Calcium plays numerous critical roles in signaling and homeostasis in eukaryotic cells. Far less is known about calcium signaling in bacteria than in eukaryotic cells, and few genes controlling influx and efflux have been identified. Previous work in Escherichia coli showed that calcium influx was induced by voltage depolarization, which was enhanced by mechanical stimulation, which suggested a role in bacterial mechanosensation. To identify proteins and pathways affecting calcium handling in bacteria, we designed a live-cell screen to monitor calcium dynamics in single cells across a genome-wide knockout panel in E. coli. The screen measured cells from the Keio collection of knockouts and quantified calcium transients across the population. Overall, we found 143 gene knockouts that decreased levels of calcium transients and 32 gene knockouts that increased levels of transients. Knockouts of proteins involved in energy production and regulation appeared, as expected, as well as knockouts of proteins of a voltage sink, F₁F_o-ATPase. Knockouts of exopolysaccharide and outer membrane synthesis proteins showed reduced transients which refined our model of electrophysiology-mediated mechanosensation. Additionally, knockouts of proteins associated with DNA repair had reduced calcium transients and voltage. However, acute DNA damage did not affect voltage, and the results suggested that only long-term adaptation to DNA damage decreased membrane potential and calcium transients. Our work showed a distinct separation between the acute and long-term DNA damage responses in bacteria, which also has implications for mitochondrial DNA damage in eukaryotes.

中文翻译：

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钙瞬变在大肠杆菌中的全基因组功能筛选确定增加的膜电位适应持久性DNA损伤。

钙在真核细胞的信号传导和体内平衡中起着至关重要的作用。对细菌中钙信号的了解远不如在真核细胞中，并且几乎没有控制潮汐和外排的基因。先前在大肠杆菌中的研究表明，电压去极化可诱导钙内流，机械刺激会增强钙的内向作用，这提示其在细菌机械感觉中的作用。为了鉴定影响细菌中钙处理的蛋白质和途径，我们设计了一个活细胞筛选器来监控整个大肠杆菌中全基因组敲除面板中单个细胞中的钙动态。。该筛查从Keio基因敲除收集的细胞中进行了测量，并对整个种群中的钙瞬变进行了定量。总体而言，我们发现143个基因敲除可降低钙瞬变水平，而32个基因敲除可增加钙瞬变水平。如预期的那样，出现了涉及能量产生和调节的蛋白质的敲除，以及电压吸收器F ₁ F _o的蛋白质的敲除。-ATP酶。外多糖和外膜合成蛋白的敲除显示减少的瞬变，完善了我们的电生理介导的机械感觉模型。此外，与DNA修复相关的蛋白质基因敲除降低了钙瞬变和电压。但是，急性DNA损伤并没有影响电压，结果表明，只有长期适应DNA损伤才能降低膜电位和钙瞬变。我们的工作表明细菌的急性和长期DNA损伤反应之间存在明显的区别，这也对真核生物的线粒体DNA损伤有影响。