Genome-Wide Functional Screen for Calcium Transients in Escherichia coli Identifies Increased Membrane Potential Adaptation to Persistent DNA Damage.

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, F1Fo-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.IMPORTANCE 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 Escherichia coli, 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.

Voltage-gated calcium flux mediates Escherichia coli mechanosensation.

Electrically excitable cells harness voltage-coupled calcium influx to transmit intracellular signals, typically studied in neurons and cardiomyocytes. Despite intense study in higher organisms, investigations of voltage and calcium signaling in bacteria have lagged due to their small size and a lack of sensitive tools. Only recently were bacteria shown to modulate their membrane potential on the timescale of seconds, and little is known about the downstream effects from this modulation. In this paper, we report on the effects of electrophysiology in individual bacteria. A genetically encoded calcium sensor expressed in Escherichia coli revealed calcium transients in single cells. A fusion sensor that simultaneously reports voltage and calcium indicated that calcium influx is induced by voltage depolarizations, similar to metazoan action potentials. Cytoplasmic calcium levels and transients increased upon mechanical stimulation with a hydrogel, and single cells altered protein concentrations dependent on the mechanical environment. Blocking voltage and calcium flux altered mechanically induced changes in protein concentration, while inducing calcium flux reproduced these changes. Thus, voltage and calcium relay a bacterial sense of touch and alter cellular lifestyle. Although the calcium effectors remain unknown, these data open a host of new questions about E. coli, including the identity of the underlying molecular players, as well as other signals conveyed by voltage and calcium. These data also provide evidence that dynamic voltage and calcium exists as a signaling modality in the oldest domain of life, and therefore studying electrophysiology beyond canonical electrically excitable cells could yield exciting new findings.