Single-Molecule Tracking of DNA Translocases in Bacillus subtilis Reveals Strikingly Different Dynamics of SftA, SpoIIIE, and FtsA
Single-Molecule Tracking of DNA Translocases in Bacillus subtilis Reveals Strikingly Different Dynamics of SftA, SpoIIIE, and FtsA
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DOI：
10.1128/aem.02610-17
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发表时间：
2018-04-01
影响因子：
4.4
通讯作者：
Graumann, Peter L.
中科院分区：
文献类型：
--
作者：
El Najjar, Nina;El Andari, Jihad;Graumann, Peter L.
像许多细菌一样，枯草芽孢杆菌具有两种DNA移位酶，它们在不同的步骤影响染色体分离。在隔膜关闭之前，未分离的DNA通过SftA移动到相对的细胞半部分中，而隔膜截留的DNA通过SpoIIIE拯救。我们使用单分子荧光显微镜和跟踪（SMT）实验来描述两种不同的DNA移位酶，细胞分裂蛋白FtsA和糖酵解酶磷酸果糖激酶（PfkA），在真实的时间的动态。SMT显示，约30%的SftA分子移动通过胞质溶胶，而70%的分数是隔膜结合和静态的。相比之下，只有35%的FtsA分子是静态的中细胞，而SpoIIIE分子扩散内的膜，并显示没有富集在隔膜。一些证据表明，FtsA在SftA的间隔募集中起作用：FtsA缺失导致间隔SftA募集显著减少，SftA分子的平均停留时间减少。FtsA可以在异源真核系统中将SftA募集到膜上，表明SftA可能通过FtsA部分募集。因此，SftA是分裂机制的一个组成部分，而SpoIIIE不是，否则它是一个自由扩散的胞质酶在体内。我们开发的SMT脚本是一个强大的技术，以确定是否低丰度蛋白质是膜结合或胞质，以检测复杂的结合和未结合/扩散蛋白质的群体的差异，并可视化的亚细胞定位的缓慢和快速移动的分子在活cells.IMPORTANCE DNA易位酶夫妇的染色体分离的细胞分裂的晚期事件，从而在细菌细胞周期中发挥重要作用。蛋白质分为两类，整合膜转位酶或非整合转位酶。我们发现，膜结合的移位酶SpoIIIE在B细胞膜上缓慢移动。枯草芽孢杆菌，并没有显示出一个明确的协会与分裂隔膜，在协议的想法，它结合膜结合的DNA，这可能会发生通过细胞分裂跨越非分离的染色体。相比之下，SftA的行为像一个可溶性蛋白质，并被招募到分裂隔膜作为一个组成部分的分裂机制。我们发现，FtsA有助于招聘的SftA，揭示了双重作用的FtsA在司机，但它不是唯一的因素，结合SftA。我们的工作是在单分子水平上对DNA移位酶进行详细的体内研究。
Like many bacteria, Bacillus subtilis possesses two DNA translocases that affect chromosome segregation at different steps. Prior to septum closure, nonsegregated DNA is moved into opposite cell halves by SftA, while septum-entrapped DNA is rescued by SpoIIIE. We have used single-molecule fluorescence microscopy and tracking (SMT) experiments to describe the dynamics of the two different DNA translocases, the cell division protein FtsA and the glycolytic enzyme phosphofructokinase (PfkA), in real time. SMT revealed that about 30% of SftA molecules move through the cytosol, while a fraction of 70% is septum bound and static. In contrast, only 35% of FtsA molecules are static at midcell, while SpoIIIE molecules diffuse within the membrane and show no enrichment at the septum. Several lines of evidence suggest that FtsA plays a role in septal recruitment of SftA: an ftsA deletion results in a significant reduction in septal SftA recruitment and a decrease in the average dwell time of SftA molecules. FtsA can recruit SftA to the membrane in a heterologous eukaryotic system, suggesting that SftA may be partially recruited via FtsA. Therefore, SftA is a component of the division machinery, while SpoIIIE is not, and it is otherwise a freely diffusive cytosolic enzyme in vivo. Our developed SMT script is a powerful technique to determine if low-abundance proteins are membrane bound or cytosolic, to detect differences in populations of complex-bound and unbound/diffusive proteins, and to visualize the subcellular localization of slow- and fast-moving molecules in live cells.IMPORTANCE DNA translocases couple the late events of chromosome segregation to cell division and thereby play an important role in the bacterial cell cycle. The proteins fall into one of two categories, integral membrane translocases or nonintegral translocases. We show that the membrane-bound translocase SpoIIIE moves slowly throughout the cell membrane in B. subtilis and does not show a clear association with the division septum, in agreement with the idea that it binds membrane-bound DNA, which can occur through cell division across nonsegregated chromosomes. In contrast, SftA behaves like a soluble protein and is recruited to the division septum as a component of the division machinery. We show that FtsA contributes to the recruitment of SftA, revealing a dual role of FtsA at the division machinery, but it is not the only factor that binds SftA. Our work represents a detailed in vivo study of DNA translocases at the single-molecule level.