Dynamic polarity control by a tunable protein oscillator in bacteria

International audienc

Multilocus ISSR Markers Reveal Two Major Genetic Groups in Spanish and South African Populations of the Grapevine Fungal Pathogen Cadophora luteo-olivacea

Cadophora luteo-olivacea is a lesser-known fungal trunk pathogen of grapevine which has been recently isolated from vines showing decline symptoms in grape growing regions worldwide. In this study, 80 C. luteo-olivacea isolates (65 from Spain and 15 from South Africa) were studied. Inter-simple-sequence repeat-polymerase chain reaction (ISSR-PCR) generated 55 polymorphic loci from four ISSR primers selected from an initial screen of 13 ISSR primers. The ISSR markers revealed 40 multilocus genotypes (MLGs) in the global population. Minimum spanning network analysis showed that the MLGs from South Africa clustered around the most frequent genotype, while the genotypes from Spain were distributed all across the network. Principal component analysis and dendrograms based on genetic distance and bootstrapping identified two highly differentiated genetic clusters in the Spanish and South African C. luteo-olivacea populations, with no intermediate genotypes between these clusters. Movement within the Spanish provinces may have occurred repeatedly given the frequent retrieval of the same genotype in distant locations. The results obtained in this study provide new insights into the population genetic structure of C. luteo-olivacea in Spain and highlights the need to produce healthy and quality planting material in grapevine nurseries to avoid the spread of this fungus throughout different grape growing regions

Regulations governing the multicellular lifestyle of Myxococcus xanthus

International audienceIn living organisms, cooperative cell movements underlie the formation of differentiated tissues. In bacteria, Myxococcus xanthus uses cooperative group movements, to predate on prey and to form multicellular fruiting bodies, where the cells differentiate into dormant spores. Motility is controlled by a central signaling Che-like pathway, Frz. Single cell studies indicate Frz regulates the frequency at which cells reverse their direction of movement by transmitting signals to a molecular system that controls the spatial activity of the motility engines. This regulation is central to all Myxococcus multicellular behaviors but how Frz signaling generates ordered patterns is poorly understood. In this review, we first discuss the genetic structure of the Frz pathway and possible regulations that could explain its action during Myxococcus development

New insights into the function of a versatile class of membrane molecular motors from studies of Myxococcus xanthus surface (gliding) motility

International audienceCell motility is a central function of living cells, as it empowers colonization of new environmental niches, cooperation, and development of multicellular organisms. This process is achieved by complex yet precise energy-consuming machineries in both eukaryotes and bacteria. Bacteria move on surfaces using extracellular appendages such as flagella and pili but also by a less-understood process called gliding motility. During this process, rodshaped bacteria move smoothly along their long axis without any visible morphological changes besides occasional bending. For this reason, the molecular mechanism of gliding motility and its origin have long remained a complete mystery. An important breakthrough in the understanding of gliding motility came from single cell and genetic studies in the delta-proteobacterium Myxococcus xanthus. These early studies revealed, for the first time, the existence of bacterial Focal Adhesion complexes (FA). FAs are formed at the bacterial pole and rapidly move towards the opposite cell pole. Their attachment to the underlying surface is linked to cell propulsion, in a process similar to the rearward translocation of actomyosin complexes in Apicomplexans. The protein machinery that forms at FAs was shown to contain up to seventeen proteins predicted to localize in all layers of the bacterial cell envelope, the cytosolic face, the inner membrane (IM), the periplasmic space and the outer membrane (OM). Among these proteins, a proton-gated channel at the inner membrane was identified as the molecular motor. Thus, thrust generation requires the transduction of traction forces generated at the inner membrane through the cell envelope beyond the rigid barrier of the bacterial peptidoglycan

The mysterious nature of bacterial surface (gliding) motility: A focal adhesion-based mechanism in Myxococcus xanthus

