Predictable expression of heterologous genes remains a key challenge in biotechnology, largely due to cellular stresses imposed on the production host. Here, we systematically dissect stress responses in E. coli MG1655 expressing diverse proteins under varying promoters and translation efficiencies. Using independent component analysis on new and existing large transcriptomic datasets, we identify distinct responses to transcriptional and translational stresses: excessive heterologous mRNA triggers a cold shock response that controls mRNA stability (cspA–I, deaD), while protein production activates a heat shock response involving proteolysis and chaperone functions. We further identify a broad adaptation response, consistently co-activated with the heat shock response during protein production, that provides stationary phase regulation (rspAB) and osmoregulation (betABIT). Targeting these latter functions through strain and media modifications significantly increases eGFP production. Other host stress responses depend on the protein being expressed; e.g. we find production of cysteine-rich proteins to uniquely activate functions regulating iron- and redox homeostasis and oxidative stress responses. This work demonstrates a holistic, systems-level view of cellular stresses to heterologous gene expression by considering transcriptional, translational, and product-specific contributions, paving the way toward predictable and optimized expression strategies.

Growing evidence indicates that soil viruses influence carbon cycling by modulating microbial metabolism and infection dynamics through auxiliary metabolic genes. Beyond host cell lysis, viral processes might regulate carbon flow and the formation of stabilized carbon pools. Integrating viral ecology into soil carbon models and restoration strategies is essential.

Protein language models (PLMs) have recently emerged as a promising approach for next-generation variant-effect prediction (VEP). Most high-performing VEP methods currently utilize PLMs combined with additional information, such as homology, protein structure and population genetics data to improve prediction accuracy. This performance gain, however, comes with added complexity or limited applicability compared to pure PLMs trained only on raw, unaligned sequences, such as evolutionary scale modeling (ESM). Here we challenge the prevailing view that sequence-only PLMs are intrinsically limited and present an efficient co-distillation approach to adapt them for high-accuracy VEP without requiring additional information beyond evolutionary signals captured during pretraining. We allow individual PLMs to self-improve by distilling the most confident predictions from multiple models of the same family and demonstrate that co-distillation of ESM models suffices to achieve state-of-the-art performance across multiple VEP benchmarks. We further show that this performance increase enables accurate quantification of the severity of variant effects on continuous clinical phenotypes in biobank data. A co-distillation framework is used to iteratively adapt sequence-only protein language models for high-accuracy variant effect prediction, without the need for additional structural or genetic data. Individual protein language models therefore self-improve by distilling the most confident predictions from multiple models, achieving state-of-the-art performance across multiple variant effect prediction benchmarks.

Insecticidal toxins from Bacillus thuringiensis (Bt) have been extensively and successfully used in genetically engineered crops for decades but continue to face challenges from the adaptive resistance in the insect population. The Bt binary toxin Vip1Ad1(Vip1) and Vip2Ag1(Vip2), a promising next-generation candidate gene combination for transgenic crops, have demonstrated high efficacy against the destructive coleopteran pest white grubs, however, their mode of action remains largely elusive. In this study, we report cryo-EM structures of the heptameric Vip1-pore and Vip2-bound Vip1-pore complex, capturing a series of putative assembly-related intermediates that suggest a binary toxin assembly and translocation pathway. Together with structure-guided mutagenesis, these data provide insights into a sequential assembly of binary complex and a sequence-independent translocation mechanism. Proof-of-principle experiments showed successful delivery of a desired protein cargo into host cells based on the mini-Vip2-Vip1 pore system, paving the way for developing much needed extracellular pesticidal protein delivery platforms. These findings not only clarify the assembly and translocation mechanism of the binary insecticidal toxin pair but also offer an excellent alternative model to investigate human-pathogenic pore-forming toxins because of its similarity and biosafety. This study reveals how the Vip1-Vip2 binary toxin assembles and enables protein translocation into target cells, providing structural insights into binary toxin action and suggesting strategies for developing next-generation bioinsecticides.

