mLife

Here, we develop CandiChrome, a multiplex labeling toolkit for Candida albicans, through combined in vitro and in vivo characterization of fluorescent proteins in a standard strain background. To this end, we screened 13 candidate fluorophores across the visible spectrum and assessed their practical performance based on brightness, stability, and usability. This analysis identified a seven-fluorophore set that achieved the most effective balance of signal strength, robustness, and compatibility. We used this optimized panel to build a modular multicolor platform that enables strain labeling, mixed-population imaging, and competition assays in C. albicans. This platform could resolve up to 21 distinct populations by flow cytometry and microscopy. Importantly, CandiChrome supported the resolution of differentially labeled populations both in vitro and in the murine host, supporting the simultaneous tracking of multiple strains in complex settings. Together, these results establish CandiChrome as a flexible platform for multiplex fungal imaging in a pathogenic species where multicolor tools remain underdeveloped.

Ionizing radiation induces DNA damage and oxidative stress; however, the genes and molecular mechanisms involved in bacterial stress responses have not been sufficiently identified. In this study, we used Limnobacter thiooxidans strain CS-K2, which is the closest relative to the bacteria detected in torus room water at the Fukushima Daiichi Nuclear Power Plant according to 16S rRNA gene sequences, and evaluated its response to {gamma}-ray irradiation using integrated transcriptomic and proteomic analyses. We identified three previously uncharacterized genes (LT3105, LT3115, and LT3126) that were strongly induced at the mRNA and protein levels. These genes exhibited low basal expression but were markedly upregulated by {gamma}-ray irradiation. Notably, LT3126 encodes a protein containing VIT (vault protein inter--trypsin) and VWA (von Willebrand factor type A) domains and showed the strongest induction. Overexpression of LT3126 increased survival after 500 Gy irradiation by approximately 200-fold compared with the control bacteria, demonstrating a direct contribution to survival under high-dose stress. Comparative genomic analysis showed that these genes are not widely conserved across bacteria but are unevenly distributed among specific lineages. Taken together, this study identified a novel set of {gamma}-ray-responsive genes and demonstrated a functional role for LT3126 in radiation resistance, providing new insights into molecular adaptation in radiation-associated environments. IMPORTANCEWe identified a novel set of {gamma}-ray-responsive genes (LT3105, LT3115, and LT3126) in the non-model bacterium Limnobacter thiooxidans. These genes are located in relatively close genomic proximity and are coordinately induced upon irradiation, suggesting a shared functional role in stress response. Overexpression of LT3126 increased survival by approximately 200-fold after 500 Gy irradiation compared with the control bacteria, demonstrating a substantial contribution to survival under high-dose stress. These genes were also induced by heat shock and oxidative stress, indicating that their function extends beyond radiation-specific responses to broader environmental stress adaptation. Consistent with this, comparative genomic analysis showed that these genes are not widely conserved across bacteria but are unevenly distributed among specific lineages. Taken together, these findings highlight previously unrecognized molecular strategies that may support bacterial survival in radiation-associated environments.

We developed a convolutional neural network-based machine learning technique to simultaneously analyze the morphology and motility of spirochetal bacteria swimming with continuous cellular deformation. Matching probabilities between experimental images and learned models realizes quantification of cell morphology and association with motility. This method can be applied to diverse transformable cells, offering critical biophysical insights into microbial dynamics.

With the rapid expansion of global food demand, aquaculture has become a critical pillar for future food security. However, aquaculture systems remain highly vulnerable to pathogenic bacteria, and rapid identification of antagonistic microbes is essential for sustainable disease control. Conventional evaluation approaches rely on fluorescence labeling or post-culture assays, limiting the ability to quantify dynamic interactions in mixed microbial populations in a real-time and label-free manner. Here, we propose a computational framework for classifying the mixing ratio of Vibrio harveyi and environmental bacteria using time-series motion features extracted from microscopy videos. We defined 24 interpretable motility descriptors and employed a Temporal Convolutional Network (TCN) to learn their temporal structure. The proposed method achieved a classification accuracy of 93.3%, outperforming conventional static statistical approaches and alternative machine learning models. These findings indicate that mixture discrimination in microbial communities is governed not by absolute motility magnitude, but by collective alignment and its temporal stability. Our study establishes a time-resolved computational framework for quantifying dynamic collective order in mixed microbial populations and highlights its potential for label-free automated screening and robotic microbiological applications.

