RMH

Phosphorus (P) availability constrains marine productivity, yet how ocean acidification interacts with intensifying phosphate limitation remains unresolved. Here we present multi-omics analyses of >1,000 generations of experimental diatom evolution to disentangle evolutionary and acclimation-driven responses to ocean acidification under different phosphate regimes. Long-term selection under elevated CO2 produced heritable increases in growth and carbon fixation that persisted irrespective of phosphate availability. In contrast, ocean acidification-driven reductions in particulate organic phosphorus were reversed after reciprocal transfers between selection treatments under phosphate limitation, indicating acclimation and environment-dependent P allocation. Multi-omics analyses indicate that the particulate organic phosphorus decline could be attributed to depletion of cellular P reserves and enrichment of non-P cell membrane lipids under combined ocean acidification and phosphate stress. Coastal mesocosm experiments revealed similarly reduced particulate organic phosphorus in natural phytoplankton at low-phosphate stations under ocean acidification. Global meta-transcriptomes further indicate widespread upregulation of gene expression in non-P cell membrane lipid pathways in high- and low-phosphate regions. Earth system models predict a 211% to 2,185% decline in particulate organic phosphorus by 2100 under the under the Shared Socioeconomic Pathway (SSP)2–4.5 or SSP5–8.5 scenarios, respectively. These results show that ocean acidification could intensify nutrient imbalance and reshape global phosphorus cycling in marine ecosystems. Ocean acidification can reduce phytoplankton phosphorus content, especially under phosphate limitation, which could reshape marine nutrient balance and phosphorus cycling, according to long-term experiments, field studies and global analyses.

Plant pathogens and pests pose persistent threats to global crop production and food security. Plant immune receptors, including pattern recognition receptors and nucleotide-binding leucine-rich repeat receptors, represent key targets for engineering disease resistance. Deeper understanding of their structural biology and activation mechanisms has driven the development of strategies to engineer, transfer and deploy these receptors, expanding recognition specificities and enabling broad-spectrum, durable resistance to pathogens and pests in plants. In this Review, we discuss advances in identifying, transferring and engineering plant immune receptors, with an emphasis on translational strategies that support sustainable agriculture. We further examine how tools such as CRISPR-based genome editing and AI-assisted protein design could progress this field. By integrating mechanistic insights with practical applications, we aim to engage bioengineers, plant scientists and agricultural biotechnologists in addressing the urgent need for environmentally sustainable crop production systems. This Review highlights advances for identifying, transferring and engineering plant immune receptors, then discusses translational strategies for developing durable, sustainable crop protection.

Microbiome homeostasis is crucial for host health and ecosystem function, yet the molecular and ecological mechanisms underlying community assembly and stability remain elusive. Here we uncover a conserved yeast–oomycete association that promotes their coexistence in the leaf microbiome. Using a continental-scale microbiome survey, we identified an asymmetric mutualistic interaction between two eukaryotic hub microbes: the yeast Dioszegia hungarica and the obligate oomycete Albugo laibachii. We show that Dioszegia facilitates Albugo colonization by supplying thiamine via a dedicated membrane permease, alleviating Albugo’s auxotrophy. Genomic and transcriptomic analyses indicate that natural selection has acted on thiamine production in Dioszegia, shaping this metabolic complementation. In planta assays further suggest that Albugo presence supports Dioszegia persistence under glasshouse conditions. Our study illustrates how the evolution of nutrient cross-feeding mediates microbial coexistence and microbiome stability. Targeting microbial nutrient flows offers new strategies for engineering microbiomes and enhancing plant resilience in natural and agricultural systems. Evolution of nutrient cross-feeding between a yeast and oomycete mediates microbial coexistence and leaf microbiome stability.

