Arias-Cartin, R. | bioRxiv Science

bioRxiv2026

Automated Optimization of Bacterial Tracking Pipelines with TrackMate 8

Quantitative analysis of bacterial dynamics in time-lapse microscopy requires robust tracking pipelines, yet selecting and optimizing algorithms for specific experiments remains challenging. Indeed, Microbiologists are confronted with numerous algorithms that must be carefully chosen and parameterized to achieve optimal tracking for their experiments. We present an automated methodology to determine optimal tracking configurations for microbiological applications. It is based on TrackMate 8, a novel version of the TrackMate Fiji plugin extended with microbiology-specific tools. Our approach systematically evaluates algorithm-parameter combinations optimizing biologically relevant metrics (e.g., cell-cycle accuracy, bacteria morphology) and includes: (1) integration of deep-learning algorithms (Omnipose, YOLO, Trackastra) adequate for bacteria images in TrackMate, (2) a TrackMate-Helper extension for parameter optimization, and (3) a tracking and segmentation editor for tracking ground-truth generation. We demonstrate the effectiveness of the methodology on two use cases showing its adaptability to diverse experimental conditions. This methodology enables microbiologists with a widely applicable, automated framework to optimize tracking pipelines, facilitating quantitative analysis in bacterial imaging.

microbiology↗

bioRxiv2026

Oral Fumarate-based drugs alter gut microbiota species via cysteine succination

Mono- and dimethyl fumarates are oral fumarate esters widely prescribed for relapsing-remitting multiple sclerosis and psoriasis. While these drugs appear to be effective, their effects on the gut microbiota and their precise bacterial targets remain unclear. In this study, we investigated how these drugs affect bacteria through a chemical modification called succination, where they react with protein thiol groups (-SH), particularly in cysteine residues. Using proteomics, enzymology and microscopy, we show how this post-translational modification disrupts several key bacterial functions and triggers oxidative and protein stress in E.coli. We also found that fumarate esters can be toxic to various gut bacteria in isolated cultures. Notably, our results demonstrate that when bacteria are studied together in microbial communities, the effect of fumarates can change, either weakening or intensifying. Our findings thus shed light on fumarate esters and microbiota interactions and allow identifying new molecular targets of succination relevant to microbiome health.

microbiology↗

bioRxiv2023

Role of the Escherichia coli ubiquinone-synthesizing UbiUVT pathway in adaptation to changing respiratory conditions

Isoprenoid quinones are essential for cellular physiology. They act as electron and proton shuttles in respiratory chains and in various biological processes. Escherichia coli and many , {beta}, and {gamma} proteobacteria possess two types of isoprenoid quinones: ubiquinone (UQ) is mainly used under aerobiosis, while (demethyl)menaquinones ((D)MK) are mostly used under anaerobiosis. Yet, we recently established the existence of an anaerobic O2- independent UQ biosynthesis pathway controlled by ubiT, ubiU, and ubiV genes. Here, we characterize the regulation of ubiTUV genes in E. coli. We show that the three genes are transcribed as two divergent operons that are both under the control of the O2 sensing Fnr transcriptional regulator. Phenotypic analyses using a menA mutant devoid of (D)MK revealed that UbiUV-dependent UQ synthesis is essential for nitrate respiration and for uracil biosynthesis under anaerobiosis, while it contributes, though modestly, to bacterial multiplication in the mouse gut. Moreover, we showed by genetic study and 18O2 labelling that UbiUV contribute to hydroxylation of ubiquinone precursors through a unique O2 - independent process. Last, we report a crucial role of ubiT in allowing E. coli to shift efficiently from anaerobic to aerobic conditions. Overall, this study uncovers a new facet of the strategy used by E. coli to adjust its metabolism upon changing O2 levels and respiratory conditions. This work links respiratory mechanisms to phenotypic adaptation, a major driver in the capacity of E. coli to multiply in gut microbiota, and of facultative anaerobic pathogens to multiply in their host. ABSTRACT IMPORTANCEEnterobacteria multiplication in the gastrointestinal tract is linked to microaerobic respiration and associated to various inflammatory bowel diseases. Our study focuses on biosynthesis of ubiquinone (UQ), a key player in respiratory chains, under anaerobiosis. The importance of this study stems from the fact that UQ usage was for long considered to be restricted to aerobic conditions. Here we investigated the molecular mechanism allowing UQ synthesis in the absence of O2 and searched for the anaerobic processes that UQ is fueling in such conditions. We found that UQ biosynthesis involves anaerobic hydroxylases, i.e. enzymes able to insert a O atom in the absence of O2. We also found that anaerobically synthesized UQ can be used for respiration on nitrate and synthesis of pyrimidine. Our findings are likely to be applicable to most facultative anaerobes, which count many pathogens (Salmonella, Shigella, Vibrio) and will help in unravelling microbiota dynamics.

microbiology↗