一種螢光色標記質體為單一質體，以螢光顏色區分，追蹤質體群內的單一質體動態。單一質體追蹤軌跡迴轉半徑分析證實細胞內多套數質體存在兩種不同的運動，我們分別稱為質體侷限運動與跳躍運動。兩種運動的平均方均根位移(mean square displacement, MSD) 曲線、擴散係數與質體位置重疊指數，表示單一質體運動受到質體群聚的質體數量影響。我們也同時應用HupA的全細胞螢光顯微術(Whole-cell fluorescence microscopy)，觀察細胞類核空間變化與質體運動的關聯。其結果表示單一質體擴散運動受到類核佔據空間排擠。綜合本次實驗結果，我們認為細胞內多套數質體的空間分布包含漂散的多套數質體與質體群聚。漂散的多套數質體分布於類核外空間進行擴散運動。細胞分裂過程類核空間結構改變，漂散的多套數質體從新的類核外空間進行長距離移動，藉此達成多套數質體分離。

關鍵字

多套數質體；質體分離機制

並列摘要

High-copy number plasmids typically present as multiple clusters at specific cellular region in bacterial cells, even during their replication and partition. However, unlike the discovered active partition system in low-copy plasmids, their essential partition mechanism is still unclear. We approach this problem in investigation of the variation of inherited plasmid copy number and single plasmid motion in clusters in bacterial cell. We co-transformed two ColE1 derivative plasmid with different fluorescent repressor-operator systems and antibiotic resistance gene, pLacOIC2.bx and pTetORK34.b, into E. coli strain BW25113. The distributions and motions of the plasmids are investigated respectively at single cell level. Our results show that the nature of competition between plasmids and the variance of the plasmid inheritance ratio for both of two plasmids from parent to daughter cells. The inheritance ratio showing more diverse distribution than simple binomial distribution. Additionally, We co-transformed the plasmid pLacOIC2.bx and pTetORK34.bp, modified from pTetORK34.b, with the synthetic replication origin into BW25113. Based on the plasmid pTetORK34.bp copy number reduction via the repression of replication primer RNAII expression which is regulated by genetic circuit of PhlF and PphlF, it provides the way to efficiently track a single plasmid motion in the plasmid clusters. According to radius of gyration of the single plasmid traces, these single plasmid motions distinguished into jump-like motion and confined motion. For the traces of these two motion, we calculated average mean square displacement, diffusivity and degree of co-localization. Our results suggest that plasmid motion determined by the plasmid clusters. Furthermore, we monitored the single plasmid motion and spatial distribution of nucleoids labeled by HupA-mTurquoise2 within a cell cycle. Our results show that the single plasmid motion was excluded by the nucleoid. The heterogeneity of partition is revealed by the variation of plasmid copy number and single plasmid motions in clusters. High-copy number plasmids are organized as both clusters and single random copies inside bacteria. We believe that partition mechanism is dominated by spatial distribution of nucleoids.