The thermophilic methanogen Methanothermobacter thermautotrophicus {Delta}H is a model microbe for hydrogenotrophic methanogenesis and an emerging platform host for metabolic engineering. Despite recent advances in its genetic accessibility, the available molecular toolbox lacks fluorescent reporter proteins that are suitable for anaerobic and thermophilic conditions. Here, we established the fluorescence-activating and absorption-shifting tag (FAST) as a reporter protein in M. thermautotrophicus. We expressed codon-optimized variants of FAST by applying established genetic tools, and evaluated the performance for two temperatures and three fluorogens. We demonstrated that FAST is functional in M. thermautotrophicus but exhibits temperature-dependent instability, which is more pronounced at 60{degrees}C compared to 50{degrees}C. Among the tested fluorogens, TFLime and TFAmber yielded comparable fluorescence intensities, while TFCoral resulted in significantly lower fluorescence intensity. Exploiting the partial thermolability of FAST, we characterized the dynamic expression profiles of several promoters, which revealed growth phase-dependent regulation patterns. Our findings challenge previous assumptions of constitutive expression for several promoters. Notably, we identified distinct expression patterns for promoters that are associated with methanogenesis and energy-converting hydrogenases. Our results establish FAST as a versatile fluorescent reporter for thermophilic methanogens and provide new insights into promoter regulation in M. thermautotrophicus. This work expands the genetic toolbox for this microbe and lays the foundation for advanced studies in archaeal cell biology and biotechnology.

Effector translocation during host-pathogen interactions is a prerequisite for the entry of Salmonella into non-phagocytic cells and establishment of a replication permissive intracellular niche. Deciphering the dynamics and kinetics of translocation and subcellular localization demands live-cell imaging and tagging approaches that do not introduce detection delays or perturb the translocation process via the type III secretion system (T3SS). Effector fusions with self-labelling enzymes (SLE), such as HaloTag, allow localization and tracking at high temporal and spatial resolution. However, interference with T3SS-dependent translocation has hampered analyses of the process of translocation and early subcellular distribution and dynamics. Herein, we report that amino acid substitutions of the HaloTag can reduce the thermodynamic stability, resulting in less steric hindrance during translocation of effector-HaloTag fusions by the T3SS in mammalian cells. The top variant, HT-SP5, showed reduced retention in Salmonella, enabling more sensitive and earlier detection of translocated effector proteins of the SPI1 and SPI2 T3SS of Salmonella and of the T3SS effector Map of enteropathogenic Escherichia coli (EPEC). We applied the improved HaloTag HT-SP5 to single molecule tracking, and to follow effector protein dynamics in living host cells early after translocation by invading and intracellular bacteria. Taken together, the improved HaloTag variant HT-SP5 represents a robust and versatile SLE tag for dynamic real-time analyses of delivery and fate of T3SS-translocated effector proteins in living cells host. Application of HT-SP5 will facilitate research on effectors throughout the entire infection process at native effector levels to understand host-pathogen interactions.

