The origin of biodiversity is arguably the most fundamental question in ecology - especially how countless microbial species coexist within a single community. A prevailing hypothesis holds that microbial species coexist by specializing on different resources, thereby reducing competition. Accordingly, increasing resource diversity is expected to promote species coexistence and boost biodiversity. Surprisingly, we observe the opposite: microbial biodiversity often declines as resource diversity increases. This unexpected result emerges from a widespread physiological response across diverse microbial taxa, in which more complex environments trigger higher overall resource uptake. This intensifies competition and drives biodiversity loss. Updating a central ecological model to include this physiological trait accurately reproduces the observed decline. Our findings demonstrate that physiological changes at the individual level can overturn classical ecological principles and scale up to restructure entire ecosystems.

All methanogens that can fix nitrogen use molybdenum (Mo) nitrogenase. Some methanogens, including Methanosarcina acetivorans, also contain alternative vanadium- and iron-nitrogenases, encoded by the vnf and anf operons, respectively. These nitrogenases are produced when there is insufficient Mo to support Mo-nitrogenase activity. The factors that control the expression of the alternative nitrogenases in response to Mo availability are unknown in methanogens. Here we show that ModE is the regulator that represses transcription of the vnf and anf operons in M. acetivorans when cells are grown with Mo. CRISPRi repression of modE results in a significant increase in the transcription of the vnf and anf operons as well as the detection of Fe-nitrogenase during nitrogen fixation in the presence of Mo. Gel shift assays with recombinant ModE demonstrated that ModE binds a specific sequence motif upstream of the vnf and anf operons, as well as other genes and operons related to nitrogen fixation and Mo transport. However, purified ModE does not contain Mo, and the addition of Mo does not alter the affinity of ModE for DNA, indicating M. acetivorans ModE may not directly bind Mo. This study shows that ModE is the primary Mo-responsive regulator of alternative nitrogenase expression in M. acetivorans, but other factor(s) are likely involved in directly sensing Mo.

De novo design of anti-freeze peptides (AFPTs) represents a formidable challenge due to the unclarified active structure of AFPTs. Here, we describe a “Site to Distance” principle for de novo design of AFPTs, in terms of understanding their structure–activity relationships. The first step is to point E, identified as the most potent ice-binding site (IBS) possessing at least 4-fold binding energy than natural IBSs, into the candidate backbones. The second step, based on the IBS (E), is to judiciously adjust the distances of sites to match the favorable number of the ice crystal lattice to achieve the strongest ice-binding, relying on a newly established low-temperature AFPT structure prediction platform. The resultant AFPTs show a substantial reduction in single ice crystal growth rates, much superior to >100 natural or designed AFPTs, including all that have been reported. Cryopreservation of therapeutic cells further confirms the accuracy of this design principle.

Protein misfolding and aggregation are central features of a wide range of diseases, including neurodegenerative disorders, systemic amyloidoses, and cancer. The identification of compounds that can modulate protein folding and aggregation is a key step toward developing effective therapies. High-throughput screening methods are essential for efficiently identifying such compounds. In this study, we optimized a previously developed high-throughput genetic screen for monitoring protein misfolding and aggregation in bacteria. This system is based on monitoring the fluorescence of Escherichia coli cells expressing fusions of human misfolding-prone and disease-related proteins (MisPs) with the green fluorescent protein. We systematically tested a variety of experimental conditions, such as overexpression conditions and MisP-GFP fusion formats, to identify key parameters that affect the sensitivity and dynamic range of the assay. Using misfolding-prone, cancer-associated variants of human p53 as a model system, we found that strong overexpression conditions, such as high copy number vectors, strong promoters, high inducer concentrations, and high overexpression temperatures, can yield optimal assay performance. These optimized assay conditions were also validated with additional MisPs, such as the Alzheimer’s disease-associated amyloid-β peptide and variants of superoxide dismutase 1 associated with amyotrophic lateral sclerosis. At the same time, we observed that certain conditions, such as inducer concentrations and overexpression temperature, may need to be precisely fine-tuned for each new MisP target to yield optimal assay performance. Our findings provide a framework for standardizing MisP-GFP screening assays, facilitating their broad application in the discovery of therapeutic agents targeting protein misfolding and aggregation.

The common fungal pathogen, Candida albicans, relies on the pore-forming toxin candidalysin to damage host cells. Cells resist other pore-forming toxins by Ca2+ dependent microvesicle shedding and annexins (cholesterol-dependent cytolysins, CDCs), or annexins and patch repair (aerolysin). However, it is unclear which Ca2+ dependent repair mechanism(s) resists candidalysin. Here, we determined the involvement of different Ca2+ dependent repair mechanisms to candidalysin and compared responses to CDCs and aerolysin using flow cytometry and high-resolution microscopy. We report that candidalysin triggered Ca2+-dependent repair, but patch repair and ceramide failed to provide significant protection. MEK-dependent repair and annexins A1, A2 and A6 contributed partially to repairing damage caused by candidalysin. However, annexin translocation after candidalysin challenge was delayed compared to CDCs or aerolysin challenge. Surprisingly, extracellular Cl- improved cell survival after candidalysin challenge, but not after challenge with a CDC or aerolysin. Finally, we found that candidalysin is removed via extracellular vesicle shedding. These findings reveal that Ca2+ dependent microvesicle shedding protects cells from candidalysin and can be engaged by multiple molecular mechanisms during membrane repair.