International audienceMotility of bacterial cells promotes a range of important physiological phenomena such as nutrient detection, harm avoidance, biofilm formation, and pathogenesis. While much research has been devoted to the mechanism of bacterial swimming in liquid via rotation of flagellar filaments, the mechanisms of bacterial translocation across solid surfaces are poorly understood, particularly when cells lack external appendages such as rotary flagella and/or retractile type IV pili. Under such limitations, diverse bacteria at the single-cell level are still able to “glide” across solid surfaces, exhibiting smooth translocation of the cell along its long axis. Though multiple gliding mechanisms have evolved in different bacterial classes, most remain poorly characterized. One exception is the gliding motility mechanism used by the Gram negative social predatory bacterium Myxococcus xanthus. The available body of research suggests that M. xanthus gliding motility is mediated by trafficked multi-protein (Glt) cell envelope complexes, powered by proton-driven flagellar stator homologues (Agl). Through coupling to the substratum via polysaccharide slime, Agl-Glt assemblies can become fixed relative to the substratum, forming a focal adhesion site. Continued directional transport of slime-associated substratum-fixed Agl-Glt complexes would result in smooth forward movement of the cell. In this review, we have provided a comprehensive synthesis of the latest mechanistic and structural data for focal adhesion-mediated gliding motility in M. xanthus, with emphasis on the role of each Agl and Glt protein. Finally, we have also highlighted the possible connection between the motility complex and a new type of spore coat assembly system, suggesting that gliding and cell envelope synthetic complexes are evolutionarily linked. (C) 2015 Elsevier Ltd. All rights reserved

bacto_tracker: a method for single-cell tracking of M. xanthus in dense and multispecies colonies

Cell motility and predation are important for the dynamics of many multi-cellular ecosystems, such as the gut or the soil. Approaches to image cell dynamics in such complex systems are scant, and high-throughput analysis methods to segment and track single-cell behaviors are currently lacking. Here, we addressed these limitations by implementing a fast fluorescence microscopy technique enabling the high-resolution acquisition of cell movement over large areas and long time periods. Next, we applied deep learning to semantically segment two different bacteria species within complex micro-environments . We implemented a method to build single cell traces by combining the cell masks from different time points to follow the dynamics of single cells with high spatial and temporal resolutions and over long periods of time. We applied and validated these methods by characterizing the dynamics of Escherichia coli predation by Myxococcus xanthus

Tol Energy-Driven Localization of Pal and Anchoring to the Peptidoglycan Promote Outer-Membrane Constriction

International audienceDuring cell division, gram-negative bacteria must coordinate inner-membrane invagination, peptidoglycan synthesis and cleavage and outer-membrane (OM) constriction. The OM constriction remains largely enigmatic, and the nature of this process, passive or active, is under debate. The proton-motive force-dependent Tol-Pal system performs a network of interactions within these three compartments. Here we confirm that the trans-envelope Tol-Pal complex accumulates at constriction site in Escherichia coli. We show that the inner-membrane complex composed of TolA, TolQ and TolR recruits the OM complex TolB-Pal to the septum, in an energy-dependent process. Pal recruitment then allows its binding to peptidoglycan and subsequently OM constriction. Our results provide evidence that the constriction of the OM is an energized process

Establishing rod shape from spherical, peptidoglycan-deficient bacterial spores

International audienceChemical-induced spores of the Gram-negative bacterium Myxococcus xanthus are peptidoglycan (PG)-deficient. It is unclear how these spherical spores germinate into rod-shaped, walled cells without preexisting PG templates. We found that germinating spores first synthesize PG randomly on spherical surfaces. MglB, a GTPase-activating protein, forms a cluster that responds to the status of PG growth and stabilizes at one future cell pole. Following MglB, the Ras family GTPase MglA localizes to the second pole. MglA directs molecular motors to transport the bacterial actin homolog MreB and the Rod PG synthesis complexes away from poles. The Rod system establishes rod shape de novo by elongating PG at nonpolar regions. Thus, similar to eukaryotic cells, the interactions between GTPase, cytoskeletons, and molecular motors initiate spontaneous polarization in bacteria

Qualitative and Quantitative Methods to Measure Antibacterial Activity Resulting from Bacterial Competition

n the environment, bacteria compete for niche occupancy and resources; they have, therefore, evolved a broad variety of antibacterial weapons to destroy competitors. Current laboratory techniques to evaluate antibacterial activity are usually labor intensive, low throughput, costly, and time consuming. Typical assays rely on the outgrowth of colonies of prey cells on selective solid media after competition. Here, we present fast, inexpensive, and complementary optimized protocols to qualitatively and quantitively measure antibacterial activity. The first method is based on the degradation of a cell-impermeable chromogenic substrate of the β-galactosidase, a cytoplasmic enzyme released during lysis of the attacked reporter strain. The second method relies on the lag time required for the attacked cells to reach a defined optical density after the competition, which is directly dependent on the initial number of surviving cells. Key features First method utilizes the release of β-galactosidase as a proxy for bacterial lysis. Second method is based on the growth timing of surviving cells. Combination of two methods discriminates between cell death and lysis, cell death without lysis, or survival to quasi-lysis. Methods optimized to various bacterial species such as Escherichia coli, Pseudomonas aeruginosa, and Myxococcus xanthus