Biochar amendments are increasingly applied to improve soil physical functioning and support carbon dioxide removal, but their effects on intrinsic soil thermal properties remain poorly characterised. We conducted the first global systematic meta-analysis of 19 independent studies, 231 control-biochar comparisons, and 529 property-specific effect sizes to test how biochar changes soil heat transfer and storage. Biochar reduced thermal conductivity by 17.6% (95% CI, -22.7 to -12.2), thermal diffusivity by 11.0% (-14.5 to -7.3), and volumetric heat capacity by 8.3% (-12.3 to -4.1). Gravimetric heat capacity showed no significant overall response (+3.3%; -7.6 to 15.4) but was supported by fewer studies. Negative responses were directionally consistent for thermal conductivity, diffusivity, and volumetric heat capacity. Moderator analyses showed that responses were most consistently associated with post-application bulk density and changes in bulk density, while application rate modulated response magnitude and soil texture constrained context dependence. Co-variation among thermal conductivity, thermal diffusivity, and volumetric heat capacity matched expected physical dependencies, indicating coordinated structural reorganisation rather than independent shifts in isolated parameters. These estimates describe intrinsic conductive and storage properties; field-scale soil temperature responses may also be modified by albedo, evaporation, vegetation, and surface energy balance. Improved integration of soil thermal measurements with moisture dynamics, structural changes, and carbon cycling is essential to accurately represent biochar effects in soil and land-surface models.

Taro (Colocasia esculenta (L.) Schott) is an important vegetable and food crop in China, but in recent years, soft rot disease has frequently occurred during its cultivation and production. This disease damages the underground corms and petiole bases of taro, causing decay in the affected parts and emitting a foul odor, leading to wilting and lodging of the entire plant. This has resulted in significant economic losses to taro production in China, along with food safety issues and ecological problems caused by excessive pesticide use, making it urgent to find a green and efficient control method. Due to its specificity and environmental safety, phage therapy exhibits advantages that chemical pesticides cannot match, representing a promising alternative to chemical pesticides for controlling pathogenic bacteria. In the preliminary work of this study, a bacterial strain was isolated from taro soft rot in Shaoguan, Guangdong, and initially identified as Pectobacterium colocasium ZXC0623. Using this strain as the host bacterium, a Pectobacterium phage was screened and named QJphage. We analyzed its physicochemical properties and obtained its biological characteristics, including optimal titer, optimal infection latency period, optimal infection multiplicity, optimal storage solvent, and resistance to ultraviolet light, pH, and chloroform. Through homologous alignment analysis, eight tail fiber proteins encoded in the QJphage genome were predicted as putative receptor-binding proteins (RBPs). To validate this prediction, the corresponding genes were cloned downstream of the egfp gene via homologous recombination, and the resulting recombinant plasmids were transformed into a prokaryotic host to express EGFP-tagged tail fiber fusion proteins. Fluorescence detection and confocal laser scanning microscopy confirmed that the protein encoded by ORF04 functions as the RBP. Furthermore, lipopolysaccharide (LPS) was knocked out in the host strain P. colocasium ZXC0623. Both{Delta} LPS1 and{Delta} LPS2 mutants formed smaller plaques compared to the wild-type strain, and the{Delta} LPS1 mutant additionally exhibited a significant reduction in plaque number, indicating that LPS serves as a receptor involved in QJphage adsorption. Finally, transcriptomic analysis during the latent period of infection focused on 20 genes predicted to be associated with phage-host receptor binding and anti-phage immune systems. The results revealed that pilin proteins act as potential reversible adsorption receptors for QJphage, while the host strain ZXC0623 also possesses a diverse repertoire of anti-phage defense systems. Collectively, QJphage exhibits stable physicochemical properties, a well-defined LPS-dependent infection mechanism, and a host with diverse defense systems, providing a foundation for the control of taro soft rot and future phage-related research. ImportancePhage therapy has emerged as a highly effective biocontrol strategy against Pectobacterium, with its specificity making it particularly valuable. A critical aspect of this approach is the identification of phage receptors. The initial step in the phage life cycle involves adsorption to the bacterial host, beginning with reversible contact followed by irreversible binding between phage receptor-binding proteins and specific bacterial surface receptors. Potential receptors include glycolipids in the Gram-negative outer membrane, capsular polysaccharides, and various membrane proteins or appendages. In this study, we first characterized the physicochemical properties of the isolated QJphage. Through integrated transcriptomic and whole-genome analyses, we demonstrated that the LPS of Pectobacterium specifically interact with the tail fiber proteins of QJphage. This research provides the first evidence revealing the molecular mechanism of interaction between Pectobacterium and its phage, establishing a foundation for developing phage-based control strategies against soft rot diseases.

