Rates of Gyrase Supercoiling and Transcription Elongation Control Supercoil Density in a Bacterial Chromosome

Gyrase catalyzes negative supercoiling of DNA in an ATP-dependent reaction that helps condense bacterial chromosomes into a compact interwound “nucleoid.” The supercoil density (σ) of prokaryotic DNA occurs in two forms. Diffusible supercoil density (σD) moves freely around the chromosome in 10 kb domains, and constrained supercoil density (σC) results from binding abundant proteins that bend, loop, or unwind DNA at many sites. Diffusible and constrained supercoils contribute roughly equally to the total in vivo negative supercoil density of WT cells, so σ = σC+σD. Unexpectedly, Escherichia coli chromosomes have a 15% higher level of σ compared to Salmonella enterica. To decipher critical mechanisms that can change diffusible supercoil density of chromosomes, we analyzed strains of Salmonella using a 9 kb “supercoil sensor” inserted at ten positions around the genome. The sensor contains a complete Lac operon flanked by directly repeated resolvase binding sites, and the sensor can monitor both supercoil density and transcription elongation rates in WT and mutant strains. RNA transcription caused (−) supercoiling to increase upstream and decrease downstream of highly expressed genes. Excess upstream supercoiling was relaxed by Topo I, and gyrase replenished downstream supercoil losses to maintain an equilibrium state. Strains with TS gyrase mutations growing at permissive temperature exhibited significant supercoil losses varying from 30% of WT levels to a total loss of σD at most chromosome locations. Supercoil losses were influenced by transcription because addition of rifampicin (Rif) caused supercoil density to rebound throughout the chromosome. Gyrase mutants that caused dramatic supercoil losses also reduced the transcription elongation rates throughout the genome. The observed link between RNA polymerase elongation speed and gyrase turnover suggests that bacteria with fast growth rates may generate higher supercoil densities than slow growing species.

DNA旋转酶（Gyrase）以ATP依赖的方式催化DNA的负超螺旋化，这一过程可帮助将细菌染色体凝聚为紧密缠绕的“类核（nucleoid）”结构。原核生物DNA的超螺旋密度（σ）存在两种形式：可扩散超螺旋密度（σD）可在染色体上以10kb的结构域范围内自由移动；约束性超螺旋密度（σC）则源于大量蛋白质在多个位点结合并弯曲、环化或解旋DNA所产生的效应。可扩散与约束性超螺旋对野生型（WT）细胞体内总负超螺旋密度的贡献大致相当，即σ = σC + σD。出人意料的是，大肠杆菌（Escherichia coli）染色体的超螺旋水平较肠炎沙门氏菌（Salmonella enterica）高出15%。为解析能够改变染色体可扩散超螺旋密度的关键调控机制，本研究以插入于基因组10个不同位点的9kb“超螺旋传感器”为工具，对沙门氏菌菌株开展了分析。该传感器包含侧翼带有同向重复解离酶结合位点的完整乳糖操纵子（Lac operon），可同时监测野生型与突变菌株的超螺旋密度及转录延伸速率。RNA转录会导致高表达基因上游区域的负超螺旋水平升高，下游区域则降低。过量的上游超螺旋会被拓扑异构酶I（Topo I）松弛，而DNA旋转酶则会补充下游损失的超螺旋，以维持动态平衡状态。携带温度敏感型DNA旋转酶突变（TS gyrase mutations）的菌株在许可温度下培养时，会出现显著的超螺旋损失，其程度从野生型水平的30%不等，直至大多数染色体位点的σD完全丧失。转录过程会影响超螺旋损失：添加利福平（Rif）后，全基因组范围内的超螺旋密度会出现反弹。引发严重超螺旋损失的DNA旋转酶突变，同时也会降低全基因组的转录延伸速率。本研究观察到的RNA聚合酶延伸速率与DNA旋转酶周转之间的关联提示，生长速率较快的细菌可能比生长缓慢的物种拥有更高的超螺旋密度。