Bacterial persistence is a phenomenon in which a small fraction of isogenic bacterial cells survives a lethal dose of antibiotics. Although the refractoriness of persistent cell populations has classically been attributed to growth-inactive cells generated before drug exposure, evidence is accumulating that actively growing cell fractions can also generate persister cells. However, single-cell characterization of persister cell history remains limited due to the extremely low frequencies of persisters. Here, we visualize the responses of over one million individual cells of wildtype Escherichia coli to lethal doses of antibiotics, sampling cells from different growth phases and culture media into a microfluidic device. We show that when cells sampled from exponentially growing populations were treated with ampicillin or ciprofloxacin, most persisters were growing before antibiotic treatment. Growing persisters exhibited heterogeneous survival dynamics, including continuous growth and fission with L-form-like morphologies, responsive growth arrest, or post-exposure filamentation. Incubating cells under stationary phase conditions increased both the frequency and the probability of survival of non-growing cells to ampicillin. Under ciprofloxacin, however, all persisters identified were growing before the antibiotic treatment, including samples from post-stationary phase culture. These results reveal diverse persister cell dynamics that depend on antibiotic types and pre-exposure history.

Robust control of DNA replication is fundamental to bacterial proliferation. In Escherichia coli, replication initiation is thought to be regulated by oscillations in DnaA activity, driven by DnaA-chromosome interactions that differ among leading models. However, direct evidence linking these oscillations to replication initiation has been lacking, and existing models fail to explain the observed decoupling of replication initiation from dnaA expression. Here, we establish a direct link between DnaA activity and replication initiation by demonstrating robust oscillations in DnaA activity, which peak precisely at replication initiation across diverse growth conditions and genetic perturbations. Notably, these oscillations persist even when dnaA transcription remains constant, suggesting a regulatory mechanism that modulates DnaA activity independently of its expression. Additionally, we propose an extrusion model in which DNA-binding proteins sense biomass-DNA imbalance and extrude DnaA from the chromosome to trigger replication, overcoming limitations of existing models. Consistent with this model, perturbation of the nucleoid-associated protein H-NS modulates DnaA activity and replication timing, supporting its mechanistic validity.

Bioprospecting algae strains with tolerance to extreme conditions such as pH, temperature, salinity, and light is crucial for advancing biotechnology and environmental applications. However, traditional screening methods often involve significant costs and labor, restricting their accessibility and practical use. In this study, we developed and validated low-cost, high-throughput screening techniques, predominantly employing agar plates and liquid culture assays, to effectively differentiate tolerance levels among various algae strains. The methodologies were optimized using the model microalga Chlamydomonas reinhardtii and its closely related species Chlamydomonas incerta and the recently discovered extremophilic Chlamydomonas pacifica. We systematically evaluated the algae for tolerance to extremes by establishing precise gradients of pH (acidic to alkaline conditions), salinity (0 to 5 M NaCl), temperature (34 - 42C), and light intensity (40 to 2977 microE/m2s1). Our results demonstrated that these cost-effective, agar plate-based methods effectively distinguished algae strains exhibiting superior tolerance to extreme environmental conditions. These screening techniques not only provided clear differentiation among the closely related strains but also delivered reproducible outcomes suitable for scaling up to larger bioprospecting efforts. Furthermore, the affordability and simplicity of these methods facilitate their implementation in resource-limited laboratories, thereby broadening participation in algae bioprospecting endeavors. This study highlights the potential of low-cost, accessible screening techniques to significantly enhance the discovery and characterization of algal strains with extreme traits. Ultimately, these methods support the development of robust algae-based resources, driving innovation in diverse industrial processes and environmental solutions.

Bacterial cooperation involves the exchange of metabolites, which can range from costless byproducts of metabolism to intentionally produced and costly molecules. These interactions occur across spatial scales, from direct cell contact to the diffusion of metabolites in the environment. Due in part to this variety of interaction modes, the impacts of mutualism on bacterial community dynamics remain unclear. Using generalized models, we derive conditions for the onset of different dynamical behaviors in communities of bacterial cooperators across these scenarios. These include exchanges of low-cost metabolites, costly cross-feeding, and cases where bacteria produce either most or only a small fraction of available metabolites. Stability depends strongly on metabolic production costs and the balance between metabolite uptake and production. We further show that perturbations to bacteria have larger impacts than to metabolites. Finally, we demonstrate that spatial metabolite diffusion drives pattern formation, emphasizing the links between local stability and spatial structures in bacterial cooperation.