Pathogens must express virulence programs while competing for limited resources and adapting to nutrient shifts. Many virulence loci exhibit atypical codon usage, but the functional consequences of this bias remain unclear. Here, we mapped unexpected rich landscapes of diverse, functionally distinct codon usage patterns across the genomes of diverse bacterial species. In Salmonella, virulence genes formed a unique signature enriched for rare tRNAs and favoring wobble decoding. This alleviated competition with highly expressed genes, at the cost of increased dependence on specific tRNA modifications for efficient and accurate decoding. In a Salmonella systemic mouse infection model, loss of i6A37 and mnm5s2U34 modifications (miaA and mnmEG mutants) abolished virulence and selectively suppressed virulence proteins. Crucially, using recoded fluorescent proteins, we showed that virulence codon bias, by avoiding codons with high turnover, sustained increased translation rates during amino-acid starvation. Together, these findings indicate that virulence codon bias is conserved across pathogens, ensures robust expression during starvation and couples tRNA modifications to pathogenic functions, highlighting tRNA modification pathways as potential targets for broad anti-virulence strategies.

To protect themselves against phage infection, bacteria employ diverse defense systems that are typically activated specifically upon infection. However, the mechanisms of activation and self versus non-self discrimination for most systems remain poorly understood. Here, we show that the bacterial immunity protein DARNA, once activated, cleaves a subset of host tRNAs, thereby inhibiting phage propagation. Although phages escape DARNA-mediated defense through mutations in the gene encoding single-stranded DNA-binding protein (SSB), we find that phage SSBs do not directly stimulate DARNA. Instead, DARNA is activated by single-stranded DNA presented by phage SSB, but not by the host SSB. The recognition of an endogenous nucleic acid signal promoted by a viral protein ensures that DARNA can detect and respond to a broad range of viruses while avoiding auto-immunity.

Trends in clean label food and the growing popularity of alcohol-free beers have led to a demand for natural compounds to prevent microbial spoilage. Oxidative coupling products of hydroxycinnamic acids and their amides, i.e. phenolamides, obtained via chemo-enzymatic synthesis and from barley rootlets, a beer brewing byproduct, were tested for their inhibitory activity against the beer spoilage yeast Saccharomyces cerevisiae subsp. diastaticus. Hydroxycinnamic acids, their dimers, and hydroxycinnamoylagmatines (HCAgms) did not inhibit the yeast. However, inhibitory activity was observed with HCAgm dimers. In particular, CouAgm-4-O-7′/3-8′-DCouAgm and FerAgm-4-O-7′/3-8′-DFerAgm were active at 46–255 μg/mL in beer. Notably, the newly synthesized agmatine amide of poacic acid, a stilbenoid dimer of ferulic acid, showed increased antiyeast activity compared to its monomeric precursors. These results demonstrate that amidation and oxidative coupling can increase the antiyeast activity of hydroxycinnamic acid derivatives, and that hydroxycinnamoylagmatine dimers possess potential as preservatives against wild yeast spoilage in beer.

Anti-CRISPRs (Acrs) are diverse proteins or RNAs that protect invading phages and plasmids from host CRISPR-Cas immunity. Most Acrs neutralize their cognate Cas proteins via direct physical interaction. Here we describe CasPRs, a particularly widespread family of DNA-binding Acrs that recognize specific sequence motifs within cas gene coding regions, thereby blocking RNA polymerase and silencing transcription. We demonstrate that eight diverse CasPRs bind to the cas8b gene to repress the type I-B CRISPR-Cas system in its native host, Listeria seeligeri. Meanwhile, a CasPR from Streptococcus dysgalactiae silences type II-A CRISPR-Cas immunity by binding to the cas9 coding sequence. We found that one CasPR is required to inhibit CRISPR immunity during lysogeny by its host prophage. Taken together, our results indicate that members of the CasPR family have diverged to silence completely unrelated CRISPR types, and suggest transcriptional repression is a common mode of phage-mediated immune antagonism.

The global rise of antimicrobial resistance has intensified the search for new microbial metabolites from underexplored environments and taxonomic groups. Extreme and geographically isolated habitats, such as Antarctic terrestrial ecosystems, represent promising reservoirs of novel biosynthetic diversity, particularly among rare and difficult-to-cultivate actinomycetes, where chemical mediators are thought to play key roles in microbial persistence and interaction under resource-limited conditions. Here, we report the characterization of kineochelins, a previously undescribed group of siderophores produced by an Antarctic isolate, Actinokineospora sp. UV203 representing difficult to cultivate actinomycetes. Structural elucidation revealed a set of closely related congener molecules with a mixed-ligand architecture consistent with metal-chelating activity. Genome mining combined with transcriptomic analysis identified the involvement of a dedicated nonribosomal peptide synthetase-encoding biosynthetic gene cluster responsible for kineochelin production. Comparative genomic analyses showed that, although kineochelin biosynthetic genes share limited homology with those of known mixed-ligand siderophores, their biosynthetic pathways differ substantially in gene content and organization, indicating a distinct evolutionary lineage. Functional characterization of kineochelins demonstrated strong and selective iron chelation, with pronounced affinity for ferric and ferrous iron. Crude culture extracts inhibited the growth of bacterial strains isolated from the same Antarctic environment, suggesting that kineochelin-associated chemistry contributes to iron-mediated competitive interactions within native microbial communities. In addition, kineochelin-enriched fractions exhibited selective inhibitory activity against the opportunistic yeast pathogen Nakaseomyces glabratus and a clinical isolate of Saccharomyces cerevisiae associated with invasive infection. Together, these findings expand the known chemical and biosynthetic diversity of the genus Actinokineospora and demonstrate that Antarctic rare actinomycetes are a valuable source of novel natural products with potential relevance for microbial ecology and biotechnology. The ecological activities of kineochelins highlight the role of iron acquisition in shaping microbial interactions in extreme environments and underscore the biotechnological potential of metabolites derived from underexplored polar microorganisms.

