Graphical Abstract Highlights d The degree of drug-tolerant cells being dormant can be measured by ''dormancy depth'' d Cellular dark foci, proved to be protein aggresomes, indicate dormancy depth d Depletion of intracellular ATP is the major force driving aggresomes formation d DnaK is vital in the disaggregation of aggresomes when a dormant cell resuscitates In this work, Pu et al. introduced a concept of ''dormancy depth'' that provides a unifying framework for understanding both persisters and viable but non-culturable cells. Subsequent mechanistic investigations revealed how ATP-dependent dynamic protein aggregation regulates cellular dormancy and resuscitation, the fine control of which facilitates bacterial drug tolerance. SUMMARYCell dormancy is a widespread mechanism used by bacteria to evade environmental threats, including antibiotics. Here we monitored bacterial antibiotic tolerance and regrowth at the single-cell level and found that each individual survival cell shows different ''dormancy depth,'' which in return regulates the lag time for cell resuscitation after removal of antibiotic. We further established that protein aggresome-a collection of endogenous protein aggregates-is an important indicator of bacterial dormancy depth, whose formation is promoted by decreased cellular ATP level. For cells to leave the dormant state and resuscitate, clearance of protein aggresome and recovery of proteostasis are required. We revealed that the ability to recruit functional DnaK-ClpB machineries, which facilitate protein disaggregation in an ATP-dependent manner, determines the lag time for bacterial regrowth. Better understanding of the key factors regulating bacterial regrowth after surviving antibiotic attack could lead to new therapeutic strategies for combating bacterial antibiotic tolerance.

Torque is generated in the rotary motor at the base of the bacterial flagellum by ion translocating stator units anchored to the peptidoglycan cell wall. Stator units are composed of the proteins MotA and MotB in proton-driven motors, and they are composed of PomA and PomB in sodium-driven motors. Strains of Escherichia coli lacking functional stator proteins produce flagella that do not rotate, and induced expression of the missing proteins leads to restoration of motor rotation in discrete speed increments, a process known as ''resurrection.'' Early work suggested a maximum of eight units. More recent indications that WT motors may contain more than eight units, based on recovery of disrupted motors, are inconclusive. Here we demonstrate conclusively that the maximum number of units in a motor is at least 11. Using back-focal-plane interferometry of 1-m polystyrene beads attached to flagella, we observed at least 11 distinct speed increments during resurrection with three different combinations of stator proteins in E. coli. The average torques generated by a single unit and a fully induced motor were lower than previous estimates. Speed increments at high numbers of units are smaller than those at low numbers, indicating that not all units in a fully induced motor are equivalent.T he flagellar motor is the mechanism of propulsion for most swimming bacteria (1-5). In Escherichia coli Ϸ40 gene products are required for motor assembly, with Ϸ20 of them being present in the final structure. Each motor drives a helical filament up to Ϸ10 m long. The flagellar motor spans the inner and outer bacterial membranes (Fig. 1). Torque is generated by interactions between the rotor protein FliG, located at the intersection between the MS and C rings, and the stator units attached to the cell wall (5). Each unit is believed to contain two copies of MotB and four copies of MotA in proton-driven motors of E. coli, two copies of PomB and four copies of PomA in sodium-driven motors of Vibrio alginolyticus (6, 7), and function as an ion channel (8, 9). Ion flux through these channels powers the motor (10, 11). Functional chimeras have been engineered containing components of proton-and sodium-driven motors (12), indicating that the structure and mechanisms of both types of motor are very similar. The torque-speed relationship of the motor has been measured by using electrorotation of tethered cells (13) and attaching varying viscous loads (14-16). A notable feature is a regime between stall and a speed of Ϸ175 Hz in WT E. coli at room temperature, over which torque falls linearly with increasing speed to Ϸ90% of the value at stall. At higher speeds torque falls more steeply, eventually to zero at a speed of Ϸ350 Hz.Successive incorporation of torque-generating units to restore rotation in paralyzed motors is known as resurrection (17). The maximum number of speed increments previously seen during resurrection of a single motor, using inducible plasmids to express missing stator proteins, was eight (18). However, in experiments w...

