DNA supercoiling (DS) is essential for life because it controls critical processes, including transcription, replication, and recombination. Current methods to measure DNA supercoiling in vivo are laborious and unable to examine single cells. Here, we report a method for high-throughput measurement of bacterial DNA supercoiling in vivo. Fluorescent evaluation of DNA supercoiling (FEDS) utilizes a plasmid harboring the gene for a green fluorescent protein transcribed by a discovered promoter that responds exclusively to DNA supercoiling and the gene for a red fluorescent protein transcribed by a constitutive promoter as the internal standard. Using FEDS, we uncovered single-cell heterogeneity in DNA supercoiling and established that, surprisingly, population-level decreases in DNA supercoiling result from a low-mean/high-variance DNA supercoiling subpopulation rather than from a homogeneous shift in supercoiling of the whole population. In addition, we identified a regulatory loop in which a gene that decreases DNA supercoiling is transcriptionally repressed when DNA supercoiling increases.

中文翻译：

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FEDS：一种基于荧光的新型高通量体内 DNA 超螺旋测量方法。


DNA 超螺旋 (DS) 对生命至关重要，因为它控制转录、复制和重组等关键过程。目前测量体内DNA 超螺旋的方法非常费力，并且无法检查单个细胞。在这里，我们报告了一种体内高通量测量细菌 DNA 超螺旋的方法。 DNA超螺旋的荧光评估 (FEDS) 利用含有由发现的启动子转录的绿色荧光蛋白基因的质粒，该启动子专门响应 DNA 超螺旋，以及由组成型启动子转录的红色荧光蛋白基因作为内部标准。使用 FEDS，我们发现了 DNA 超螺旋的单细胞异质性，并令人惊讶地发现，群体水平的 DNA 超螺旋下降是由低均值/高方差的 DNA 超螺旋亚群引起的，而不是整个群体超螺旋的同质变化造成的。此外，我们还发现了一个调节环，其中当 DNA 超螺旋增加时，减少 DNA 超螺旋的基因会受到转录抑制。