Core promoters comprise multiple elements whose interaction with RNA polymerase initiates transcription. Despite decades of research, substantial sequence and length variation of promoter elements has hindered efforts to elucidate their function and the evolutionary diversity of transcriptional regulation. Combining massively parallel assays, biophysical modeling, and functional validation, we systematically dissected the promoter architecture upstream of experimentally determined transcription start sites in 49 phylogenetically diverse bacterial genomes (GC content: 27.8%–72.1%). We identified a conserved 3-bp promoter element, termed “start,” which dictates transcription start site selection and enhances transcription. We uncovered a four-region organization within the variable spacer element, whose sequence composition modulates transcription by up to 600-fold. We showed that the discriminator element is conserved in Terrabacteria but diverse in Gracilicutes, the two major bacterial clades. High discriminator sequence diversity in Gracilicutes likely reflects diversifying evolution, enabling promoter-encoded regulation to orchestrate global gene expression in response to growth rate changes. Together, our findings reveal broad conservation of bacterial promoter organization while highlighting regulatory divergence of promoter elements and RNA polymerase between Terrabacteria and Gracilicutes. Sequence and functional similarities between bacterial promoter elements and their archaeal and eukaryotic counterparts further suggest a shared evolutionary origin of promoter architecture.

Bacterial flagella are known for facilitating motility to support nutrient acquisition and predator evasion, but can also serve as receptors for phages. Here, we characterize a dual role of flagella during infection of Yersinia enterocolitica by phage X1: functioning as a conduit for phage DNA entry and as a trigger for bacterial defense. X1 employs a “contraction-driven” mechanism, using its contractile tail to inject DNA into Yersinia flagella. Unlike other characterized flagellotropic phages that rely exclusively on flagella or less efficiently on secondary surface receptors, X1 can infect cells via lipopolysaccharides with even higher efficiency. Furthermore, certain Yersinia strains can detect X1 invasion and activate a flagellum-dependent toxin–antitoxin (Flag-TA) system, which requires the flagellar motor stator proteins MotAB to activate the abortive infection response. Our findings reveal a novel phage infection strategy and uncover a new bacterial defense that are both based on the flagellar machinery.

ArsR-based whole-cell biosensors offer sensitive colorimetric detection of arsenite [As(III)], yet their broad reactivity toward Group 15 metalloids—especially antimonite [Sb(III)]—limits field specificity. The recently identified ant operon from Comamonas testosteroni JL40 confers Sb(III)-selective resistance via the efflux ATPase AntA, the metallochaperone AntC, and the regulator AntR, providing genetic parts to suppress Sb(III) cross-talk. We systematically introduced antA, antC, and three antR homologs into an ArsR regulator coupled with a deoxyviolacein reporter chassis (pJ23119-K12). Co-expression of AntA and AntC under a moderate constitutive promoter (PceuR) shifted the Sb(III) limit of detection (LOD) from 0.073 µM to 0.586 µM, with a modest increase in the As(III) LOD to 0.018 µM. Subsequent integration of AntR1 not only maintained the As(III) LOD at 0.018 µM but also unexpectedly amplified the As(III) signal, extending the linear range to 36 nM–37.5 µM (R² = 0.991). It suggests that AntR1 may modulate the transcriptional circuitry via cross-regulation, warranting further mechanistic inquiry. The modified biosensor TOP10/pJ23119-antACR1 exhibited high selectivity for As(III) over divalent metals (Cd, Pb, Cu, Hg, Mn, Mg) and tolerated Sb(III) up to 1 µM. Performance was retained in 90% freshwater and 50% seawater matrices, enabling accurate quantification of 0–2.5 µM As(III) in deionized, tap, surface, and marine samples. By coupling Sb(III)-specific ant efflux/sequestration components with an ArsR-based sensing module, we developed a portable, low-cost biosensor that overcomes longstanding As(III)/Sb(III) cross-reactivity and performs robustly in complex environmental waters.