Biological ethanol production is important for the circular carbon economy and makes up 73% of the U.S. biological fuels market. Previously, we produced ethanol by reversing methanogenesis and capturing methane by cloning methyl-coenzyme M reductase (Mcr) from an unculturable population of anaerobic methanotrophic archaea; this process was predicated on the generation of the intermediate acetate and its conversion by the methanogenic host to ethanol. Moreover, methanogens are generally thought to be detrimental for converting acetate to ethanol and are usually intentionally inhibited. Here, we demonstrate that direct growth on acetate as the sole carbon and energy source by the methanogen Methanosarcina acetivorans C2A results in 40% of the metabolized acetate becoming ethanol and that there is 430% more ethanol produced, compared to growth on methane via Mcr. In addition, we found growth on methanol results primarily in methane generation and low levels of ethanol. Therefore, acetate may be readily converted by the methanogen M. acetivorans to ethanol at high yields.

High level of protein expression is usually welcomed in industry and research, and codon optimization is widely used to achieve high expression. Methods of implementing codon optimization can be divided into two branches, one is classical methods which develop cost functions based on empirical law, another is AI methods which learn the codon choice principles from endogenous genes with neural networks. Here we develop two codon optimization tools based on two branches respectively, namely OptimWiz 2.1 and OptimWiz 3.0. Results of fusion protein fluorescence detection indicate that both OptimWiz 2.1 and OptimWiz 3.0 are superior to all the other commercially available codon optimization tools. Principles of codon optimization are revealed in the process of machine learning on both tools.

Extracellular nucleic acids (eNA) are central components of bacterial biofilms, contributing to structural integrity, antibiotic tolerance, and emerging functions such as extracellular electron transfer and peroxidase-like catalysis. While extracellular DNA has traditionally been assumed to adopt the canonical B-DNA conformation, biofilms are now known to contain non-canonical structures, including Z-DNA/RNA (Z-NA), G-quadruplex DNA/RNA (G4-NA), and substantial amounts of extracellular RNA. Conventional nucleic acid-binding dyes are widely used for rapid eNA detection, yet their specificity for these diverse structures has not been systematically evaluated. Here, we compare the fluorescence properties of eleven cyanine monomer and dimer dyes (TOTO, BOBO, YOYO, and POPO series, SYTOX Green, SYTOX Red, and propidium iodide) against synthetic B-DNA, Z-DNA, G4-DNA, A-RNA, Z-RNA, and G4-RNA oligonucleotides, with Z-NA stabilised through brominated guanosine analogues synthesised in-house. A clear pattern emerged: green-fluorescent dyes preferentially bound canonical B-DNA, whereas red-fluorescent counterparts displayed broader specificity that extended to non-canonical structures. TOTO-3 and SYTOX Red bound G4-NA with higher fluorescence than B-DNA, and propidium iodide showed an unexpected preference for A-RNA over B-DNA. These observations were validated in Staphylococcus aureus biofilms by parallel immunolabelling with structure-specific antibodies. TOTO-3, YOYO-3, BOBO-3, POPO-3, and propidium iodide reproduced the eNA distribution at the bacterial cell surface. Finally, we introduce poly-A tailing with fluorescently labelled ATP as a stringent, RNA-specific imaging method for biofilms. Together, these results provide practical guidelines for visualising the structural diversity of eNA in biofilms.

