Laboratoire de Chimie Bactérienne

Fast skeletal muscle fibers from zebrafish share a number of functional properties with mammalian twitch muscle fibers, making this vertebrate a precious model to investigate the pathophysiology of neuromuscular disorders. We previously reported that action potentials (APs) from zebrafish fast fibers exhibit low amplitude and require unusually strong negative resting membrane voltage levels to be elicited. In this study, using voltage-clamp and current-clamp techniques, we explored the properties of voltage-gated Na+ channels (NaV) responsible for initiation and propagation of AP in isolated adult zebrafish fast skeletal muscle fibers and compared them to mouse fast-twitch muscle fibers using the same experimental conditions. We found that kinetics of activation and inactivation of NaV were faster in zebrafish fibers and, overall, that the voltage-dependence of inactivation was shifted by 25 mV toward negative voltages as compared to mouse fibers, yielding a mean half-inactivation potential of -90 mV. In agreement with these findings, recording of APs at various resting membrane potentials indicated that APs vanished for resting membrane potentials less negative than -80 mV in zebrafish, whereas APs could still be elicited from resting membrane potentials as low as -60 mV in mice. In addition, Ca2+ transients induced by field stimulation were insensitive to Na+ current blockade in zebrafish but not in mouse fibers. Fluorescence labeling of nicotinic acetylcholine receptors showed that zebrafish fast fibers were multi-innervated with a mean distance between extra-synaptic sarcolemma and motor endplates of 14 µm, expected to lead to negligible attenuation of depolarization propagated from endplates. Finally, knock out of the two genes encoding pore-forming NaV subunits in zebrafish muscles did not induce any change in locomotion and escape behavior of the animals. Taken together, these data question the role of NaV and the occurrence of APs in zebrafish fast muscle.

ABSTRACT Motility enables cells to adapt to environmental cues, explore surfaces, and coordinate collective behaviors. Many organisms encode multiple motility systems, but whether these act independently or can operate together within the same cell has remained unclear. The predatory bacterium Myxococcus xanthus has long been thought to segregate its two propulsion systems: focal adhesions for gliding (A-motility) and type IV pili for twitching (S-motility), with each acting in distinct contexts. Here we overturn this view by providing the first direct demonstration that both systems are co-deployed and active within individual cells. Using dual-color high-resolution imaging, quantitative single-cell tracking, and agent-based simulations, we show that calcium, a key regulator of pili activity, tunes their relative engagement, shifting cells from adhesion- to pili-dominated propulsion with intermediate states combining both. This dynamic co-propulsion allows wild-type cells to move faster and explore more effectively than single-motility mutants, revealing a synergistic mode of motility integration shaped by environmental context.

IntroductionRespiratory diseases have a substantial impact on swine production worldwide. Understanding the relationship between gross lung lesions and the presence of infectious agents is crucial for developing effective disease control strategies that target both primary and secondary pathogens.Material and MethodsA cross-sectional study was conducted on 22 pig farms in western Poland. Cranioventral pulmonary consolidation (CVPC) in slaughtered pigs was assessed, and 20 lung tissue samples were collected from each herd. The presence of common bacterial and viral respiratory pathogens was identified using PCR-based methods.ResultsThe disorder was observed in 79.3% (95% confidence interval 75.3–82.8) of slaughtered pigs across all examined herds. The most frequently detected pathogens at both the herd and individual animal levels were Glaesserella parasuis, Mycoplasma hyopneumoniae and porcine circovirus 2. Co-infections involving two or more respiratory pathogens were prevalent, occurring in 100% of herds and 87.7% of individual pigs. Mean CVPC scores were significantly higher in pigs infected with Mycoplasma hyopneumoniae, Mycoplasma hyorhinis and porcine reproductive and respiratory syndrome virus 1.ConclusionThese findings highlight the multifactorial nature of respiratory infections in pigs. Effective control measures should consider the high prevalence of co-infections and their impact on lung lesion severity to improve overall herd health and productivity.

In bacterial chemosensing, environmental cues are typically sensed by bacterial transmembrane receptors known as methyl-accepting chemotaxis proteins (MCPs). MCPs form highly organized arrays using the bacterial membrane as a scaffold. These arrays amplify the signals and transduce them into a cellular response. The FrzCD cytoplasmic receptor from Myxococcus xanthus is unique due to its ability to bind DNA and use the nucleoid as a scaffold to form arrays. In this study, we identified two HAMP (histidine kinase, adenylyl cyclase, MCP, and phosphatase) domains located between the DNA binding and signaling domains of FrzCD. In vitro experiments demonstrate that the di-HAMP domain restricts FrzCD to a dimeric form in solution and modulate FrzCD affinity for DNA, whereas the signaling domain stabilizes higher-order oligomeric assemblies upon DNA binding. Through fluorescence microscopy and analyses of M. xanthus social behavior, we demonstrate that the impact of the FrzCD HAMP domains on DNA binding and oligomerization significantly influences the formation of Frz clusters on the nucleoid as well as group motility and development. Our results suggest that the di-HAMP domain might have roles not only in signal transduction but also in the plasticity of chemosensory arrays. These observations illustrate mechanisms of regulation of a DNA-bound cytoplasmic array formed by a diffusible MCP.IMPORTANCEOur study identifies the presence of a di-HAMP domain in a cytoplasmic chemoreceptor, FrzCD, from Myxococcus xanthus, and highlights its role in dynamic receptor oligomerization on a DNA scaffold. By controlling receptor oligomerization and subsequently the array formation on the nucleoid, the di-HAMP domain imparts plasticity to receptor arrays. Such plasticity governs cellular responses to external signals and dictates bacterial social behaviors such as group motility and multicellular structure formation.