Efficient and scalable isolation of specific cell populations remains a central bottleneck for genome engineering, pooled screening, and cell therapy manufacturing. Here, we present DASIT (Destabilized-nanobody Antigen Selection and Identification Tool), a protein-based circuit for antigen-specific cell selection. DASIT uses a destabilized nanobody fused to an antibiotic resistance protein. In cells expressing the target antigen, binding of the nanobody fusion to the cognate antigen stabilizes DASIT, thereby coupling the presence of an antigen to a selectable signal. We developed DASIT circuits that enable robust selection of antigen-expressing cells and show that they can be designed to target distinct antigen classes and perform across cell types. Because DASIT operates at the protein level, it supports both stable integration and transient delivery, enabling recyclable selection without permanent genomic integration of resistance markers. We demonstrate scalable, FACS-free enrichment in three challenging applications: multiplexed, logic-gated integration of landing pads in human iPSCs, high-throughput CRISPR screening, and phenotypic selection of in vitro-derived neurons at transplantation scale. By decoupling selection from vector integration, DASIT establishes an automation-compatible architecture for multistep genome engineering, high-throughput library screening and large-scale cell manufacturing.

Directed evolution enables the rapid creation of biomolecules with new functions, yet linking genotype and phenotype often requires laborious cell-based workflows. Here we integrate a boosted in vitro transcription-translation system capable of protein expression from single-copy DNA with ultrahigh-throughput fluorescence-activated droplet sorting, developing a simple one-day workflow for efficient protein evolution. Using this system, we evolved SP6 RNA polymerase into a highly robust variant that functions in multiple environments, including cell-free reactions and mammalian cells. We further engineered the evolved enzyme into a proximity-dependent split RNA polymerase that converts molecular interactions into transcriptional activity with minimal background. The resulting biosensors detect diverse molecular targets in vitro, including proteins, peptides, RNAs, and molecular glues. This cell-free platform provides versatile routes for evolving diverse functional biomolecules, accelerating the development of advanced biotechnologies such as diagnostics and therapeutics.

The origin of functional proteins remains a fundamental biological enigma. Although Anfinsen's dogma established sequence as the determinant of structure, and deep learning models can predict structures with high fidelity, the physical principles governing protein genesis itself, from prebiotic condensation to functional protein emergence, remain unresolved. This gap leaves a critical disconnect between mechanistic biological insights and artificial intelligence. Herein, we introduce a unified methodological framework ProtGenesis that recasts genesis of protein as a structured, deterministic navigation within a discrete structural space. We identify three universal principles governing this hierarchical organization: the Assembly Principle directs amino acids condensation into multilayer fractal-like architectures; the Emergence Principle ensures nascent peptides' emergence follow deterministic spatial trajectories; and the Phase-Transition Principle describes wherein incremental residue accrual or mutations drives precise topological phase shifts from short-range to long-range order. By quantifying these trajectories with novel tripartite spatial metrics, we reveal that protein genesis is not an abstract continuum but a principle-governed physical process with measurable coordinates. ProtGenesis thus provides an universal interpretable mathematical foundation for decoding "black-box" of deep learning models and establishes a rigorous basis for exploring, understanding, and engineering the molecular blueprint of life.

Multi-drug resistant bacteria (MDR) pose a major public health challenge. Their ability to exchange resistance genes through Horizontal Gene Transfer (HGT) promotes the appearance of resistant strains, limiting antibiotic treatments for infections caused by these MDR bacteria. Among alternative approaches, phage therapy stands out as a promising strategy that utilizes bacteriophages to specifically target and effectively eliminate bacteria. This narrative review provides an overview of the current knowledge on the use of whole bacteriophages as antimicrobial agents in human and veterinary medicine, as well as in the food industry whether used alone, in cocktails, or combined with antimicrobials. While whole phages offer high specificity and an efficient elimination of bacteria, their application is associated with several limitations, including their contribution to HGT, the emergence of bacterial resistance, their narrow host range, the immune recognition, and the difficulties posed by their regulation. To address these challenges, this review focuses on phage-derived enzymatically active proteins, such as endolysins and depolymerases, as alternative antimicrobial tools, used alone or in combination. These phage components, being smaller and structurally simpler than whole phages, behave more similarly to conventional antimicrobial compounds. They have so far presented a low risk of bacterial resistance appearance and less chance of immune response. In addition, their classification as antimicrobial enzymes or conventional biologics could facilitate regulatory approval by aligning with existing regulatory frameworks. A total of 40 studies were included in this narrative review, highlighting the outcomes of applications involving whole bacteriophages (n = 11) and phage-derived enzymes, including endolysins and depolymerases (n = 27).