Search

Abstract: The bacterial flagellar motor is a rotary motor in the cell envelope of bacteria that couples ion flow across the cytoplasmic membrane to torque generation by independent stators anchored to the cell wall. The recent observation of stepwise rotation of a Na+-driven chimeric motor in Escherichia coli promises to reveal the mechanism of the motor in unprecedented detail. We measured torque–speed relationships of this chimeric motor using back focal plane interferometry of polystyrene beads attached to flagellar filaments in the presence of high sodium-motive force (85 mM Na+). With full expression of stator proteins the torque–speed curve had the same shape as those of wild-type E. coli and Vibrio alginolyticus motors: the torque is approximately constant (at ∼2200 pN nm) from stall up to a “knee” speed of ∼420 Hz, and then falls linearly with speed, extrapolating to zero torque at ∼910 Hz. Motors containing one to five stators generated ∼200 pN nm per stator at speeds up to ∼100 Hz/stator; the knee speed in 4- and 5-stator motors is not significantly slower than in the fully induced motor. This is consistent with the hypothesis that the absolute torque depends on stator number, but the speed dependence does not. In motors with point mutations in either of two critical conserved charged residues in the cytoplasmic domain of PomA, R88A and R232E, the zero-torque speed was reduced to ∼400 Hz. The torque at low speed was unchanged by mutation R88A but was reduced to ∼1500 pN nm by R232E. These results, interpreted using a simple kinetic model, indicate that the basic mechanism of torque generation is the same regardless of stator type and coupling ion and that the electrostatic interaction between stator and rotor proteins is related to the torque–speed relationship. [Copyright &y& Elsevier]

The bacterial flagellar filament is an extracellular tubular protein structure that acts as a propeller for bacterial swimming motility. It is connected to the membrane-anchored rotary bacterial flagellar motor through a short hook. The bacterial flagellar filament consists of approximately 20,000 flagellins and can be several micrometers long. In this article, we reviewed the experimental works and models of flagellar filament construction and the recent findings of flagellar filament ejection during the cell cycle. The length-dependent decay of flagellar filament growth data supports the injection-diffusion model. The decay of flagellar growth rate is due to reduced transportation of long-distance diffusion and jamming. However, the filament is not a permeant structure. Several bacterial species actively abandon their flagella under starvation. Flagellum is disassembled when the rod is broken, resulting in an ejection of the filament with a partial rod and hook. The inner membrane component is then diffused on the membrane before further breakdown. These new findings open a new field of bacterial macro-molecule assembly, disassembly, and signal transduction. [ABSTRACT FROM AUTHOR]

• Bacterial Flagellar Motor is a natural rotary molecular machines for motility and chemotaxis. • Bacterial Flagellar Motor can be used as a single cell voltmeter. • Bacterial Flagellar Motor is a mechanosensor. • Potential application as a multimodal biosensor. Bacterial Flagellar Motor is one of nature's rare rotary molecular machines. It enables bacterial swimming and it is the key part of the bacterial chemotactic network, one of the best studied chemical signalling networks in biology, which enables bacteria to direct its movement in accordance with the chemical environment. The network can sense down to nanomolar concentrations of specific chemicals on the time scale of seconds. Motor's rotational speed is linearly proportional to the electrochemical gradients of either proton or sodium driving ions, while its direction is regulated by the chemotactic network. Recently, it has been discovered that motor is also a mechanosensor. Given these properties, we discuss the motor's potential to serve as a multifunctional biosensor and a tool for characterising and studying the external environment, the bacterial physiology itself and single molecular motor biophysics. [ABSTRACT FROM AUTHOR]

Bacterial flagella are nanomachines that drive bacteria motility and taxis in response to environmental changes. Whether flagella are permanent cell structures and, if not, the circumstances and timing of their production and loss during the bacterial life cycle remain poorly understood. Here we used the single polar flagellum of Vibrio alginolyticus as our model and implementing in vivo fluorescence imaging revealed that the percentage of flagellated bacteria (PFB) in a population varies substantially across different growth phases. In the early‐exponential phase, the PFB increases rapidly through the widespread production of flagella. In the mid‐exponential phase, the PFB peaks at around 76% and the partitioning of flagella between the daughter cells are 1:1 and strictly at the old poles. After entering the stationary phase, the PFB starts to decline, mainly because daughter cells stop making new flagella after cell division. Interestingly, we observed that bacteria can actively abandon flagella after prolonged stationary culturing, though cell division has long been suspended. Further experimental investigations confirmed that flagella were ejected in V. alginolyticus, starting from breakage in the rod. Our results highlight the dynamic production and loss of flagella during the bacterial life cycle. Importance: Flagella motility is critical for many bacterial species. The bacterial flagellum is made up of about 20 different types of proteins in its final structure and can be self‐assembled. The current understanding of the lifetime and durability of bacterial flagella is very limited. In the present study, we monitored Vibrio alginolyticus flagellar assembly and loss by in vivo fluorescence labeling, and found that the percentage of flagellated bacteria varies substantially across different growth phases. The production of flagella was synchronized with cell growth but stopped when cells entered the stationary phase. Surprisingly, we observed that bacteria can actively abandon flagella after prolonged stationary culturing, as well as in the low glucose buffering medium. We then confirmed the ejection of flagella in V. alginolyticus started with breakage of the rod. Our results highlight the dynamic production and loss of flagella during the bacterial life cycle. [ABSTRACT FROM AUTHOR]