Nonhomologous end-joining repairs chromosomal double strand breaks, but it is unknown whether both strands are repaired by this pathway, and if one strand break’s repair path impacts the other. Here, we show that nonhomologous end-joining employs both of two a priori possible strategies. Strand breaks that can be directly ligated are joined near-simultaneously, with no effect of one strand break’s repair path on the other. More complex end structures require obligatorily ordered repair. The first strand to be repaired is used as template for repair of the opposite/second strand break, with the latter repair reaction occurring fastest when also coupled to nonhomologous end-joining. Enforced asymmetry in repair of each strand break can extend to the gap-filling polymerase employed, and whether the polymerases incorporate RNA or DNA. Our results resolve questions about pathway mechanism and identify a requirement for flexibility of the nonhomologous end-joining machinery for efficient repair of both strand breaks within diverse cellular double strand breaks. Breakage of both chromosomal DNA strands creates unique problems for DNA repair. Here, Luthman et al. show that for some broken ends, the two strand breaks are repaired in parallel, while for other ends one strand must be repaired before the other.

Advances in long-read sequencing (LRS) and assembly algorithms have made it possible to create highly complete genome assemblies for humans, animals and plants. However, ongoing development is needed to improve accessibility, affordability, and assembly quality and completeness. ‘Cornetto’ is a new strategy in which we use programmable selective nanopore sequencing to focus LRS data production onto the unsolved regions of a nascent assembly. This improves assembly quality and streamlines the process, both for humans and non-human vertebrates. Cornetto enables us to generate highly complete diploid human genome assemblies using only nanopore LRS data, surpassing the quality of previous efforts at a fraction of the cost. Cornetto enables genome assembly from challenging sample types like human saliva. Finally, we obtain accurate assemblies for clinically-relevant repetitive loci at the extremes of the genome, demonstrating valid approaches for genetic diagnosis in facioscapulohumeral muscular dystrophy (FSHD) and MUC1-autosomal dominant tubulointerstitial kidney disease (MUC1-ADTKD). Long-read sequencing enables high-quality genome assemblies, but challenges remain. Here, the authors introduce Cornetto, a method that improves assembly quality, enables genome sequencing from saliva, and accurately resolves medically-relevant repetitive genes.

CRISPR activation is a powerful tool to upregulate a vast array of genes in many different contexts. However, there are few dynamic CRISPR transcriptional programs, which limit its usage in the creation of living biosensors, self-regulating microbial factories, or conditional therapeutics. Here, we address this limitation by embedding a molecular switch directly into a guide RNA to create a combined sensor–actuator called a metabolite-responsive scaffold RNA (MR-scRNA). We demonstrate the regulatory potential for MR-scRNAs by conditionally activating genes in three different kingdoms of life. We create MR-scRNAs responsive to two distinct metabolites, theophylline and tryptophan, by swapping the molecular switch used. MR-scRNAs respond quickly in a dose-dependent manner specifically to their target metabolite and enhance biochemical production when used as a dynamic regulator of pathway enzyme expression. The broad functionality and ease of design of the MR-scRNAs offer a promising tool for dynamic cellular regulation.

Advances in CRISPR technologies have led to the use of diverse CRISPR-associated (Cas) proteins in diagnostic applications. Herein, we present a CRISPR/Cas12a2-based amplification-free RNA detection method that exhibits sub-attomolar sensitivity and substrate versatility. Cas12a2, a recently characterized RNA-guided nuclease, uniquely integrates bimolecular recognition through CRISPR RNA (crRNA)-target complementarity and protospacer flanking sequence identification, enabling highly specific trans-cleavage of single-stranded DNA, double-stranded DNA, and RNA. We have optimized key biochemical parameters, including pH, ionic strength, and temperature, to enhance the catalytic efficiency of Cas12a2. Based on the optimal activity conditions of Cas12a2, we have achieved ultra-sensitive viral RNA detection with a limit of detection of 46.7 aM through the strategic design and cooperative activation of crRNAs targeting conserved regions of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome. The diagnostic accuracy of the Cas12a2-based assay has been demonstrated for 26 SARS-CoV-2 variants, and it has further resulted in the definitive diagnosis of 317 clinical samples. This work establishes Cas12a2 as a promising molecular diagnostic tool that provides an amplification-free, rapid, and versatile solution for RNA detection. The adaptability and simplicity of the platform render it particularly well suited for point-of-care applications, paving the way for next-generation CRISPR diagnostics.