Oxygen consumption is a direct functional readout of bacterial respiration and metabolic state, yet existing methods for quantifying oxygen dynamics are limited in throughput and temporal resolution. Here, we establish a high-throughput platform for real-time profiling of bacterial respiration by adapting the Resipher, a non-invasive oxygen quantification system, for use in bacterial cultures. Measurements obtained with the Resipher were comparable to those generated using a Clark-type electrode-based high-resolution respirometer, validating its quantitative accuracy. Across Gram-negative (Escherichia coli, Francisella novicida) and Gram-positive (Enterococcus faecalis, Staphylococcus aureus) species, the Resipher generated reproducible measurements under both growth-permissive and growth-limited conditions, enabling assessment of respiration, independent of proliferation. Functional profiling revealed that oxygen consumption responds dynamically to nutrient availability and electron transport chain perturbation, including species-specific inhibition by benzarone. Notably, oxygen consumption profiles distinguished bactericidal and bacteriostatic antibiotics, with bactericidal agents transiently increasing respiration and bacteriostatic agents suppressing metabolic activity. Together, these findings establish oxygen consumption as a sensitive physiological readout and highlight the potential utility of respiratory profiling for mechanistic studies.

A challenge in using plant biomass is its highly recalcitrant nature, which makes it economically infeasible to utilize. In natural environments, various microbes, including bacteria and fungi, are reported to decompose plant cell wall materials such as cellulose and hemicellulose, and there may be undescribed microbes that contribute to the degradation of plant biomass. We focused on isolating novel plant biomass-degrading bacteria and screened more than 100 isolates from the Tomakomai experimental forest in Hokkaido, Japan. Among them, one novel Bacillus species was chosen for whole-genome sequencing. Comparative genomics and a carbon source utilization assay indicated that the isolate belongs to a subspecies of Bacillus subtilis, which we named B. sp. TTS1. Glucose, cellobiose, xylose, xylan, mannose, or mannan was used as the sole carbon source in the minimum medium, and the growth of this bacterium was determined. Furthermore, a proteomic analysis of B. sp. TTS1 was performed using culture supernatants from various polysaccharide-containing media. In the present study, several key enzymes involved in plant biomass degradation were identified, namely {beta}-1,4-mannanase and xylanase, and they were highly enriched in all tested polysaccharides.

3',5'- Cyclic adenosine monophosphate (cAMP) serves as a global regulatory hub in Mycobacterium tuberculosis (Mtb), which dedicates approximately 1% of its genome to the synthesis, degradation and sensing of this secondary messenger. Despite cAMPs central role in Mtb virulence and metabolic adaptation, we currently do not have any tools to monitor cAMP dynamics in real time at single-cell resolution. Here, we report the development and validation of rmgCarvi, a genetically encoded biosensor adapted from eukaryotic systems for use in mycobacteria. By utilizing cAMP-responsive cpGFP and constitutive mCherry fluorescence, rmgCarvi provides a high-fidelity ratiometric readout of intracellular cAMP levels, revealing that carbon source and growth state act as primary determinants of cAMP dynamics. Real-time single-cell imaging using rmgCarvi demonstrated robust correlations between cAMP and growth rate, with pronounced cAMP spikes coinciding with cell division. In starved Mtb populations, a bimodal distribution in cAMP levels dictates a distinct hysteresis during regrowth upon nutrient supplementation. Low-cAMP subpopulations are associated with strong induction in cAMP levels and a longer lag before initiation of growth, while high cAMP cells do not exhibit significant induction in cAMP and have recovery times uncorrelated with prior cAMP levels. Finally, use of rmgCarvi in infection studies captured host species-specific responses, including a strong cAMP induction upon internalization followed by a decline in murine, but not human, macrophages. By resolving cAMP dynamics with unprecedented cellular and temporal resolution, rmgCarvi provides a framework for understanding how Mtb tunes this crucial metabolite to navigate the complex microenvironments in the host. Significance StatementWe report the evaluation of a ratiometric genetic biosensor, rmgCarvi, for real-time single-cell monitoring of cAMP dynamics in mycobacteria. This biosensor reveals how Mycobacterium tuberculosis precisely tunes cAMP within a narrow range ("Goldilocks zone") to coordinate metabolic adaptation, and growth across complex nutrient environments. In nutrient-starved populations, we identify a bimodal cAMP distribution that functions as a phenotypic memory, dictating resuscitation kinetics upon nutrient restoration. As the first cAMP biosensor validated in any mycobacterial species, rmgCarvi facilitates the discovery of host-specific signaling dynamics during infection and provides a platform for screening of compounds that perturb cAMP homeostasis. This study provides a framework to explore how Mtb utilizes this secondary messenger molecule to survive and adapt within complex host environments.

