Department of Molecular Cellular Developmental Biology, University of Colorado, Boulder, Colorado, USA.
BioFrontiers Institute, University of Colorado, Boulder, Colorado, USA.
J Bacteriol. 2021 Jan 11;203(3). doi: 10.1128/JB.00509-20.
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 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 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, FF-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. All eukaryotic cells use calcium as a critical signaling molecule. There is tantalizing evidence that bacteria also use calcium for cellular signaling, but much less is known about the molecular actors and physiological roles. To identify genes regulating cytoplasmic calcium in , we created a single-cell screen for modulators of calcium dynamics. The genes uncovered in this screen helped refine a model for voltage-mediated bacterial mechanosensation. Additionally, we were able to more carefully dissect the mechanisms of adaptation to long-term DNA damage, which has implications for both bacteria and mitochondria in the face of unrepaired DNA.
钙在真核细胞的信号转导和动态平衡中发挥着许多关键作用。与真核细胞相比，人们对细菌中的钙信号转导知之甚少，并且仅鉴定出少数控制流入和流出的基因。之前的研究表明，钙内流是由电压去极化诱导的，机械刺激增强了钙内流，这表明其在细菌机械感受中起作用。为了鉴定影响细菌钙处理的蛋白和途径，我们设计了一个活细胞筛选实验，以监测全基因组敲除面板中单个细胞的钙动态。该筛选实验测量了来自 Keio 敲除库的细胞，并对群体中的钙瞬变进行了量化。总的来说，我们发现 143 个基因敲除降低了钙瞬变的水平，32 个基因敲除增加了钙瞬变的水平。如预期的那样，与能量产生和调节相关的蛋白以及电压汇 FF-ATPase 的蛋白的敲除出现了。胞外多糖和外膜合成蛋白的敲除导致钙瞬变减少，这完善了我们的电生理学介导的机械感受模型。此外，与 DNA 修复相关的蛋白的敲除导致钙瞬变和电压降低。然而，急性 DNA 损伤不会影响电压，结果表明只有长期适应 DNA 损伤才会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分离，这也对真核生物中线粒体 DNA 损伤具有启示意义。所有真核细胞都将钙用作关键的信号分子。有诱人的证据表明，细菌也将钙用于细胞信号转导，但对分子作用物和生理作用知之甚少。为了鉴定调控细菌细胞质钙的基因，我们创建了一个用于钙动力学调节剂的单细胞筛选。该筛选中发现的基因有助于完善电压介导的细菌机械感受模型。此外，我们能够更仔细地剖析长期 DNA 损伤适应的机制，这对于面对未修复的 DNA 的细菌和线粒体都具有重要意义。
