Lighting Up Clostridium Difficile: Reporting Gene Expression Using Fluorescent Lov Domains.
Lighting Up Clostridium Difficile: Reporting Gene Expression Using Fluorescent Lov Domains.
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DOI：
10.1038/srep23463
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发表时间：
2016-03-21
影响因子：
4.6
通讯作者：
Douce GR
中科院分区：
文献类型：
--
作者：
Buckley AM;Jukes C;Candlish D;Irvine JJ;Spencer J;Fagan RP;Roe AJ;Christie JM;Fairweather NF;Douce GR
从绿色荧光蛋白衍生的荧光报告的使用已被证明对在有氧条件下生长的细菌的生物过程的可视化是无价的。然而，它们对氧气的需求限制了它们在专性厌氧菌（如艰难梭菌）中的应用。来自光、氧或电压传感（LOV）结构域的荧光蛋白已被证明可以克服这一限制，但它们作为翻译融合物在严格的厌氧细菌中监测蛋白质表达和定位的用途尚未报道。在这里，我们展示了phiLOV在三种梭状芽孢杆菌中的应用，并通过与细胞分裂蛋白FtsZ的遗传融合，将其作为实时蛋白质翻译和动态的标记物。分裂细胞的延时显微镜显示，Z环组装是通过从圆周上的一点开始的FtsZ弧的延伸而产生的。此外，通过将phiLOV整合到鞭毛亚基（FliC）中，我们展示了细菌基于lovv的融合蛋白成功输出到细胞外环境的潜力。
The uses of fluorescent reporters derived from green fluorescent protein have proved invaluable for the visualisation of biological processes in bacteria grown under aerobic conditions. However, their requirement for oxygen has limited their application in obligate anaerobes such as Clostridium difficile. Fluorescent proteins derived from Light, Oxygen or Voltage sensing (LOV) domains have been shown to bridge this limitation, but their utility as translational fusions to monitor protein expression and localisation in a strict anaerobic bacterium has not been reported. Here we demonstrate the utility of phiLOV in three species of Clostridium and its application as a marker of real-time protein translation and dynamics through genetic fusion with the cell division protein, FtsZ. Time lapse microscopy of dividing cells suggests that Z ring assembly arises through the extension of the FtsZ arc starting from one point on the circumference. Furthermore, through incorporation of phiLOV into the flagella subunit, FliC, we show the potential of bacterial LOV-based fusion proteins to be successfully exported to the extracellular environment.