Phage therapy offers a promising solution to the antimicrobial resistance crisis. However, a major concern preventing the adoption of phage therapy is the potential for unintended consequences of phage release; both in regard to preventing the spread of phage resistance, and the proliferation of a non-endemic virus into the microbial ecosystem. Conditional replication (biocontainment) of phages through bioengineering may address these concerns, but the impact on bactericidal efficacy is unknown. Here, we created a biocontained T7 phage (T7{Delta}capsid) lacking the major structural capsid gene, gp10AB, that can only replicate on Escherichia coli strains expressing gp10AB in trans, and assessed its bactericidal efficacy compared with wild-type T7. Congruent with model predictions, T7{Delta}capsid was only able to clear a well-mixed culture of E. coli at a multiplicity of infection (MOI) of 10 or higher, whereas wild-type T7 prohibited growth at an MOI of 0.1. The reduction in efficacy was more evident in a complex structured environment within a microfluidic device, where phage success depends on its ability to penetrate a microbial niche via propagation. In this environment, T7{Delta}capsid was unable to propagate into the bacterial population and unlike wild-type T7, had no impact on the population's growth. This study shows that whilst biocontainment of phages may improve the biosafety of phage therapy, it comes at the cost of its propagation efficacy and niche penetration in relevant environments.

Plant growth-promoting bacteria are gaining significant attention as promising biofertilizers. However, the inconsistency between in vitro plant growth-promoting traits and actual field performance remains a challenge, driven partly by a limited understanding of in situ colonization. This study characterized the colonization patterns of Citrobacter sedlakii CESi7, a novel plant growth-promoting bacterium, isolated from oil palm waste compost, during Brassica rapa cultivation. The in situ behavior of CESi7 was observed in both sterilized medium and non-sterilized soil using fluorescence in situ hybridization with a strain-targeting probe. The results revealed that CESi7 can establish both epiphytic and endophytic populations that transiently colonize roots. In a sterilized medium, CESi7 was widely distributed throughout the root tissues. Conversely, in non-sterilized soil, the bacterium formed dense aggregates specifically at the root tips. This study provides direct microscopic evidence of the colonization strategy of CESi7, offering crucial insights for its development as an effective biofertilizer.