Diatoms exhibit high competitive capacity in nitrogen assimilation, but the underlying mechanisms remain unclear. Here, we identify a non-ribosomal peptide synthase-like gene (PtNRPS1) with an atypical domain structure (A-T-R1-R2) in the marine diatom Phaeodactylum tricornutum, crucial for short-term nitrogen assimilation. In vitro enzyme assays show PtNRPS1 catalyzes conversion of L-tryptophan to tryptophanol, a tryptophan-derived indole compound that promotes diatom growth at concentrations far lower than indole-3-acetic acid. Transcriptomic, metabolomic analyses, and stable-isotope analyses indicate tryptophanol enhances short-term nitrogen assimilation. CRISPR-Cas9 knockout of PtNRPS1 abolishes tryptophanol biosynthesis and reduces nitrogen-assimilation enzymes activities, which are restored by exogenous tryptophanol. PtNRPS1 overexpression results in delayed but sustained enzyme elevation. Global distribution of PtNRPS1 homologues in stramenopiles positively correlates with nitrogen-assimilation gene abundance. Our findings suggest tryptophanol, synthesized by a diatom NRPS, accelerates nitrogen assimilation, providing a competitive edge in oceanic nitrogen acquisition. . Here the authors show that marine diatoms produce tryptophanol, a molecule that at extremely low concentrations rapidly activates nitrogen assimilation genes and enzymes. This facilitates acquisition of nitrogen, a vital ocean nutrient.

In all kingdoms of life, the regulation of membrane-bound enzyme function via oligomerization is a fundamental aspect of cell physiology. Often, the mechanistic role of oligomerization is unclear, due to a lack of structure-function comparisons between constituent forms of the enzyme. Here, we elucidate the structural underpinnings of enzyme regulation and oligomerization in the quinol-dependent nitric oxide reductase (qNOR) from Neisseria meningitidis, by high-resolution structural analyses of the less active monomeric form (2.25 Å) and the highly active dimeric form (1.89 Å). The comparison revealed that broad helical flexibility near the dimer interface of the monomer causes a conformational change in a critical amino acid near the active site, located apart from the dimer interface. We demonstrate that the crosstalk between the dimer interface and catalytic site in qNOR allows enhanced activation of the enzyme via dimerization. Given Neisseria meningitidis’ dependence on qNOR to detoxify the host’s immune response of nitric oxide, our results pave a way for new strategies to combat bacterial infections, via the inactivation of qNOR by monomerization. More broadly, this provides new insights into the role of membrane protein oligomerization and its influence on regulating activity. Dimer-monomer structural and functional comparison of quinol-dependent nitric oxide reductase from Neisseria meningitidis reveals insights into the interlinking role of dimer stability and high catalytic activity.

RNA molecules display remarkable heterogeneity in structure, dynamics, and function, yet methods for their precise visualization in living cells remain limited. While CRISPR-based RNA imaging holds great potential, existing systems often suffer from high background fluorescence due to constitutive signal emission or non-specific binding. To overcome these challenges, we developed CtDeg (CRISPR–dCas13–tDeg), a modular RNA imaging platform that links fluorescence activation directly to target RNA recognition while leveraging degron-mediated degradation to suppress background signals. By engineering the crRNA scaffold to embed the Pepper RNA motif, CtDeg ensures that fluorescence is present only upon binding to the native RNA target. We systematically optimized C-terminal tDeg variants to maximize the signal-to-noise ratio and demonstrated that CtDeg achieves substantially lower background and higher specificity than conventional fluorescent protein–CRISPR-based RNA imaging approaches. Using CtDeg, we captured real-time paraspeckle assembly dynamics and visualized early-stage SARS-CoV-2 genomic RNA transport. Remarkably, CtDeg provided the first direct imaging evidence of virus-induced NEAT1_2 lncRNA accumulation, revealing a host-virus regulatory interaction. Beyond these applications, CtDeg is compatible with multiple Cas13 orthologs and fluorescent proteins, establishing a versatile, target-induced platform for probing RNA localization, dynamics, and function in living cells, with broad applications in synthetic biology and RNA biology.

Homarine (N-methylpicolinic acid) is a ubiquitous marine metabolite produced by phytoplankton and noted for its infochemical properties among marine animals, yet its microbial degradation pathways are uncharacterized. Here we identify a conserved operon (homABCDER) that mediates homarine catabolism in bacteria using comparative transcriptomics, mutagenesis and targeted knockouts. Phylogenetic and genomic analyses show that this operon is distributed across abundant bacterial clades, including coastal copiotrophs (for example, Rhodobacterales) and open-ocean oligotrophs (for example, SAR11, SAR116), and in genomes from non-marine environments. High-resolution mass spectrometry revealed N-methylglutamic acid and glutamic acid as key metabolic products of homarine in both model and natural systems, with N-methyl-glutamate dehydrogenase catalysing their conversion. Metatranscriptomics showed responsive and in situ expression of hom genes aligned with homarine availability. These findings uncover the genetic and metabolic basis of homarine degradation, establish its ecological relevance and highlight homarine as a versatile growth substrate that feeds into central metabolism via glutamic acid in diverse marine bacteria. Homarine is a ubiquitous, phytoplankton-derived metabolite that is broken down by widely distributed and diverse marine bacteria containing a conserved homABCDER operon.

