Biology subjects

Sadler, N.

Publications and source records attributed to Sadler, N..

6 recordsLinked to original sources

bioRxiv2026

Five Rotational Registers Define Modular Portal-Capsid Assembly in Cyanophage P-SSP7

The virus P-SSP7 infects the cyanobacterium Prochlorococcus marinus, one of the most abundant photosynthetic microbes in the ocean, making this pairing a useful model for studying host-virus interactions. Infection proceeds through a portal-tail complex that attaches to the viral protein shell at a single specialized vertex, where the two structures have mismatched symmetries. Previous studies described this region only at coarse resolution which continues to limit understanding of how the virus assembles and injects its genome. Using cryo-electron microscopy of high-quality samples, we determined the first near-atomic-resolution structure of the complete P-SSP7 virion and its portal-tail complex. We captured 3D classes where the shell and portal-tail complex connect in five distinct arrangements. Fitting and modeling 2 of these states helped resolve and establish modular attachment in five rotational registers as the basis for capsid-portal symmetry mismatch in P-SSP7 assembly.

microbiology↗

bioRxiv2026

Extreme genome reduction selectively retains modular regulatory architecture in Prochlorococcus MED4: conserved transcriptional modules reveal core physiological regulatory programs

Prochlorococcus MED4 is a minimal photoautotroph whose extreme genome streamlining extends to its transcriptional regulatory architecture, yet it dominates high-light oligotrophic surface waters and drives marine carbon cycling. Despite ecological significance, MED4 remains genetically intractable, lacking molecular tools to characterize regulatory mechanisms and construct a transcriptome-wide regulatory map. To address this, we assembled an RNA-seq compendium of 253 samples, including 207 new samples capturing transcriptional responses across three classes of experiments: diverse environmental perturbations, a 24-hour circadian cycle, and phage infection. Using independent component analysis (ICA) applied to 247 quality-filtered samples, we identify 32 independently regulated gene set modules in MED4 (iModulons). By comparison, we previously identified 78 iModulons in the model cyanobacterium Synechococcus elongatus PCC 7942, revealing how dramatically genome reduction has simplified MED4s regulatory architecture. Of the 32 iModulons in MED4, 13 are conserved modules that correlate with experimentally validated transcriptional regulons in PCC 7942, identifying regulatory programs that resisted elimination under extreme selective pressure. These conserved modules reveal regulatory programs governing photosynthesis and light responses (RpaB), circadian rhythms (RpaA), and nutrient assimilation (NtcA, PhoB). Known regulator-specific DNA-binding motifs upstream of genes in conserved modules independently support their identification as regulatory targets. Notably, RpaA governs circadian rhythms through three temporally distinct modules in MED4 versus one in PCC 7942, and the RpaB photoprotection module similarly splits into two. This work uncovers the minimal regulatory core governing photosynthesis, circadian rhythms, and C/N/P metabolism in a globally critical but genetically intractable photoautotroph. This approach offers a generalizable framework for regulatory inference beyond model organisms. ImportanceThe minimal photoautotroph Prochlorococcus MED4 possesses only 28 transcriptional regulators, versus 150+ found in most cyanobacteria, reflecting a genome streamlined by billions of years of natural selection. This streamlining minimized metabolic costs, enabling MED4 to dominate nutrient-depleted oceans today. This raises a fundamental question: which regulatory programs did nature choose to keep? The answer matters, but MED4 cannot be studied by conventional genetics, placing its molecular machinery beyond direct experimental reach. Instead, we use large-scale computational methods to define groups of co-regulated genes in MED4. By comparing MED4 with a genetically tractable model cyanobacterium, we can distinguish which regulatory programs nature preserved from those it discarded. This work reveals the minimal regulatory architecture sufficient to sustain the smallest oxygenic photoautotroph on Earth. The principles uncovered here, distinguishing essential from dispensable regulatory programs in a naturally streamlined organism, inform the design of minimal photosynthetic platforms for biotechnology.