Biological control using beneficial bacteria is a promising strategy for managing pea Ascochyta blight (AB), yet the underlying mechanisms remain poorly understood. In this study, we identified ten bacterial strains from four genera, including Bacillus, Paenibacillus, Peribacillus, and Pseudomonas, that significantly reduced the severity of AB caused by Didymella pinodes under greenhouse conditions. Most strains inhibited D. pinodes in vitro, suggesting antibiosis as a primary mode of action. To further elucidate the biocontrol mechanisms, we used Pseudomonas protegens Pf-5, which produces eight known antimicrobial compounds, as a model. While wild-type Pf-5 strongly inhibited D. pinodes in cultures and controlled AB in planta, a derivative ({Delta}8-fold mutant) lacking all eight compounds showed significantly compromised biocontrol efficacy. Individual complementation of biosynthetic genes for rhizoxin, 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, or hydrogen cyanide partially restored inhibitory activity, confirming their roles in inhibition of D. pinodes. Notably, restoring rhizoxin and DAPG biosynthesis recovered the disease control capability of the {Delta}8-fold mutant in greenhouse trials. These results demonstrate that rhizoxin and DAPG are key metabolites driving the biocontrol activity of P. protegens against D. pinodes. SIGNIFICANCEAn advanced understanding of how beneficial bacteria control plant diseases can help us better use these microorganisms in agriculture. In this study, beneficial bacteria isolated from pea roots and soils effectively mitigated damages of pea Ascochyta blight caused by the fungal pathogen Didymella pinodes. Most of the identified beneficial bacteria inhibited the fungal pathogen in cultures, indicating antimicrobial compounds were likely produced by the bacteria to control the disease. Using the soil beneficial bacterium Pseudomonas protegens Pf-5 as a model, we demonstrated that four bacteria-derived antimicrobial compounds, rhizoxin and 2,4-diacetylphloroglucinol (DAPG), pyrrolnitrin, and hydrogen cyanide play important roles in inhibiting D. pinodes growth. This study also showed that rhizoxin and DAPG produced by Pf-5 contribute to the suppression of AB development. These findings provided new insights into the molecular basis of beneficial bacteria-mediated disease suppression of pea Ascochyta blight.

Acinetobacter baumannii is a top-priority, ESKAPE pathogen that poses a major challenge to human health. The pathogen is difficult to combat due to its extensive arsenal of antibiotic resistance and its protective polysaccharide capsule. In addition, A. baumannii isolates are highly heterogeneous, which complicates the development of rapid detection methods or novel targeted therapeutic approaches. Here, we discovered and characterized a new biotechnological tool, the nanobody H7 (NbH7), along with its conserved target, the surface-exposed Omp25 protein of A. baumannii, and elucidated their interaction at the molecular level. Moreover, we demonstrate that NbH7-functionalized magnetic beads enable selective and efficient capture of A. baumannii from bacterial mixtures, including non-pathogenic intestinal bacteria. This provides proof of concept for a new targeting system that remains effective across diverse A. baumannii clinical isolates and capsule types and holds potential for use in diagnostic cell enrichment and targeted therapies.

In the plant phyllosphere, many bacteria produce pigments to mitigate oxidative stress, including pink-pigmented facultative methylotrophs (PPFMs). PPFMs are prominent members of the phyllosphere microbiota, yet their secondary metabolism remains underexplored. This limits our understanding of how these bacteria interact with other phyllosphere constituents at the molecular level. Here, we develop a screen for PPFM biosynthetic gene clusters and identify listianol, a previously undescribed metabolite that inhibits the pigmentation and growth of diverse bacteria. Listianol blocks carotenoid biosynthesis by targeting the desaturases CrtN and CrtI, revealing a previously unrecognized inhibitory mechanism for a secondary metabolite. This activity sensitizes normally pigmented bacteria to UVB radiation in vitro, and a listianol-producing strain reshapes the composition of a synthetic bacterial community on Arabidopsis thaliana under UVB exposure. Together, these findings establish a platform for exploring PPFM biosynthetic potential and uncover a new metabolite with potential to impact the phyllosphere microbiota composition.

Antibiotic tolerance enables populations of microbes to survive normally lethal antibiotic concentrations, increasing the likelihood of reinfection and facilitating the evolution of resistance. Tolerance measurements typically involve quantifying viable cells after antibiotic exposure. Existing methods range from accessible but low-throughput approaches, such as plate counting, to higher-throughput but semi-quantitative techniques, such as the TDtest. Here, we develop a new system for rapid, precise and high-throughput tolerance measurements. We utilize Surface Patterned Omniphobic Tiles (SPOTs) to discretize cell suspensions into nano-to microliter droplets and estimate the viable cell concentrations following antibiotic exposure from the proportion of empty droplets using Poissonian statistics. We apply the platform to monitor Klebsiella pneumoniae tolerance to meropenem over time as a proof of concept. The resulting assay is accessible, compatible with multiple media, and boasts a large dynamic range, sufficient resolution, and rapid handling.