Automated high-content image-based characterization of microorganism behavioral diversity and distribution.

Microorganisms have evolved complex systems to respond to environmental signals. Gradients of particular molecules and elemental ions alter the behavior of microbes and their distribution within their environment. Microdevices coupled with automated image-based methods are now employed to analyze the instantaneous distribution and motion behaviors of microbial species in controlled environments at small temporal scales, mimicking, to some extent, macro conditions. Such technologies have so far been adopted for investigations mainly on individual species. Similar versatile approaches must now be developed for the characterization of multiple and complex interactions between a microbial community and its environment. Here, we provide a comprehensive step-by-step method for the characterization of species-specific behavior in a synthetic mixed microbial suspension in response to an environmental driver. By coupling accessible microfluidic devices with automated image analysis approaches, we evaluated the behavioral response of three morphologically different telluric species (Phytophthora parasitica, Vorticella microstoma, Enterobacter aerogenes) to a potassium gradient driver. Using the TrackMate plug-in algorithm, we performed morphometric and then motion analyses to characterize the response of each microbial species to the driver. Such an approach enabled to confirm the different morphological features of the three species and simultaneously characterize their specific motion in reaction to the driver and their co-interaction dynamics. By increasing the complexity of suspensions, this approach could be integrated in a framework for phenotypic analysis in microbial ecology research, helping to characterize how key drivers influence microbiota assembly at microbiota host-environment interfaces.

Coordination of two opposite flagella allows high-speed swimming and active turning of individual zoospores.

Phytophthora species cause diseases in a large variety of plants and represent a serious agricultural threat, leading, every year, to multibillion dollar losses. Infection occurs when their biflagellated zoospores move across the soil at their characteristic high speed and reach the roots of a host plant. Despite the relevance of zoospore spreading in the epidemics of plant diseases, individual swimming of zoospores have not been fully investigated. It remains unknown about the characteristics of two opposite beating flagella during translation and turning, and the roles of each flagellum on zoospore swimming. Here, combining experiments and modeling, we show how these two flagella contribute to generate thrust when beating together, and identify the mastigonemes-attached anterior flagellum as the main source of thrust. Furthermore, we find that turning involves a complex active process, in which the posterior flagellum temporarily stops, while the anterior flagellum keeps on beating and changes its gait from sinusoidal waves to power and recovery strokes, similar to Chlamydomonas's breaststroke, to reorient its body to a new direction. Our study is a fundamental step toward a better understanding of the spreading of plant pathogens' motile forms, and shows that the motility pattern of these biflagellated zoospores represents a distinct eukaryotic version of the celebrated 'run-and-tumble' motility class exhibited by peritrichous bacteria.

Microorganisms of the Phytophthora genus are serious agricultural pests. They cause diseases in many crops, including potato, onion, tomato, tobacco, cotton, peppers, and citrus. These diseases cause billions of dollars in losses each year. Learning more about how the tiny creatures disseminate and reach host plants could help scientists develop new ways to prevent such crop damage. The spore cells of Phytophthora, also known as zoospores, have two appendages called flagella on their bodies. A tinsel-shaped flagellum is near the front of the creature and a long smooth filament-like flagellum is near the posterior. Zoospores use their flagella to swim at high speeds through liquid toward potential plant hosts. Their complex swimming patterns change in response to different physical, chemical, and electrical signals in the environment. But exactly how they use their flagella to generate these movements is not clear. Tran et al. reveal new details about zoospore locomotion. In the experiments, Tran et al. recorded the movements of zoospores in a tiny 'swimming pool' of fluid on top of a glass slide and analyzed the movements using statistical and mathematical models. The results uncovered coordinated actions of the flagella when zoospores swim in a straight line and when they turn. The tinsel-like front flagellum provides most of the force that propels the zoospore forward. To do this, it beats with an undulating wave pattern. It shifts the beating to a breast-stroke pattern to change direction. The posterior flagellum provides a smaller forward thrust and temporarily pauses during turns. The study provides new details about zoospore's movements that may help scientists develop new strategies to control these pests. It also offers more information about how flagella coordinate their actions to switch speeds or change directions that may be of interest to other scientists studying organisms that use flagella to move.

Spatiotemporal pattern formation in E. coli biofilms explained by a simple physical energy balance.

