Simon Rainville

The Motility of Bacteria in an Anisotropic Liquid Environment

Although the influence on bacterial motility of many genetic and biochemical factors has been extensively studied, there have been limited studies of the impact of physical parameters. Indeed, despite the fact that natural environments are often asymmetric (such as stretched supramolecular structures), the majority of behavioral experiments with bacteria have been done in isotropic liquid solutions. In the present work, we show that the behavior of living microorganisms is dramatically different in media that are asymmetric. The example of Escherichia coli bacteria swimming in a bulk uniaxial liquid environment is used to demonstrate this phenomenon. The results of our study provide insight into the behavior of bacteria in conditions encountered in real environments and open new avenues for the control of their movements.

Bacterial flagella grow through an injection-diffusion mechanism

The bacterial flagellum is a self-assembling nanomachine. The external flagellar filament, several times longer than a bacterial cell body, is made of a few tens of thousands subunits of a single protein: flagellin. A fundamental problem concerns the molecular mechanism of how the flagellum grows outside the cell, where no discernible energy source is available. Here, we monitored the dynamic assembly of individual flagella using in situ labelling and real-time immunostaining of elongating flagellar filaments. We report that the rate of flagellum growth, initially ∼1,700 amino acids per second, decreases with length and that the previously proposed chain mechanism does not contribute to the filament elongation dynamics. Inhibition of the proton motive force-dependent export apparatus revealed a major contribution of substrate injection in driving filament elongation. The combination of experimental and mathematical evidence demonstrates that a simple, injection-diffusion mechanism controls bacterial flagella growth outside the cell. DOI: http://dx.doi.org/10.7554/eLife.23136.001

Recent advances and future prospects in bacterial and archaeal locomotion and signal transduction

Unraveling the structure and function of two-component and chemotactic signaling along with different aspects related to motility of bacteria and archaea are key research areas in modern microbiology. Escherichia coli is the traditional model organism to study chemotaxis signaling and motility. However, the recent study of a wide range of bacteria and even some archaea with different lifestyles has provided new insight into the eco-physiology of chemotaxis, which is essential for the host establishment of different pathogens or beneficial bacteria. The expanded range of model organisms has also permitted the study of chemosensory pathways unrelated to chemotaxis, multiple chemotaxis pathways within an organism, and new types of chemoreceptors. This research has greatly benefitted from technical advances in the field of cryo-microscopy that continues to reveal with increasing resolution the complexity and diversity of large protein complexes like the flagellar motor or chemoreceptor arrays. In addition, sensitive instruments now allow for an increasing number of experiments to be conducted at the single-cell level, thereby revealing information that is beginning to bridge the gap between individual cells and population behavior. Evidence has also accumulated showing that bacteria have evolved different mechanisms for surface sensing, which appears to be mediated by flagella and possibly type IV pili, and that the downstream signaling involves chemosensory pathways and two-component system based processes. Herein we summarize the recent advances and research tendencies in this field as presented at the latest Bacterial Locomotion and Signal Transduction (BLAST XIV) conference.

Variability in bacterial flagella re-growth patterns after breakage

Many bacteria swim through liquids or crawl on surfaces by rotating long appendages called flagella. Flagellar filaments are assembled from thousands of subunits that are exported through a narrow secretion channel and polymerize beneath a capping scaffold at the tip of the growing filament. The assembly of a flagellum uses a significant proportion of the biosynthetic capacities of the cell with each filament constituting ~1% of the total cell protein. Here, we addressed a significant question whether a flagellar filament can form a new cap and resume growth after breakage. Re-growth of broken filaments was visualized using sequential 3-color fluorescent labeling of filaments after mechanical shearing. Differential electron microscopy revealed the formation of new cap structures on broken filaments that re-grew. Flagellar filaments are therefore able to re-grow if broken by mechanical shearing forces, which are expected to occur frequently in nature. In contrast, no re-growth was observed on filaments that had been broken using ultrashort laser pulses, a technique allowing for very local damage to individual filaments. We thus conclude that assembly of a new cap at the tip of a broken filament depends on how the filament was broken.

Application of a microrheology technique to measure the viscosity of disodium cromoglycate liquid crystal

ABSTRACT Although the science of rheology is well established, some important challenges still persist to measure the viscoelastic properties of complex fluids, such as biological solutions and liquid crystals (LC). In this work, we present a method, based on the calculation of the step length of Brownian particles, to measure the effective local viscosity sensed by microscopic objects in the LC host. This approach allowed us to quantify the anisotropy of the viscosity, and it could also be extended to measure the local viscosity in other nonhomogeneous media. We also present a new guided light dark-field microscopy technique that was used to track particles during our experiments.

Transient locking of the hook procures enhanced motility to flagellated bacteria

Flagellated bacteria often proliferate in inhomogeneous environments, such as biofilms, swarms and soil. In such media, bacteria are observed to move efficiently only if they can get out of “dead ends” by changing drastically their swimming direction, and even to completely reverse it. Even though these reorientations are ubiquitous, we have only recently begun to describe and understand how they happen. In the present work, we visualized the flagella of bacteria swimming in a soft agar solution. The surprising observation that the filaments do not rotate while being flipped from one side of the cell to the other suggests that reversals are driven directly by the motor rather than by the thrust created by the rotating filament. This was confirmed by observing bacteria in a liquid crystal, where the linear movement of bacteria greatly simplifies the analysis. These observations suggest that the reversal and reorientation processes involve a temporary locking of the flagellum’s hook, which is the normally flexible joint between the rotary motor and the long helical filament that propels the cell. This newly described locked-hook mode occurs only when the motor switches to a clockwise rotation. That correlates with other phenomena that are triggered by a switch in one direction and not the other.

In the search of anisotropic biocompatible liquid environments for bacterial motility studies

Several experimental approaches are explored to introduce the E. coli bacteria in a liquid anisotropic host. Fonctionalization of the bacterial surface is experimented with 2 different molecules. The 5CB is first used as host and it is shown that, while the bacteria survive at short term in such an environment, they aggregate into colonies. Water solution of the cromolyn sodium salt is also explored with success and the time stability of corresponding sandwich-like structures is characterized.

Taking control of the flagellar motor

Numerous types of bacteria swim in their environment by rotating long helical filaments. At the base of each filament is a tiny rotary motor called the bacterial flagellar motor. A lot is already known about the structure, assembly and function of this splendid molecular machine of nanoscopic dimensions. Nevertheless many fundamental questions remain open and the study of the flagellar motor is a very exciting area of current research. We are developing an in vitro assay to enable studies of the bacterial flagellar motor in precisely controlled conditions and to gain direct access to the inner components of the motor. We partly squeeze a filamentous E. coli bacterium inside a micropipette, leaving a working flagellar motor outside. We then punch a hole through the cell wall at the end of the bacterium located inside the micropipette using a brief train of ultrashort (~60 fs) laser pulses. This enables us to control the rotation of the motor with an external voltage (for at least 15 minutes). In parallel, new methods to monitor the speed of rotation of the motor in the low load (high speed) regime are being developed using various nanoparticules.