systems biology↗

bioRxiv2026

Spatiotemporal 4D Whole-cell Modeling of a Minimal Autotroph Reveals Central Carbon Metabolism Regulated Locally by Protein Megacomplexes via Post-translational Modifications under Light Disturbance

Photosynthetic microorganisms rely on multiple pathways in central carbon metabolism to adapt to fluctuating light and energy availability across diel cycles. Mechanistic insight into the regulatory dynamics of this adaptation requires integrating processes spanning disparate timescales, from rapid redox-dependent post-translational modifications (PTMs) to slower changes in protein expression and metabolic pathway usage. To address this complexity beyond genome-based inference and traditional modeling, we develop a whole-cell four-dimensional (3D + time) model of the marine cyanobacterium Prochlorococcus marinus MED4 that explicitly represents the spatial organization of enzymatic and molecular processes in central carbon metabolism under light perturbation. We employ a perturbation-based research design to experimentally generate time-series, multi-omics measurements that provide molecular descriptors and cryo-ET images as constraints for this dynamic 4D framework. The integration of experiments and modeling across defined light regimes enables quantitative validation of system-level responses and forecasting under distinct light disturbances. We test the hypothesis that light-dependent redox PTMs regulating the structural assembly of a protein megacomplex, the "dark complex," modulate metabolic flux at a conserved regulatory node of the Calvin-Benson cycle (CBC) in cyanobacteria. Our model shows that subcellular spatial organization buffers rapid light-induced changes in thylakoid reaction rates, which are followed by redox-PTM-mediated sequestration or release of CBC enzymes in the dark complex, ultimately impacting carbon fixation dynamics within carboxysomes. Comparison with an equivalently parameterized well-mixed stochastic model demonstrates that post-translational regulation not only buffers transcriptional noise and diffusion-driven fluctuations but also stabilizes phenotypic outcomes, underscoring the importance of spatial heterogeneity in phenotypic robustness. This ability to probe adaptive, spatiotemporally resolved mechanisms in photosynthetic machinery and central carbon metabolism addresses a critical gap in genotype-to-phenotype inference and expands modeling and design capabilities for understudied or genetically intractable autotrophs such as P. marinus MED4. Significance StatementThis work advances 4D whole-cell modeling by presenting the first spatiotemporal simulation of a photosynthetic autotroph using the Lattice Microbes platform. Using Prochlorococcus marinus MED4, we show that subcellular spatial organization of organelles, diffusion constraints, and redox regulation collectively shape central carbon metabolism across orders of magnitude in space and time. Through a perturbation-based strategy that generates multi-omics data sets over time, we construct and validate a spatially and temporally resolved model of MED4, constrained by high-resolution (10 nm) cryo-electron tomography. Our results highlight the importance of localized biochemical reactions and redox-dependent post-translational modification of enzymes in regulating carbon fixation in a noisy environment under light disturbance. This study establishes a spatiotemporal, whole-cell physiology modeling framework as a transformative tool for uncovering multiscale regulatory responses to environmental gradients.

systems biology↗

bioRxiv2026

Systematic Scale-Up and Enhanced Purification of Marine Cyanophage P-SSP7

Cyanophages represent important models for understanding virus-host interactions, yet high-resolution structural studies remain relatively few due to challenges with preparing enough sample of sufficient quality for cryo-EM and functional multi-omics studies. Here we developed an integrated methodology for scaling production of the model cyanophage P-SSP7 from laboratory maintenance volumes (5-100 mL) to production scales (up to 40 L) while dramatically improving the quality of phage preparation for structural applications. Our systematic approach integrates host cultivation using adaptation to local seawater to reduce production costs, optimized infection protocols to maximize infectious titer yields, and multi-stage purification workflows specifically designed for cryo-EM quality requirements. The final methodology consistently produces infectious phage titers exceeding 3x1012 units/mL with recoverable yields of 1013 total infectious units and >95% purity validated by cryo-electron microscopy at each optimization step. Most significantly, this approach achieves a 60-fold reduction in cryo-EM data collection time by increasing usable particles per field of view for single particle analysis. Overall, our final preparations demonstrate robust phage stability, retaining 68% infectivity after 3 months and 23% after 6 months at 4{degrees}C. This workflow moves cyanophage culturing and downstream structural studies from specialized, resource-intensive endeavors toward routine research capability and establishes an adaptable framework for scaling production that can be applied to other host-virus systems.

