Significance: Bacteria cope with changing external environments and must therefore sense and respond to their local conditions. In addition to sensing numerous chemical cues, in this paper we show that Escherichia coli can sense the local mechanical environment through voltage-induced calcium flux. This mechanism is similar to sensory neurons in vertebrates, suggesting an ancient process of voltage-regulated mechanotransduction. Mechanical contact drives pathogenicity in several species of bacteria and we now propose a potential mechanism for this sensation.

Aminoglycosides are broad-spectrum antibiotics whose mechanism of action is under debate. It is widely accepted that membrane voltage potentiates aminoglycoside activity, which is ascribed to voltage-dependent drug uptake. In this paper, we measured the response ofEscherichia colitreated with aminoglycosides and discovered that the bactericidal action arises not from the downstream effects of voltage-dependent drug uptake, but rather directly from dysregulated membrane potential. In the absence of voltage, aminoglycosides are taken into cells and exert bacteriostatic effects by inhibiting translation. However, cell killing was immediate upon re-polarization. The hyperpolarization arose from altered ATP flux, which induced a reversal of the F1Fo-ATPase to hydrolyze ATP and generated the deleterious voltage. Heterologous expression of an ATPase inhibitor completely eliminated bactericidal activity, while loss of the F-ATPase reduced the electrophysiological response to aminoglycosides. Our data support a model of voltage-induced death, and separates aminoglycoside bacteriostasis and bactericide inE. coli.

Counting viable cells is a universal practice in microbiology. The colony-forming unit (CFU) assay has remained the gold standard to measure viability across disciplines, but it is time-intensive and resource-consuming. Here we describe the geometric viability assay (GVA) that replicates CFU measurements over 6 orders of magnitude while reducing over 10-fold the time and consumables required. GVA computes a sample’s viable cell count on the basis of the distribution of embedded colonies growing inside a pipette tip. GVA is compatible with Gram-positive and Gram-negative planktonic bacteria (Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis), biofilms and fungi (Saccharomyces cerevisiae). Laborious CFU experiments such as checkerboard assays, treatment time-courses and drug screens against slow-growing cells are simplified by GVA. The ease and low cost of GVA evinces that it can replace existing viability assays and enable viability measurements at previously impractical scales.

Addition of glucose to starved Saccharomyces cerevisiae initiates collective NADH dynamics termed glycolytic oscillations. Numerous questions remain about the extent to which single cells can oscillate, if oscillations occur in natural conditions, and potential physiological consequences of oscillations. In this paper, we report sustained glycolytic oscillations in single cells without the need for cyanide. Glucose addition to immobilized cells induced pH oscillations that could be imaged with fluorescent sensors. A population of cells had oscillations that were heterogeneous in frequency, start time, stop time, duration and amplitude. These changes in cytoplasmic pH were necessary and sufficient to drive changes in NADH. Oscillators had lower mitochondrial membrane potentials and budded more slowly than non-oscillators. We also uncovered a new type of oscillation during recovery from H₂O₂ challenge. Our data show that pH in S. cerevisiae changes over several time scales, and that imaging pH offers a new way to measure glycolytic oscillations on individual cells.

We examine the structural changes during the primary photoreaction in blue-absorbing proteorhodopsin (BPR), a light-driven retinylidene proton pump, using low-temperature FTIR difference spectroscopy. Comparison of the light-induced BPR difference spectrum recorded at 80 K to that of green-absorbing proteorhodopsin (GPR) reveals that there are several differences in the BPR and GPR primary photoreactions despite the similar structure of the retinal chromophore and all-trans → 13-cis isomerization. Strong bands near 1700 cm⁻¹ assigned previously to a change in hydrogen bonding of Asn230 in GPR are still present in BPR. However, additional bands in the same region are assigned on the basis of site-directed mutagenesis to changes occurring in Gln105. In the amide II region, bands are assigned on the basis of total ¹⁵N labeling to structural changes of the protein backbone, although no such bands were previously observed for GPR. A band at 3642 cm⁻¹ in BPR, assigned to the OH stretching mode of a water molecule on the basis of H₂¹⁸O substitution, appears at a different frequency than a band at 3626 cm⁻¹ previously assigned to a water molecule in GPR. However, the substitution of Gln105 for Leu105 in BPR leads to the appearance of both bands at 3642 and 3626 cm⁻¹, indicating the waters assigned in BPR and GPR exist in separate distinct locations and can coexist in the GPR-like Q105L mutant of BPR. These results indicate that there exist significant differences in the conformational changes occurring in these two types proteorhodopsin during the initial photoreaction despite their similar chromophore structures, which might reflect a different arrangement of water in the active site as well as substitution of a hydrophilic for hydrophobic residue at residue 105.

Changes in bacterial physiology necessarily precede cell death in response to antibiotics. Herein we investigate the early disruption of Ca²⁺ homeostasis as a marker for antibiotic response. Using a machine learning framework, we quantify the temporal information encoded in single-cell Ca²⁺ dynamics. We find Ca²⁺ dynamics distinguish kanamycin sensitive and resistant cells before changes in gross cell phenotypes such as cell growth or protein stability. The onset time (pharmacokinetics) and probability (pharmacodynamics) of these aberrant Ca²⁺ dynamics are dose and time-dependent, even at the resolution of single-cells. Of the compounds profiled, we find Ca²⁺ dynamics are also an indicator of Polymyxin B activity. In Polymyxin B treated cells, we find aberrant Ca²⁺ dynamics precedes the entry of propidium iodide marking membrane permeabilization. Additionally, we find modifying membrane voltage and external Ca²⁺ concentration alters the time between these aberrant dynamics and membrane breakdown suggesting a previously unappreciated role of Ca²⁺ in the membrane destabilization during Polymyxin B treatment. In conclusion, leveraging live, single-cell, Ca²⁺ imaging coupled with machine learning, we have demonstrated the discriminative capacity of Ca²⁺ dynamics in identifying antibiotic-resistant bacteria.