Real-Time Deduction of Mechanisms and Kinetics Underlying Photocatalytic Water Disinfection: Cell Motility and Particle Tracking.

The current methods used to study photocatalysis-assisted water disinfection at a laboratory scale may not lead to process scale-up for large-scale implementation. These methods do not capture the process complexity and address all the factors underlying disinfection kinetics, including the physical characteristics (e.g., shape and size) of the photocatalyst, the light intensity, the form of the catalyst (e.g., free-floating and immobilized), and the photocatalyst-microorganism interaction mode (e.g., collision mode and constant contact mode). This drawback can be overcome using in situ methods to track the interaction between the photocatalysts and the microorganisms (e.g., Escherichia coli) and thereby engineering the resulting disinfection kinetics. Contextually, this study employed microscopy and particle-tracking algorithms to quantify in situ cell motility of E. coli undergoing titanium dioxide (TiO2) nanowire-assisted photocatalysis, which was observed to correlate with cell viability closely. This experimentation also informed that the E. coli bacterium interacted with the photocatalysts through collisions (without sustained contact), which allowed for phenomenological modeling of the observed first-order kinetics of E. coli inactivation. Addition of fluorescent-tagging assays to microscopy revealed that cell membrane integrity loss is the primary mode of bacterial inactivation. This methodology is independent of the microorganism or the photocatalyst type and hence is expected to be beneficial for engineering disinfection kinetics.

Indole modulates cooperative protein-protein interactions in the flagellar motor.

Indole is a major component of the bacterial exometabolome, and the mechanisms for its wide-ranging effects on bacterial physiology are biomedically significant, although they remain poorly understood. Here, we determined how indole modulates the functions of a widely conserved motility apparatus, the bacterial flagellum. Our experiments in Escherichia coli revealed that indole influences the rotation rates and reversals in the flagellum's direction of rotation via multiple mechanisms. At concentrations higher than 1 mM, indole decreased the membrane potential to dissipate the power available for the rotation of the motor that operates the flagellum. Below 1 mM, indole did not dissipate the membrane potential. Instead, experiments and modeling indicated that indole weakens cooperative protein interactions within the flagellar complexes to inhibit motility. The metabolite also induced reversals in the rotational direction of the motor to promote a weak chemotactic response, even when the chemotaxis response regulator, CheY, was lacking. Experiments further revealed that indole does not require the transporter Mtr to cross the membrane and influence motor functions. Based on these findings, we propose that indole modulates intra- and inter-protein interactions in the cell to influence several physiological functions.

Mechanosensitive recruitment of stator units promotes binding of the response regulator CheY-P to the flagellar motor.

Reversible switching of the bacterial flagellar motor between clockwise (CW) and counterclockwise (CCW) rotation is necessary for chemotaxis, which enables cells to swim towards favorable chemical habitats. Increase in the viscous resistance to the rotation of the motor (mechanical load) inhibits switching. However, cells must maintain homeostasis in switching to navigate within environments of different viscosities. The mechanism by which the cell maintains optimal chemotactic function under varying loads is not understood. Here, we show that the flagellar motor allosterically controls the binding affinity of the chemotaxis response regulator, CheY-P, to the flagellar switch complex by modulating the mechanical forces acting on the rotor. Mechanosensitive CheY-P binding compensates for the load-induced loss of switching by precisely adapting the switch response to a mechanical stimulus. The interplay between mechanical forces and CheY-P binding tunes the chemotactic function to match the load. This adaptive response of the chemotaxis output to mechanical stimuli resembles the proprioceptive feedback in the neuromuscular systems of insects and vertebrates.

Protein expression-independent response of intensity-based pH-sensitive fluorophores in Escherichia coli.

Fluorescent proteins that modulate their emission intensities when protonated serve as excellent probes of the cytosolic pH. Since the total fluorescence output fluctuates significantly due to variations in the fluorophore levels in cells, eliminating the dependence of the signal on protein concentration is crucial. This is typically accomplished with the aid of ratiometric fluorescent proteins such as pHluorin. However, pHluorin is excited by blue light, which can complicate pH measurements by adversely impacting bacterial physiology. Here, we characterized the response of intensity-based, pH-sensitive fluorescent proteins that excite at longer wavelengths where the blue light effect is diminished. The pH-response was interpreted in terms of an analytical model that assumed two emission states for each fluorophore: a low intensity protonated state and a high intensity deprotonated state. The model suggested a scaling to eliminate the dependence of the signal on the expression levels as well as on the illumination and photon-detection settings. Experiments successfully confirmed the scaling predictions. Thus, the internal pH can be readily determined with intensity-based fluorophores with appropriate calibrations irrespective of the fluorophore concentration and the signal acquisition setup. The framework developed in this work improves the robustness of intensity-based fluorophores for internal pH measurements in E. coli, potentially extending their applications.

Biphasic chemotaxis of Escherichia coli to the microbiota metabolite indole.

Bacterial chemotaxis to prominent microbiota metabolites such as indole is important in the formation of microbial communities in the gastrointestinal (GI) tract. However, the basis of chemotaxis to indole is poorly understood. Here, we exposed Escherichia coli to a range of indole concentrations and measured the dynamic responses of individual flagellar motors to determine the chemotaxis response. Below 1 mM indole, a repellent-only response was observed. At 1 mM indole and higher, a time-dependent inversion from a repellent to an attractant response was observed. The repellent and attractant responses were mediated by the Tsr and Tar chemoreceptors, respectively. Also, the flagellar motor itself mediated a repellent response independent of the receptors. Chemotaxis assays revealed that receptor-mediated adaptation to indole caused a bipartite response-wild-type cells were attracted to regions of high indole concentration if they had previously adapted to indole but were otherwise repelled. We propose that indole spatially segregates cells based on their state of adaptation to repel invaders while recruiting beneficial resident bacteria to growing microbial communities within the GI tract.