Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut, USA.
Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, Connecticut, USA
mBio. 2020 Jul 28;11(4):e01053-20. doi: 10.1128/mBio.01053-20.
DNA supercoiling (DS) is essential for life because it controls critical processes, including transcription, replication, and recombination. Current methods to measure DNA supercoiling are laborious and unable to examine single cells. Here, we report a method for high-throughput measurement of bacterial DNA supercoiling luorescent valuation of NA upercoiling (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. DNA represents the chemical support of genetic information in all forms of life. In addition to its linear sequence of nucleotides, it bears critical information in its structure. This information, called DNA supercoiling, is central to all fundamental DNA processes, such as transcription and replication, and defines cellular physiology. Unlike reading of a nucleotide sequence, DNA supercoiling determinations have been laborious. We have now developed a method for rapid measurement of DNA supercoiling and established its utility by identifying a novel regulator of DNA supercoiling in the bacterium as well as behaviors that could not have been discovered with current methods.
DNA 超螺旋（DS）对生命至关重要，因为它控制着包括转录、复制和重组在内的关键过程。目前测量 DNA 超螺旋的方法既繁琐又无法检测单细胞。在这里，我们报告了一种用于高通量测量细菌 DNA 超螺旋的方法，即荧光评估 DNA 超螺旋（FEDS）。该方法利用携带绿色荧光蛋白基因的质粒，该基因由一个独特响应 DNA 超螺旋的发现启动子转录，以及一个由组成型启动子转录的红色荧光蛋白基因作为内参。使用 FEDS，我们揭示了 DNA 超螺旋的单细胞异质性，并令人惊讶地发现，DNA 超螺旋水平的群体降低是由于低均值/高方差的 DNA 超螺旋亚群，而不是整个种群的超螺旋均匀变化。此外，我们确定了一个调控回路，其中一个降低 DNA 超螺旋的基因在 DNA 超螺旋增加时被转录抑制。DNA 是所有生命形式中遗传信息的化学载体。除了其核苷酸的线性序列外，它还具有结构中的关键信息。这种信息称为 DNA 超螺旋，对转录和复制等所有基本 DNA 过程都至关重要，并定义了细胞生理学。与阅读核苷酸序列不同，DNA 超螺旋的测定一直很繁琐。我们现在已经开发出一种快速测量 DNA 超螺旋的方法，并通过在细菌中识别一种新的 DNA 超螺旋调节剂以及当前方法无法发现的行为，证明了其用途。