Many bacterial species swim using flagella. The flagellar motor couples ion flow across the cytoplasmic membrane to rotation. Ion flow is driven by both a membrane potential (V(m)) and a transmembrane concentration gradient. To investigate their relation to bacterial flagellar motor function we developed a fluorescence technique to measure V(m) in single cells, using the dye tetramethyl rhodamine methyl ester. We used a convolution model to determine the relationship between fluorescence intensity in images of cells and intracellular dye concentration, and calculated V(m) using the ratio of intracellular/extracellular dye concentration. We found V(m) = -140 +/- 14 mV in Escherichia coli at external pH 7.0 (pH(ex)), decreasing to -85 +/- 10 mV at pH(ex) 5.0. We also estimated the sodium-motive force (SMF) by combining single-cell measurements of V(m) and intracellular sodium concentration. We were able to vary the SMF between -187 +/- 15 mV and -53 +/- 15 mV by varying pH(ex) in the range 7.0-5.0 and extracellular sodium concentration in the range 1-85 mM. Rotation rates for 0.35-microm- and 1-microm-diameter beads attached to Na(+)-driven chimeric flagellar motors varied linearly with V(m). For the larger beads, the two components of the SMF were equivalent, whereas for smaller beads at a given SMF, the speed increased with sodium gradient and external sodium concentration.

The bacterial flagellar motor is a large rotary molecular machine that propels swimming bacteria, powered by a transmembrane electrochemical potential difference. It consists of an ∼50-nm rotor and up to ∼10 independent stators anchored to the cell wall. We measured torque-speed relationships of single-stator motors under 25 different combinations of electrical and chemical potential. All 25 torque-speed curves had the same concave-down shape as fully energized wild-type motors, and each stator passes at least 37 ± 2 ions per revolution. We used the results to explore the 25-dimensional parameter space of generalized kinetic models for the motor mechanism, finding 830 parameter sets consistent with the data. Analysis of these sets showed that the motor mechanism has a "powerstroke" in either ion binding or transit; ion transit is channel-like rather than carrier-like; and the rate-limiting step in the motor cycle is ion binding at low concentration, ion transit, or release at high concentration. where V m is the membrane voltage and Δμ = k B T ln(C in /C out ) and q are the transmembrane chemical potential difference and charge of the ions, respectively, with C in and C out the internal and external ion concentrations. In most species the primary form of biological free energy is the proton-motive force (PMF), the IMF for H + ions (1). Physiological PMF is typically in the range −150 mV to −200 mV, with the inside electrically negative and slightly alkaline relative to the outside. Some organisms use sodium-motive force (SMF) to drive numerous cellular processes, such as bacterial motility (2), ATP synthesis (3), and active membrane transport (4). Arguably the most important process driven by IMF is ATP synthesis, which generates cellular ATP by forced rotation of the F 1 part of F 1 F O ATP-synthase. F 1 is mechanically coupled to and rotated by F O , which like the bacterial flagellar motor (BFM) is an ion-driven rotary motor. Understanding the mechanism of these and other ion-driven molecular machines is a fundamental challenge in cellular energetics and biophysics. The BFM (Fig. 1A) is a rotary molecular machine that propels many species of swimming bacteria. It couples ion flow, for example protons (H + ) in Escherichia coli or sodium ions (Na + ) in Vibrio alginolyticus, to the rotation of extracellular helical flagellar filaments at hundreds of revolutions per second (Hz) (2, 5, 6). Torque is generated by interactions between stator complexes (containing the proteins MotA and MotB in E. coli and PomA and PomB in V. alginolyticus) and the rotor protein FliG (7). In E. coli, each motor can be powered by any number between 1 and at least 11 functionally independent stators (8), which exchange with a membrane-bound pool of "spare" stators on a timescale of minutes (9).The most important biophysical method for studying the torque-generating mechanism of the BFM has been to measure its torque-speed curves. This has been done using varying viscous load (10-14) or external torque (15, 16) to control the speed. Fu...