Efflux-mediated antibiotic resistance poses a significant global threat, affecting diverse bacterial species. Clinicians recognize the danger of efflux mechanisms during antibiotic treatment, yet precise diagnostic tools remain unavailable. The antibiogram currently infers abnormal efflux pump activity in clinical isolates, which is subsequently confirmed through transcriptomic or genomic analysis. This study harnesses the colorimetric, fluorescence, and solubility properties of phenazinium derivatives to develop a rapid protocol for detecting bacterial efflux. Among several synthesized phenazinium compounds, the compound demonstrating differential MIC inStaphylococcusefflux mutants and exhibiting appropriate physicochemical properties is selected. A diagnostic protocol is developed using the selected compound to assess efflux levels, categorized as no, weak, or strong, through colorimetry and spectroscopy techniques. Testing on Gram-positive efflux mutants and clinical Staphylococcus isolates further validates the approach. In-silico docking analysis confirms the interaction between the chosen compound and the NorA efflux pump inS. aureus. Flow cytometry is employed to re-evaluate the detection assays. The developed molecule and protocol represent the first known method to evaluate efflux levels in any Gram-positive species through a streamlined and user-friendly process. This pioneering test significantly advances the epidemiological analysis of efflux mechanisms and enables more informed therapeutic decision-making, leading to more effective treatment.

Cell movement on surfaces relies on focal adhesion complexes (FAs), which connect cytoskeletal motors to the extracellular matrix to produce traction forces. The soil bacterium Myxococcus xanthus uses a bacterial FA (bFA), for surface movement and predation. The bFA system, known as Agl-Glt, is a complex network of at least 17 proteins spanning the cell envelope. Despite understanding the system dynamics, its molecular structure and protein interactions remain unclear. In this study, we utilize AlphaFold to generate models based on the known interactions and dynamics of gliding motility proteins. This approach provides us with a comprehensive view of the interactions across the entire complex. Our structural insights show the connection of essential functional modules throughout the cell envelope and offer an inspiring view of the force transduction mechanism from the inner molecular motor to the exterior of the cell.

The magnetotactic bacterium MO-1 possesses a pair of unique flagellar apparatuses. Each apparatus comprises seven flagella and 24 thin fibrils enclosed in a sheath. Using these apparatuses, the cells swim at high speed through solutions, tracing helical trajectories. The mechanism by which MO-1 cells utilize their two flagellar apparatuses to propel the cell body remains unclear. Due to the short length of the flagellar apparatus, direct observation of individual movements under a microscope is technically challenging. In this study, we performed numerical simulations based on the boundary element method to investigate the swimming motility of MO-1 cells. Our cell models successfully reproduced the helical trajectories of active MO-1 cells. The two flagellar apparatuses were positioned at the front and back of the cell body relative to the direction of movement. The cell body was pulled by the anterior flagellar apparatus and pushed by the posterior apparatus. The swimming motion of the cell model exhibited minimal changes near a wall. This unique swimming behavior of MO-1 cells could only be achieved through the rotation of both flagellar apparatuses. Based on the revolution rate of active MO-1 cells, we estimated the rotational speed of the flagellar apparatus to be approximately 1200 s-1. This rapid rotation may result from the tight packing of flagellar filaments within the sheath. Our study elucidates the mechanism by which active MO-1 cells swim in helical trajectories.

Phosphorus is an essential component of numerous macromolecules and is vital for life. Its availability significantly influences primary production, particularly in oligotrophic environments. Marine diazotrophic cyanobacteria, which play key roles in biogeochemical cycles through nitrogen fixation (N2 fixation), have adapted to thrive in phosphate (Pi)-poor areas. However, the molecular mechanisms that facilitate their adaptation to such conditions remain incompletely understood. Bacteria have evolved various strategies to cope with Pi limitation, including detecting Pi availability, utilising high-affinity Pi transporters, and hydrolyzing dissolved organic phosphorus (DOP) with various enzymes. This review synthesises current knowledge regarding how cyanobacteria adapt to Pi scarcity, with particular emphasis on subtropical marine free-living diazotrophs and their ability to utilise diverse DOP molecules. Omics approaches, such as (meta)genomics and (meta)transcriptomics, reveal the resilience of marine diazotrophs in the face of Pi scarcity and highlight the need for further research into their molecular adaptive strategies. Adaptation to Pi limitation is often intertwined with the broader response of cyanobacteria to multiple limitations and stresses. This underscores the importance of understanding Pi adaptation to assess the ecological resilience of these crucial microorganisms in dynamic environments, particularly in the context of global climate change.

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Abstract InMyxococcus xanthus, cell polarity and motility are regulated by the small GTPase MglA and its associated regulators, MglB, a potential GTPase Activating Protein and the RomRX complex, a potential Guanine nucleotide Exchange Factor. However, recent results have questioned the exact function of these regulators. Using a new type of fluorescent nucleotides for fluorescence-based anisotropy, we first demonstrate that RomRX does not function as a GEF but as an effector that binds strictly to MglAGTP. Secondly, based on an enzymology model, we found that MglB functions as a weak catalyst, unlike typical GAPs, and requires additional factors to dissociate from MglA. We demonstrate that RomRX, and specifically RomR, effectively competes with MglA by sequestering MglB. This low affinity interaction is still permissive for GAP activity but sufficient to partition these proteinsin vivoas indicated by a biomimetic assay in oil emulsion droplets reconstituting these interactions in a cell-sized environment. These results suggest a new model for cell polarity where RomRX exerts a dual function, targeting MglAGTPto the pole via its effector function and preventing its accumulation at the lagging pole by destabilizing the MglA-MglB complex.