Dispensability of extrinsic DnaA regulators in Escherichia coli cell-cycle control

Proceedings of the National Academy of Sciences, Journal Year: 2024, Volume and Issue: 121(33)

Published: Aug. 6, 2024

Investigating a long-standing conceptual question in bacterial physiology, we examine why DnaA, the master replication initiator protein, exists both ATP and ADP forms, despite only form being essential for initiation. We engineered Δ4 Escherichia coli strain, devoid of all known external elements facilitating DnaA-ATP/ADP conversion found that these cells display nearly wild-type behaviors under nonoverlapping cycles. However, during rapid growth with overlapping cycles, exhibit initiation instability. This aligns our model predictions, suggesting intrinsic ATPase activity DnaA alone is sufficient robust control E. regulatory extend robustness to multifork replication, indicating an evolutionary adaptation. Moreover, experiments revealed constant concentrations steady-state cell elongation cells. These insights not advance understanding cell-cycle regulation but also highlight fundamental divergence from eukaryotic controls, emphasizing protein copy-number sensing bacteria versus programmed concentration oscillations eukaryotes.

Language: Английский

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Tools and methods for high-throughput single-cell imaging with the mother machine

eLife, Journal Year: 2023, Volume and Issue: 12

Published: July 26, 2023

Despite much progress, image processing remains a significant bottleneck for high-throughput analysis of microscopy data. One popular platform single-cell time-lapse imaging is the mother machine, which enables long-term tracking microbial cells under precisely controlled growth conditions. While several machine pipelines have been developed in past years, adoption by non-expert audience challenge. To fill this gap, we implemented our own software, MM3, as plugin multidimensional viewer napari. napari-MM3 complete and modular pipeline data, takes advantage high-level interactivity Here, give an overview test it against well-designed widely used pipelines, including BACMMAN DeLTA. Researchers often analyze data with custom scripts using varied methods, but quantitative comparison output different has lacking. end, show that key physiological parameter correlations distributions are robust to choice method. However, also find small changes thresholding parameters can systematically alter extracted from experiments. Moreover, explicitly deep learning-based segmentation, 'what you put what get' (WYPIWYG) - is, pixel-level variation training cell segmentation propagate model bias spatial temporal measurements. Finally, while primary purpose work introduce software over last decade lab, provide information those who want implement machine-based methods their research.

Language: Английский

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Microbial life in slow and stopped lanes

Trends in Microbiology, Journal Year: 2023, Volume and Issue: 32(7), P. 650 - 662

Published: Dec. 19, 2023

Microbes in nature often lack nutrients and face extreme or widely fluctuating temperatures, unlike microbes growth-optimized settings laboratories that much of the literature examines. Slowed suspended lives are norm for microbes. Studying them is important understanding consequences climate change addressing fundamental questions about life: there limits to how slowly a cell's life can progress, long cells remain viable without self-replicating? Recent studies began these with single-cell-level measurements mathematical models. Emerging principles govern slowed – including dormant spores at temperatures re-defining discrete cellular states as continuums revealing intracellular dynamics new timescales. Nearly inactive, lifeless-appearing transforming our life.

Language: Английский

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Segmentation of dense and multi-species bacterial colonies using models trained on synthetic microscopy images

PLoS Computational Biology, Journal Year: 2025, Volume and Issue: 21(4), P. e1012874 - e1012874

Published: April 4, 2025

The spread of microbial infections is governed by the self-organization bacteria on surfaces. Bacterial interactions in clinically relevant settings remain challenging to quantify, especially systems with multiple species or varied material properties. Quantitative image analysis methods based machine learning show promise overcome this challenge and support development novel antimicrobial treatments, but are limited a lack high-quality training data. Here, experimental techniques for high-fidelity single-cell segmentation bacterial colonies developed. Machine learning-based models trained solely using synthetic microscopy images that processed look realistic state-of-the-art image-to-image translation method (cycleGAN), requiring no biophysical modeling. Accurate achieved densely packed single-species multi-species common pathogenic bacteria, even under suboptimal imaging conditions both brightfield confocal laser scanning microscopy. resulting data provide quantitative insights into soft Thanks their high adaptability relatively simple implementation, these greatly facilitate descriptions environments, may be used rapid diagnostic tools clinical settings.

Language: Английский

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Antimicrobial efflux and biofilms: an interplay leading to emergent resistance evolution

Trends in Microbiology, Journal Year: 2025, Volume and Issue: unknown

Published: May 1, 2025

The biofilm mode of growth and drug efflux are both important factors that impede the treatment bacterial infections with antimicrobials. Decades work have uncovered mechanisms involved in biofilm-mediated antimicrobial tolerance, but links between these phenomena only recently been discovered. Novel findings show how impacts global cellular physiology antibiotic underpinned by phenotypic heterogeneity. In addition can mediate cell-to-cell interactions, relevant biofilms, via including signaling molecules metabolites, using pump components establishment local gradients pumping. These recent suggest tolerance closely coupled, synergistic effects leading to evolution resistance environment.

Language: Английский

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Scaling of stochastic growth and division dynamics: A comparative study of individual rod-shaped cells in the Mother Machine and SChemostat platforms

Molecular Biology of the Cell, Journal Year: 2024, Volume and Issue: 35(6)

Published: April 10, 2024

Microfluidic platforms enable long-term quantification of stochastic behaviors individual bacterial cells under precisely controlled growth conditions. Yet, quantitative comparisons physiological parameters and cell different microorganisms in experimental device modalities is not available due to experiment-specific details affecting physiology. To rigorously assess the effects mechanical confinement, we designed, engineered, performed side-by-side experiments otherwise identical conditions Mother Machine (with confinement) SChemostat (without confinement), using latter as ideal comparator. We established a protocol cultivate suitably engineered rod-shaped mutant

Language: Английский

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Efficient Absorbance-Based Assay for Rapid Antibiotic Susceptibility Testing of Enterobacterales

Antibiotics, Journal Year: 2024, Volume and Issue: 13(9), P. 852 - 852

Published: Sept. 6, 2024

It is increasingly important to rapidly receive information on the antimicrobial susceptibility of bacteria due rise in resistance worldwide. However, traditional phenotypic methods are time-consuming. Thus, objective this study was develop a rapid assay that can detect antibiotic-resistant bacterial isolates phenotypically less than 2 h. The microplate used based absorbance measurements pure grown liquid media with and without antibiotics. Absorbance measured at beginning after 90 min incubation 37 °C. Susceptibility calculated for that, absence antibiotics, exhibited more 50% growth increase by comparing value culture presence an antibiotic its initial value. Here, we show it possible screen Enterobacterales Acinetobacter spp. three different antibiotics min. test demonstrated accuracy 98.8%, sensitivity 97.6%, specificity 99.6%. false rates were 0.2% 1.0%. This simple has proven suitable screening large number strains high sensitivity. method be performed specialized costly materials and/or equipment, which makes highly laboratories limited resources.

Language: Английский

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Engineering plasmid copy number heterogeneity for dynamic microbial adaptation

Nature Microbiology, Journal Year: 2024, Volume and Issue: 9(8), P. 2173 - 2184

Published: June 18, 2024

Language: Английский

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Live Imaging of Fission Yeast Single-Cell Lineages Using a Microfluidic Device

Methods in molecular biology, Journal Year: 2024, Volume and Issue: unknown, P. 61 - 76

Published: Nov. 11, 2024

Language: Английский

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