While the biofilm growth mode conveys notable thriving advantages to bacterial populations, the mechanisms of biofilm formation are still strongly debated. Here, we investigate the remarkable spontaneous formation of regular spatial patterns during the growth of an Escherichia coli biofilm. These patterns reported here appear with non-motile bacteria, which excludes both chemotactic origins and other motility-based ones. We demonstrate that a minimal physical model based on phase separation describes them well. To confirm the predictive capacity of our model, we tune the cell-cell and cell-surface interactions using cells expressing different surface appendages. We further explain how F pilus-bearing cells enroll their wild type kindred, poorly piliated, into their typical pattern when mixed together. This work supports the hypothesis that purely physicochemical processes, such as the interplay of cell-cell and cell-surface interactions, can drive the emergence of a highly organized spatial structure that is potentially decisive for community fate and for biological functions.

Guidance of zoospores by potassium gradient sensing mediates aggregation.

The biflagellate zoospores of some phytopathogenic Phytophthora species spontaneously aggregate within minutes in suspension. We show here that Phytophthora parasitica zoospores can form aggregates in response to a K+ gradient with a particular geometric arrangement. Using time-lapse live imaging in macro- and microfluidic devices, we defined (i) spatio-temporal and concentration-scale changes in the gradient, correlated with (ii) the cell distribution and (iii) the metrics of zoospore motion (velocity, trajectory). In droplets, we found that K+-induced aggregates resulted from a single biphasic temporal sequence involving negative chemotaxis followed by bioconvection over a K+ gradient concentration scale [0-17 mM]. Each K+-sensing cell moved into a region in which potassium concentration is below the threshold range of 1-4 mM, resulting in swarming. Once a critical population density had been achieved, the zoospores formed a plume that migrated downward, with fluid advection in its wake and aggregate formation on the support surface. In the microfluidic device, the density of zoospores escaping potassium was similar to that achieved in droplets. We discuss possible sources of K+ gradients in the natural environment (zoospore population, microbiota, plant roots, soil particles), and implications for the events preceding inoculum formation on host plants.

The inducible chemical-genetic fluorescent marker FAST outperforms classical fluorescent proteins in the quantitative reporting of bacterial biofilm dynamics.

To increase our understanding of bacterial biofilm complexity, real- time quantitative analyses of the living community functions are required. To reach this goal, accurate fluorescent reporters are needed. In this paper, we used the classical fluorescent genetic reporters of the GFP family and demonstrated their limits in the context of a living biofilm. We showed that fluorescence signal saturated after only a few hours of growth and related this saturation to the reduction of oxygen concentration induced by bacterial consumption. This behaviour prevents the use of GFP-like fluorescent proteins for quantitative measurement in living biofilms. To overcome this limitation, we propose the use of a recently introduced small protein tag, FAST, which is fluorescent in the presence of an exogenously applied fluorogenic dye, enabling to avoid the oxygen sensitivity issue. We compared the ability of FAST to report on biofilm growth with that of GFP and mCherry, and demonstrated the superiority of the FAST:fluorogen probes for investigating dynamics in the complex environment of a living biofilm.

Bacterial biofilm under flow: First a physical struggle to stay, then a matter of breathing.

Bacterial communities attached to surfaces under fluid flow represent a widespread lifestyle of the microbial world. Through shear stress generation and molecular transport regulation, hydrodynamics conveys effects that are very different by nature but strongly coupled. To decipher the influence of these levers on bacterial biofilms immersed in moving fluids, we quantitatively and simultaneously investigated physicochemical and biological properties of the biofilm. We designed a millifluidic setup allowing to control hydrodynamic conditions and to monitor biofilm development in real time using microscope imaging. We also conducted a transcriptomic analysis to detect a potential physiological response to hydrodynamics. We discovered that a threshold value of shear stress determined biofilm settlement, with sub-piconewton forces sufficient to prevent biofilm initiation. As a consequence, distinct hydrodynamic conditions, which set spatial distribution of shear stress, promoted distinct colonization patterns with consequences on the growth mode. However, no direct impact of mechanical forces on biofilm growth rate was observed. Consistently, no mechanosensing gene emerged from our differential transcriptomic analysis comparing distinct hydrodynamic conditions. Instead, we found that hydrodynamic molecular transport crucially impacts biofilm growth by controlling oxygen availability. Our results shed light on biofilm response to hydrodynamics and open new avenues to achieve informed design of fluidic setups for investigating, engineering or fighting adherent communities.