microbiology↗

bioRxiv2025

Modular cyanobacterial regulatory architecture enables dynamic graded responses to oxidative stress

A fundamental paradox of oxygenic photosynthesis: growth-essential energy machinery generates reactive oxygen species (ROS) threatening survival, yet the systems-level regulatory networks balancing the growth-survival trade-off remain unclear. Through integrative experimental-computational analysis combining steady-state transcriptomics with independent component analysis across 0-78.4% oxygen, we decoded the regulatory architecture driving progressive transitions in Synechococcus elongatus PCC 7942 from ROS sensing through defense to growth shutdown. Integration of 407 transcriptome samples identified 78 regulatory modules (iModulons) explaining 72.3% of expression variance and revealed calibrated responses: low stress triggers metalloregulators (SufR, PerR) for ROS sensing and primary antioxidants; moderate stress activates RpaB operating through four distinct regulatory states redirecting metabolism to defense; severe stress induces growth arrest via stringent response pathway convergence. This quantitative regulatory framework enables precise growth-defense calibration through modular network architecture: RpaB coordinates genome-wide resource reallocation (RpaB[~]P growth-promoting, RpaB ROS defense-activating, RpaABC circadian-integrating, RpaB ycf46 checkpoint activation), offering systematic strategies for engineering stress-tolerant bioplatforms and predictive models for environmental stress responses.

systems biology↗

bioRxiv2024

Environmental matrix and moisture are key determinants of microbial phenotypes expressed in a reduced complexity soil-analog

Soil moisture and porosity regulate microbial metabolism by influencing factors such as redox conditions, substrate availability, and soil connectivity. However, the inherent biological, chemical, and physical heterogeneity of soil complicates laboratory investigations into microbial phenotypes that mediate community metabolism. This difficulty arises from challenges in accurately representing the soil environment and in establishing a tractable microbial community that limits confounding variables. To address these challenges in our investigation of community metabolism, we use a reduced-complexity microbial consortium grown in a soil analog using a glass-bead matrix amended with chitin. Long-read and short-read metagenomes, metatranscriptomes, metaproteomes, and metabolomes were analyzed to test the effects of soil structure and moisture on chitin degradation. Our soil structure analog system greatly altered microbial expression profiles compared to the liquid-only incubations, emphasizing the importance of incorporating environmental parameters, like pores and surfaces, for understanding microbial phenotypes relevant to soil ecosystems. These changes were mainly driven by differences in overall expression of chitin-degrading Streptomyces species and stress-tolerant Ensifer. Our findings suggest that the success of Ensifer in a structured environment is likely related to its ability to repurpose carbon via the glyoxylate shunt while potentially using polyhydroxyalkanoate granules as a C source. We also identified traits like motility, stress resistance, and biofilm formation that underlie the degradation of chitin across our treatments and inform how they may ultimately alter carbon use efficiency. Together our results demonstrate that community functions like decomposition are sensitive to environmental conditions and more complex than the multi-enzyme pathways involved in depolymerization. ImportanceSoil moisture and porosity are critical mediators of microbial metabolism by influencing factors such as redox conditions, substrate availability, and soil connectivity. However, identifying how microbial community metabolism shifts in response to varying levels of moisture and porosity remains a challenging frontier. This difficulty arises from challenges in accurately representing the soil environment and in establishing tractable microbial communities that limit confounding variables. Moreover, inferring phenotypes based on "key" genes often fails to predict complex phenotypes that arise from cellular interactions. Here, we establish a tractably complex microbial community in a soil analog system amended with chitin and leverage it to understand how microorganisms respond to changes in porosity and moisture. By using genome-resolved metagenomics, metatranscriptomics, and metaproteomics, we report on the microbial lifestyle strategies that underpin changes in community expression like carbon conservation, biofilm production, and stress response.

microbiology↗