CRISPR–Cas12a nucleases typically induce double-strand breaks (DSBs) to initiate genome editing. However, reliance on DSBs risks chromosomal rearrangements and unintended insertions or deletions at off-target sites. Therefore, engineering nickase variants that cleave only a single DNA strand might enable precise editing platforms. We describe an unbiased in vivo screen in E. coli to identify LbCas12a nickase variants. We targeted the RuvC Lid domain for mutagenesis, hypothesizing that altering this region would disrupt the coordination of strand cleavage. While the screen produced many nickase variants with targeted point substitutions (Lid1), it also serendipitously yielded a variant (Lid2) with an unprecedented Lid domain in terms of size and sequence. Biochemical characterization confirms that Lid2 robustly nicks the non-target strand (NTS) with minor cleavage activity observed for the target strand (TS), accumulating predominantly nicked substrates. Cryo-electron microscopy (cryo-EM) at 2.5 Å resolution reveals that, in Lid2, the native α-helical Lid is replaced, uncoupling the concerted cleavage mechanism. Specifically, the remodeled Lid appears to stabilize the NTS in the active site, whereas it hampers the conformational transitions required for subsequent TS cleavage. This robust nickase-phenotype of this Cas12a variant expands the CRISPR–Cas toolbox and offers a new component for precise genome editing applications.

Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger that coordinates lifestyle transitions, virulence, and developmental processes in bacteria. In Clostridioides difficile, elevated c-di-GMP levels inhibit sporulation, but the underlying mechanism remained unclear. Here, we identified a conserved family of small, membrane-associated proteins encoded by c-di-GMP riboswitch-regulated genes. Transcriptomic analyses revealed that c-di-GMP represses these genes, and reporter assays demonstrated riboswitch-dependent transcriptional regulation via premature termination mechanism. Overexpression of a single gene, CD1980.2, was sufficient to trigger the transcriptional activation of sporulation genes, including sigma factors and their regulons, and to increase spore formation. Conversely, sporulation efficiency decreased proportionally with the number of deleted small-protein genes, and the strain lacking all seven genes displayed a severe sporulation defect, underscoring their cumulative and functionally redundant roles. Elevating c-di-GMP levels in the deletion mutant did not further reduce sporulation, supporting a model in which c-di-GMP inhibits spore formation by repressing the expression of this small-protein family. Thus, our work establishes these small proteins, encoded by c-di-GMP riboswitch-regulated genes, as important regulators of spore formation in C. difficile, a process essential for pathogen persistence and transmission. The second messenger c-di-GMP inhibits sporulation in the pathogenic bacterium Clostridioides difficile through unclear mechanisms. Here, Badilla Lobo et al. show that the expression of sporulation genes is modulated by a family of small membrane-associated proteins encoded by genes that are controlled by c-diGMP-regulated riboswitches.

Efficient use of limited cellular resources is fundamental to metabolism. Although flux optimization is widely recognized as a central objective of metabolic networks, how flux efficiency influences the allocation of the metabolic proteome remains unclear and lacks direct validation. Here, we derive a simple analytical relationship linking the equilibrium constant (K) and the catalytic-abundance quotient (CAQ) of reactions within a pathway that defines the condition for maximal efficiency. By integrating reaction thermodynamics, enzyme kinetics, transcriptomic, and proteomic data, we compared enzyme allocation in glycolysis and the TCA cycle with this K-CAQ relationship across evolutionarily distant species, and showed that deviation from it can be explained by a trade-off between maximizing flux efficiency and concentrating flux control. Moreover, the drive to optimize glycolytic efficiency is strengthened under oncogenic signaling and a limited cellular budget for glycolytic enzymes. These findings establish a principle of Pareto optimality governing enzyme allocation in metabolic pathways and reveal key determinants of efficiency optimality in glycolysis.

In this work, we show how exploration of genomic sequence space yields an unexpected evolutionary ‘fix’ that allows the cell to bypass the phenotypic defects associated with a dysfunctional enzyme. We identified a transposon (Tn) insertion in the gene (argA) encoding N-acetylglutamate synthase (NAGS) that leads to arginine auxotrophy in Pseudomonas aeruginosa. This Tn insertion generates a truncated version of the ArgA protein (ArgA188-432) that retains NAGS activity but, unlike the wild-type protein, is unrestrained by arginine feedback inhibition. However, the Tn::argA mutant readily re-acquired a prototrophic phenotype through the acquisition of spontaneous secondary mutations outside of the argA locus. Whole-genome sequencing and complementation analyses of one such ‘bypass mutant’ allowed us to trace this epistatic effect to an Ala128→Val substitution in AmbA, a LysE-type transmembrane transporter. Using a variety of genetic approaches, we show that the ambAAla128Val mutation gives rise to a change/expansion of function in the encoded gene product and that this mutation is directly or indirectly responsible for bypassing the auxotrophy associated with the original Tn::argA mutation. Further analysis of the Tn::argA ambAAla128Val mutant revealed that, compared with the ‘wild-type’ progenitor, the mutant exhibited large-scale changes in its proteome and metabolome consistent with elevated intracellular arginine and enhanced polyamine degradation. We conclude that the ambAAla128Val mutation overcomes the phenotypic impact of the dysregulated ArgA188-432 enzyme, possibly by enabling efflux of toxic polyamines (which we also show can competitively inhibit NAGS).