Speed of evolution in large asexual populations with diminishing returns.

The adaptive evolution of large asexual populations is generally characterized by competition between clones carrying different beneficial mutations. Interference slows down the adaptation speed and makes the theoretical description of the dynamics more complex with respect to the successional occurrence and fixation of beneficial mutations typical of small populations. A simplified modeling framework considering multiple beneficial mutations with equal and constant fitness advantage is known to capture some of the essential features of laboratory evolution experiments. However, in these experiments the relative advantage of a beneficial mutation is generally dependent on the genetic background. In particular, the general pattern is that, as mutations in different loci accumulate, the relative advantage of new mutations decreases, a trend often referred to as "diminishing return" epistasis. Here, we propose a phenomenological model that generalizes the fixed-advantage framework to include this negative epistasis in a simple way. We evaluate analytically as well as with direct simulations the quantitative consequences of diminishing returns on the evolutionary dynamics. The speed of adaptation decreases in time and reaches a limit value corresponding to neutral evolution in the long time limit. This corresponds to an increase of the diversity in terms of "classes of mutation" in the population. Finally, we show how the model can be compared with dynamic data on fitness and number of beneficial mutations from laboratory evolution experiments.

Long-term diversity and genome adaptation of Acinetobacter baylyi in a minimal-medium chemostat.

Laboratory-based evolution experiments on microorganisms that do not recombine frequently show two distinct phases: an initial rapid increase in fitness followed by a slower regime. To explore the population structure and the evolutionary tree in the later stages of adaptation, we evolved a very large population (~3 × 10(10)) of Acinetobacter baylyi bacteria for approximately 2,800 generations from a single clone. The population was maintained in a chemostat at a high dilution rate. Nitrate in limiting amount and as the sole nitrogen source was used as a selection pressure. Analysis via resequencing of genomes extracted from populations at different generations provides evidence that long-term diversity can be established in the chemostat in a very simple medium. To find out which biological parameters were targeted by adaptation, we measured the maximum growth rate, the nitrate uptake, and the resistance to starvation. Overall, we find that maximum growth rate could be a reasonably good proxy for fitness. The late slow adaptation is compatible with selection coefficients spanning a typical range of 10(-3)-10(-2) per generation as estimated by resequencing, pointing to a possible subpopulations structuring.

Propagation of fluorescent viruses in growing plaques.

To study virus propagation, we have developed a method by which the propagation of the Lambda bacteriophage can be observed and quantified. This is done by creating a fusion protein of the capsid protein gpD and the enhanced yellow fluorescent protein (EYFP). We show that this fusion allows capsid formation and that the modified viruses propagate on a surface covered with host bacteria thus forming fluorescent plaques. The intensity of fluorescence in a growing plaque determines the distribution of phages. This provides a new tool to study the propagation of infection at the microscopic level.

Unravelling the mechanism of RNA-polymerase forward motion by using mechanical force.

Polymerases form a class of enzymes that act as molecular motors as they move along their nucleic acid substrate during catalysis, incorporating nucleotide triphosphates at the end of the growing chain and consuming chemical energy. A debated issue is how the enzyme converts chemical energy into motion [J. Gelles and R. Landick, Cell 93, 13 (1998)]. In a single molecule assay, we studied how an opposing mechanical force affects the translocation rate of T7 RNA polymerase. Our measurements show that force acts as a competitive inhibitor of nucleotide binding. This result is interpreted in the context of possible models, and with respect to published crystal structures of T7 RNA polymerase. The transcribing complex appears to utilize only a small fraction of the energy of hydrolysis to perform mechanical work, with the remainder being converted to heat.

Rotational drag on DNA: a single molecule experiment.

Within a single-molecule configuration, we have studied rotational drag on double stranded linear DNA by measuring the force during mechanical opening and closing of the double helix at different rates. The molecule is cranked at one end by the effect of unzipping and is free to rotate at the other end. In this configuration the rotational friction torque tau on double-stranded DNA leads to an additional contribution to the opening force. It is shown that the effect of rotational drag increases with the length of the molecule, is approximately proportional to the angular velocity of cranking, and we estimate that the torque tau is of the order of 1k(B)T for 10 000 base pairs of DNA cranked at 2000 turns per second.