Coral reefs are increasingly threatened by marine heatwaves, prompting the need for proactive interventions that enhance coral thermal tolerance. Assisted evolution, which aims to accelerate natural adaptation rates, has emerged as a promising approach. However, programmes of assisted evolution must outpace the escalating frequency and intensity of marine heatwaves. Here, we present a Roadmap for accelerating progress towards using assisted evolution to enhance coral thermal tolerance. We highlight advances in coral biology across cellular, organismal, and ecological scales that support the feasibility of assisted evolution in coral populations. We compare current experimental gains in thermal tolerance via assisted evolution with projected temperatures, finding that these are unlikely to keep pace with predicted climate change. We identify key knowledge gaps that hinder timely development of assisted evolution and propose a comprehensive research agenda to address these gaps. This agenda will be catalysed by large-scale, multi-institutional field hubs increasing experimental scope and statistical power, support for long-term research at these hubs, spanning coral generations, and development and application of methodologies that safeguard broodstock and experimental corals from disturbances. By implementing these proposals, scientists can realize the potential of assisted evolution and help to safeguard a future for coral reefs. Ongoing and projected climate changes are bringing increased marine heatwave frequency and intensity, threatening the health and survival of coral reefs. This Roadmap outlines the potential for assisted evolution methods to increase thermal tolerance in corals and describes ways to accelerate research and development for enhancing coral adaptation rates.

Bacteriophages are key drivers of microbial ecology, co-existing and co-evolving with bacteria across diverse environments. Limitations in culturing, alongside advances in sequencing and bioinformatics, have driven the use of metagenomics to explore viral diversity. Viral-specific analysis of >3000 food metagenomes from cFMD produced the FVGC, comprising ~3400 metagenome-assembled viruses, most of which belong to novel Caudoviricetes lineages (n = 91), with only ~15% represented in IMG/VR v4. Together, these findings reveal extensive uncharacterized viral diversity in food systems. Beyond serving as a reference, the FVGC facilitates detailed investigation of virus–host interactions. Viral sequences were pervasive across microbial genomes, with several bacterial families exhibiting near-universal associations with viral elements. Bacterial antiviral defence systems were abundant and taxonomically diverse, dominated by restriction–modification systems, while CRISPR–Cas systems showed pronounced lineage-specific distributions; in contrast, viral anti-defence genes were detected at low frequency (<10% of MAVs). Host prediction linked MAVs to clinically relevant taxa, including expanded ESKAPE pathogens such as Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, and Enterobacter spp., highlighting the ecological connectivity between food-associated viruses and clinically important bacteria. Antimicrobial resistance signals were scarce, suggesting minimal phage-mediated AMR dissemination in food environments. This new publicly available viral database represents a valuable resource for further exploration of viral diversity.

Microplastics (MPs) in wastewater treatment plants (WWTPs) represent novel ecological niches rather than passive contaminants. They rapidly acquire distinct microbial biofilms, forming the “Plastisphere”. This Perspective examines the underexplored role of quorum sensing (QS) within these complex synthetic microenvironments. We highlight how the intrinsic properties of MPs, such as surface hydrophobicity, chemical leachates, and aging states, create altered signaling landscapes. These distinct environments may decouple QS responses from classical population-density thresholds. Consequently, these dynamic interactions potentially enhance cooperative microbial metabolism and may indirectly facilitate the horizontal gene transfer of antibiotic resistance genes by fostering dense cellular proximity. Despite substantial methodological challenges in detecting active signaling in situ, MPs must be reconceptualized as quorum-modulating microscopes capable of reprogramming microbial communication. Advancing this field requires integrating high-resolution spatial imaging and functional genomics under operationally relevant conditions to bridge theoretical insights with empirical validation, ultimately informing future treatment designs and environmental risk assessments. Microplastics intensify quorum-sensing-mediated gene exchange in wastewater treatment plants, enabling their reconceptualization as quorum-modulating microscopes that reprogram microbial communication and function, based on their interplay with biofilms and quorum sensing within wastewater systems