Porins mediate the passage of hydrophilic nutrients and antibiotics across the outer membrane but might contribute to proton leak from the periplasm, suggesting that their conductance could be regulated. Here we show, using single-cell imaging, that porin permeability in Escherichia coli is controlled by changes in periplasmic H+ and K+ concentration. Conductance through porins increases with low periplasmic H+ caused by starvation, promoting nutrient uptake, and decreases with periplasmic acidification during growth in lipid media, limiting proton loss. High metabolic activity during growth in glucose media, however, activates the inner membrane voltage-gated potassium channel, Kch, increasing periplasmic potassium and enhancing porin permeability to dissipate reactive oxygen species. This metabolic control of porin permeability explains the observed increase in ciprofloxacin resistance of bacteria catabolizing lipids and clarifies the impact of mutations in central metabolism genes on drug resistance, identifying Kch as a therapeutic target to improve bacterial killing by antibiotics. The permeability of bacterial porins is dynamically regulated by periplasmic proton and potassium concentrations, altering antibiotic resistance.

Efficient cargo delivery is essential for plant trait engineering, yet existing methods are often species-specific and ineffective across diverse habitats. Here, we develop core-shell microneedles for targeted delivery of biomolecular cargoes and active microorganisms into both terrestrial and aquatic plants. The microneedle architecture is rationally engineered to resist water exposure and release cargo upon contact with plant interstitial fluid, enabling controlled delivery into tissues and cells. We demonstrate that these core-shell microneedles can efficiently transport diverse cargoes, from nanoscale biomolecules such as functional nucleic acids, proteins, and plant hormones to microscale bioactive Agrobacterium, leading to strong protein expression and enhanced plant growth. Underwater delivery of salt-tolerance genes into submerged freshwater plants further demonstrates the platform’s utility for engineering stress resilience in challenging environments. By facilitating the cellular uptake of diverse cargoes into intact plants across different habitats, this amphibious microneedle strategy offers a versatile cargo delivery tool to advance plant biotechnology and environmental applications. An efficient cargo delivery tool is essential for plant trait engineering. Here, the authors repot a core-shell microneedles for targeted delivery of nucleic acids, proteins, phytohormones, and Agrobacterium to both terrestrial and aquatic plants.

Mass spectrometry is recognized as the gold standard for glycan analysis, yet the complexity of the generated data hampers progress in glycobiology, as existing tools lack full automation, requiring extensive manual effort. We introduce GlycoGenius, an open-source program offering an automated workflow for glycomics data analysis, featuring an intuitive graphical interface. With algorithms tailored to reduce manual workload, it allows for data visualization and automatically constructs search spaces, identifies, scores, and quantifies glycans, filters results, and annotates fragment spectra of N- and O-glycans, glycosaminoglycans and more. It seamlessly guides researchers of all expertise levels from raw data to publication-ready figures. Our findings demonstrate that GlycoGenius achieves results comparable to manual analysis or competing tools, identifying more glycans, including novel ones, while significantly reducing processing time. This groundbreaking tool represents a significant advancement in the study of glycoconjugates, empowering researchers to focus on insights rather than data processing. Researchers present GlycoGenius, an open-source tool that automates complex glycomics data analysis. It streamlines workflows, identifies known and previously unreported glycans, and enables faster, more accessible insights into glycobiology.

Designing effective mRNA sequences for therapeutics remains a formidable challenge. Inspired by successes in protein design, language models (LMs) are now being applied to RNA, but progress is often impeded by the lack of comprehensive training data. Existing models are frequently limited to UTR or CDS regions, restricting their application for complete mRNA sequences. We introduce mRNABERT, a robust, all-in-one mRNA designer pre-trained on the largest available mRNA dataset. To enhance performance, we propose a dual tokenization scheme with a cross-modality contrastive learning framework to integrate semantic information from protein sequences. On a comprehensive benchmark, mRNABERT demonstrates state-of-the-art performance, outperforming previous models in the majority of tasks for 5’ UTR and CDS design, RNA-binding protein (RBP) site prediction, and full-length mRNA property prediction. It also surpasses large protein models in several related tasks. In conclusion, mRNABERT’s superior performance across these diverse tasks signifies a substantial leap forward in mRNA research and therapeutic development. Designing complete mRNA sequences for new vaccines and therapies is a complex challenge. Here, the authors develop mRNABERT, a foundational AI model that designs entire mRNA sequences and demonstrates superior performance across comprehensive benchmarks.