Pseudomonas aeruginosa is a ubiquitous, Gram-negative bacterium that forms biofilms and is responsible for antibiotic-resistant hospital-acquired infections in humans. The P. aeruginosa BqsRS two-component system regulates biofilm formation and dispersal by sensing extracytoplasmic Fe2+, but the mechanistic details of this process are poorly understood. In this work, we report the crystal and solution structures of the PaBqsR response regulator receiver domain, comprising a (βα)5 response regulator assembly, and the DNA-binding domain, comprising a helix-turn-helix motif. Consistent with its cognate stimulus being Fe2+, we show that BqsR binds directly to the promoter region of the feo operon that encodes the bacterial Fe2+ transport system FeoABC. Corroborating these in vitro results, transcriptional studies show that BqsR is a global regulator controlling many important genes in PAO1, including the feo operon. Intriguingly, promoter-based assays reveal that BqsR is a dynamic regulator that responds to bioavailable Fe2+, likely through the ability of BqsR to bind Fe2+ directly via a His-rich motif, independent of the BqsS membrane His kinase. To our knowledge, this mode of regulation has not been reported previously among OmpR-like response regulators but represents an important level of control over Fe2+ acquisition in P. aeruginosa that could be an attractive therapeutic target to treat hospital-acquired infections. The BqsRS two-component system regulates biofilms in the infectious pathogen Pseudomonas aeruginosa. Here, the authors show that the global response regulator BqsR controls Fe2+ acquisition in PAO1 through a previously unrecognized regulatory mechanism.

Artificial intelligence (AI) is transforming scientific research, including proteomics. In this Perspective, we highlight key mass spectrometry (MS)-based proteomics areas where AI is driving innovation, ranging from protein identification to building AI virtual cells. These include improving peptide and protein identification and quantification; characterizing protein-protein interactions and protein complexes; advancing spatial and perturbation proteomics; integrating multi-omics data; and, ultimately, enabling AI virtual cells. Finally, we call for global collaboration among data producers, data consumers and other stakeholders to establish an AI-friendly ecosystem for MS-based proteomics, laying the foundation for transformative advancements in proteomics driven by AI. This Perspective highlights key research areas within mass spectrometry-based proteomics where AI is poised to drive significant advances.

Methane (CH4) from ruminants is a major source of agricultural greenhouse gas and represents a loss of dietary energy. 3-nitrooxypropanol (3-NOP) is a known methanogenesis inhibitor, but its hydrophilic nature may limit the cellular accessibility to methanogens. Here, we systematically evaluated 1,3-propanediol dinitrate (1,3-PDN), a more hydrophobic derivative of 3-NOP, for its anti-methanogenic potential and underlying mode of action using ruminal fermentation, pure-culture assays, multi-omics analyses, and molecular docking. Intracellular accumulation assays indicated greater cellular accumulation of 1,3-PDN than 3-NOP in rumen-derived methanogen Methanobrevibacter olleyae. Ruminal fermentation assays showed that 1,3-PDN reduced CH4 production by ~55%, and altered hydrogen (H2) metabolism, leading to 11-fold increase in H2 accumulation. Metatranscriptomic profiling revealed that 1,3-PDN significantly altered the active archaeal community, with a notable reduction in Methanobrevibacter_A and suppression of hydrogenotrophic methanogenesis. Molecular docking suggested that 1,3-PDN may bind to the conserved active site of methyl-coenzyme M reductase (MCR), potentially contributing to MCR-associated inhibition. Proteomic analysis further indicated that 1,3-PDN supplementation downregulated key MCR subunits and simultaneously affected other redox-sensitive methanogenesis-related processes, including tetrahydromethanopterin S-methyltransferase (MTR) subunits and proteins involved in the biosynthesis of cofactors F430 and cobalamin. Nitrogen-equivalent assay suggested partial contribution of nitrite to the methanogenesis inhibition and oxidative effects induced by 1,3-PDN. Together, these findings identify 1,3-PDN as an effective inhibitor of ruminal methanogenesis with enhanced cellular enrichment, providing mechanistic insights for the rational development of new CH4 inhibitors.

GapR is a pleiotropic α-proteobacterial nucleoid-associated protein (NAP) reported to either directly regulate transcription of AT-rich DNA or to regulate transcription indirectly through sensing DNA topology and modulating topoisomerase activity. We use single-DNA micromechanics, biolayer interferometry (BLI), and computational analysis to study GapR transcriptional regulation. Micromechanics experiments show that GapR overtwists DNA and shortens its contour length. GapR binding also substantially increases DNA bending persistence length and twist stiffness. For DNA tension ∼0.5 pN, as occurs in supercoiled domains, GapR also promotes DNA strand separation, a transition not observed for lower forces. Nonequilibrium binding experiments show GapR–DNA complexes to be extremely stable, with essentially no dissociation on hour-long time scales. Strikingly, our BLI experiments show that GapR has high affinity for AT-rich DNA, while our micromechanics show that GapR binding is enhanced by pre-twisting of DNA, validating GapR affinity for both forms. By analyzing published GapR binding data, we reveal that overtwisted DNA primarily determines GapR localization. We demonstrate that these two binding modes have opposing impacts on gene expression, with AT-rich binding activating and overtwisted DNA binding repressing transcription. Together, our findings demonstrate how the biophysical activities and topological sensitivity of a single NAP generate context-specific behavior.

Bioiontronics is an interdisciplinary field emerging from a confluence of breakthroughs in iontronics and bioengineering. Bioiontronics promotes communication with living matter through ions and biological molecules, thereby mediating the detection and modulation of biological activities. This process can function at abiotic–biotic interfaces in an autonomous fashion or in integral, modular components of biomedical devices. The clinical need to use specific biomolecular information, such as ion concentrations and biomarker levels, for personalized diagnostics and treatments has driven the rapid evolution of bioiontronics, through the convergence of fundamental science and application-oriented engineering. However, several challenges remain, including control of ion transport, device miniaturization, biocompatible encapsulation for long-term implantation and tools to decipher and then harness multimodal biological signals. In this Review, we discuss the underlying mechanisms and recent prototypes of bioiontronic devices and describe challenges in the fabrication and application of such systems. Bioiontronics enables communication with living systems through controlled ion and biomolecule transport at abiotic–biotic interfaces. This Review outlines the mechanisms and emerging device prototypes driving the field and discusses key challenges in ion control, miniaturization, biocompatible fabrication, as well as diagnostic and therapeutic applications.

Protein function annotation is crucial for understanding biological processes and mechanisms. Traditionally, annotations rely on sequence homology, providing valuable insights but often leaving gaps even in well-characterized organisms. With AlphaFold enabling rapid generation of protein structural models, we can now infer function from three-dimensional shape. Here, we present WASP, a pipeline leveraging structural homology to enhance protein annotation prediction at scale, providing a more comprehensive understanding of protein functions across various organisms. WASP relies on network topology for better accuracy and more robust statistical power. We show that WASP achieves superior F1 scores compared to state-of-the-art sequence-based tools when recovering hidden annotations. On 20 industrially relevant organisms, WASP retrieves annotations for 20-30% of previously uncharacterised proteins. We further demonstrate utility in genome-scale metabolic model curation, identifying native candidates for 75-100% of orphan reactions. WASP highlights how structural homology can systematically discover annotations missed by sequence-based approaches. WASP predicts protein functions from AlphaFold structures using network-based structural homology, retrieving annotations for 20-30% of uncharacterised proteins and filling metabolic model gaps by mapping 75-100% of orphan reactions.