The turnover number (kcat) is crucial for understanding enzyme kinetics, yet its experimental determination remains costly and low-throughput. Existing deep learning approaches often rely on single substrate assumptions and overlook full reaction contexts or language-informed semantic representations, limiting their accuracy and generalization across diverse reactions. To address these challenges, we propose Katalyst, a knowledge-guided semantic alignment framework for kcat prediction based on contrastive learning. Katalyst integrates enzyme sequences, protein textual descriptions, substrates and products into a shared latent space using multi-channel encoders and contrastive alignment. An interaction-aware fusion module is designed to capture complementary crossmodal information. Experimental results demonstrate that Katalyst achieved the best overall performance, with the RMSE of 0.9880, R2 of 0.5829, and SRCC of 0.7603. Compared with the State-Of-The-Art (SOTA) baselines, Katalyst reduced RMSE by 3.01% and improved R2 and SRCC by 4.72% and 2.31%, respectively. Moreover, ablation studies and evaluations across sequence scales further confirmed the effectiveness of semantic alignment and multimodal feature fusion. Overall, Katalyst provides a robust and accurate framework for kcat prediction.

In Streptococcus pneumoniae, the RpiR transcriptional regulator NanR (SpNanR) senses sialic acid in the environment and upregulates transcription of the nan and sia A operons to increase uptake and metabolism of sialic acid. The molecular basis of this activation is unknown. Here, we demonstrate that SpNanR binds N-acetylmannosamine-6-phosphate, a metabolite of sialic acid catabolism. SpNanR exists in a dimer–tetramer equilibrium, and N-acetylmannosamine-6-phosphate binding strongly stabilizes the tetramer. Crystal structures and site-specific substitutions demonstrate that N-acetylmannosamine-6-phosphate bridges and stabilizes the SpNanR tetramer. SpNanR binds its DNA recognition sequence with nanomolar affinity. Notably, the effector N-acetylmannosamine-6-phosphate does not affect the affinity of SpNanR for DNA. The DNA-binding domains are not structurally coupled to the sugar isomerase domains, explaining why N-acetylmannosamine-6-phosphate binding does not affect DNA binding. Structural analysis reveals that sequence specificity arises through distortion of B-DNA and an unusual π-stack formed by two arginine residues in the minor groove, while affinity is driven by backbone contacts. We propose a mechanism by which S. pneumoniae regulates sialic acid metabolism, consistent with our biophysical experiments and in vivo regulatory behavior. These findings define a unique activation mechanism for an RpiR regulator and provide new insights into carbohydrate-responsive gene regulation in pneumococci.

Broad-host-range IncA and IncC (A/C) conjugative plasmids propagate multidrug resistance in bacteria and mobilize chromosomal resistance islands, including Salmonella Genomic Island 1 (SGI1), across genera. A/C plasmids mobilize SGI1 at very high frequencies and are inhibited by SGI1. Here, we show that SGI1 and kin encode a fertility inhibitor, which we named CtiC (for ‘Conjugative Transfer Inhibition of IncC’), that hampers A/C plasmid transfer. Fertility inhibition facilitates the propagation of SGI1, which is known to be destabilized by coresident A/C plasmids. ctiC expression is both constitutive and activated during conjugative transfer of A/C plasmids. Structural predictions suggest that CtiC resembles the C-terminus of the MOBH12-family relaxase TraI of A/C plasmids. Bacterial two-hybrid assays and relaxase domain substitutions show that CtiC and the C-terminus of TraI interact with the plasmid-encoded mobilization factor MobI, which recognizes and binds the origin of transfer (oriT) of A/C plasmids. Hence, CtiC acts as a decoy that competes with TraI for binding to MobI, freeing TraI for efficient SGI1 mobilization, and precluding efficient processing of the plasmid’s oriT. Our work reveals a mechanism of fertility inhibition that prevents relaxosome assembly and uncovers a functional domain in MOBH12 relaxases.