For in vivo, single-cell imaging bacterial cells are commonly immobilised via physical confinement or surface attachment. Different surface attachment methods have been used both for atomic force and optical microscopy (including super resolution), and some have been reported to affect bacterial physiology. However, a systematic comparison of the effects these attachment methods have on the bacterial physiology is lacking. Here we present such a comparison for bacterium Escherichia coli, and assess the growth rate, size and intracellular pH of cells growing attached to different, commonly used, surfaces. We demonstrate that E. coli grow at the same rate, length and internal pH on all the tested surfaces when in the same growth medium. The result suggests that tested attachment methods can be used interchangeably when studying E. coli physiology. [ABSTRACT FROM AUTHOR]

Bacterial flagellar motor (BFM) is a protein complex used for bacterial motility and chemotaxis that involves in energy transformation, torque generation and switching. FliL is a single-transmembrane protein associated with flagellar motor function. We performed biochemical and biophysical approaches to investigate the functional roles of FliL associated with stator-units. Firstly, we found the periplasmic region of FliL is crucial for its polar localization. Also, the plug mutation in stator-unit affected the polar localization of FliL implying the activation of stator-unit is important for FliL recruitment. Secondly, we applied single-molecule fluorescent microscopy to study the role of FliL in stator-unit assembly. Using molecular counting by photobleaching, we found the stoichiometry of stator-unit and FliL protein would be 1:1 in a functional motor. Moreover, the turnover time of stator-units are slightly increased in the absence of FliL. By further investigation of protein dynamics on membrane, we found the diffusions of stator-units and FliL are independent. Surprisingly, the FliL diffusion rate without stator-units is unexpectedly slow indicating a protein-complex forming event. Our results suggest that FliL plays a supporting role to the stator in the BFM. [ABSTRACT FROM AUTHOR]

Background Flagellar motility of Helicobacter pylori has been shown to be important for the bacteria to establish initial colonization. The ferric uptake regulator (Fur) is a global regulator that has been identified in H. pylori which is involved in the processes of iron uptake and establishing colonization. However, the role of Fur in H. pylori motility is still unclear. Materials and Methods Motility of the wild-type, fur mutant, and fur revertant J99 were determined by a soft-agar motility assay and direct video observation. The bacterial shape and flagellar structure were evaluated by transmission electron microscopy. Single bacterial motility and flagellar switching were observed by phase-contrast microscopy. Autoinducer-2 ( AI-2) production in bacterial culture supernatant was analyzed by a bioluminescence assay. Results The fur mutant showed impaired motility in the soft-agar assay compared with the wild-type J99 and fur revertant. The numbers and lengths of flagellar filaments on the fur mutant cells were similar to those of the wild-type and revertant cells. Phenotypic characterization showed similar swimming speed but reduction in switching rate in the fur mutant. The AI-2 production of the fur mutant was dramatically reduced compared with wild-type J99 in log-phase culture medium. Conclusions These results indicate that Fur positively modulates H. pylori J99 motility through interfering with bacterial flagellar switching. [ABSTRACT FROM AUTHOR]

PomA and PomB form the stator complex, which functions as a Na+ channel, in the Na+-driven flagellar motor of Vibrio alginolyticus. The plug region of PomB is thought to regulate the Na+ flow and to suppress massive ion influx through the stator channel. In this study, in order to measure the Na+ conductivity of the unplugged stator, we over-produced a plug-deleted stator of the Na+-driven flagellar motor in Escherichia coli. The over-production of the plug-deleted stator in E. coli cells caused more severe growth inhibition than in Vibrio cells and that growth inhibition depended on the Na+ concentration in the growth medium. Measurement of intracellular Na+ concentration by flame photometry and fluorescent analysis with a Na+ indicator, Sodium Green, revealed that over-production of the plug-deleted stator increased the Na+ concentration in cell. Some mutations in the channel region of PomB or in the cytoplasmic region of PomA suppressed both the growth inhibition and the increase in intracellular Na+ concentration. These results suggest that the level of growth inhibition correlates with the intracellular Na+ concentration, probably due to the Na+ conductivity through the stator due to the mutations. [ABSTRACT FROM AUTHOR]

Using thermoplastic polymers as substrate material is an attractive approach to develop low-cost, disposable microfluidic devices. This study investigates a simple and rapid polymer replication method of fabricating microchannels by a hot roller embossing process. The hot roller embosser used in this study was modified from a commercially available film laminator, and the roller micromold was fabricated by spin coating an SU-8 layer on a flexible copper sheet. A straight microchannel measuring 5 cm long, 200 μm wide, and 41.4 μm deep was used to evaluate the imprinting performance on cyclic olefin copolymer and polyvinylchloride film. This study also investigates the effects of hot roller embossing temperature, rolling speed, and embossing pressure on the microchannel depth and geometry transfer efficiency. [ABSTRACT FROM AUTHOR]

We experimentally investigate the collective dynamics in bacterial carpet microfluidic channel. The microfluidic channel is composed of single polar flagellated Vibrio alginolyticus deposited glass substrates. The individual flagellum swimming speed is tuned by varying buffer sodium concentration. Hydrodynamic coupling strength is tuned by varying buffer viscosity. The attached bacteria constantly perform two major modes in flagellum motion, namely, the local rotation and large angle flick. Particle tracking statistics shows high flagellum rotational rate and strong hydrodynamic coupling strength lead to collective sub-diffusive dynamics. The observed effect is strongly correlated to hydrodynamic coupling of flick motions between nearby bacteria. [ABSTRACT FROM AUTHOR]

Summary Cell 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. Graphical Abstract Highlights • The degree of drug-tolerant cells being dormant can be measured by "dormancy depth" • Cellular dark foci, proved to be protein aggresomes, indicate dormancy depth • Depletion of intracellular ATP is the major force driving aggresomes formation • 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. [ABSTRACT FROM AUTHOR]