Siderophores are classically viewed as shared iron-scavenging public goods, yet their ecological roles in multispecies communities remain poorly defined. Here, we establish a synthetic microbial community to dissect how different siderophores, their uptake compatibility and spatial structure shape iron competition. Using Corynebacterium glutamicum as a model, we show that this siderophore non-producer accesses diverse xenosiderophores, including enterobactin secreted by E. coli. However, exploitation was constrained and co-cultures converged to stable compositions. Dose-response experiments combined with mathematical modelling indicated that the producer retains more effective access to enterobactin than the exploiter. Presence of Pseudomonas putida altered this interaction, as it exploited enterobactin while producing pyoverdine, a siderophore inaccessible to the other community members that restricted their iron access. Across different cultivation scales, community dynamics was strongly influenced by spatial organization and initial composition. These findings identify siderophores as context-dependent iron-allocation agents that can promote microbial coexistence or exclusion.

The evolutionary origin of nitrogen-fixing symbiosis has been a long-standing question. To address this, we focused on Bradyrhizobium, a globally abundant bacterial genus that includes classic symbiotic lineages, which rely on the common Nod factor signaling pathway to form nodules, and close relatives capable of fixing nitrogen in a free-living state. We isolated 88 strains carrying the key genes for nitrogen fixation (nif) from non-legume environments and analyzed them alongside 586 public Bradyrhizobium genomes harboring these genes to reconstruct a robust phylogeny of nif genes. Analysis reveals that the earliest-diverging nif lineages are members capable of free-living nitrogen fixation, establishing this as the ancestral state. The Nod factor-dependent symbiotic lineages are polyphyletic, demonstrating at least three independent origins via horizontal acquisition of symbiosis islands. This evolutionary history is reflected in a genomic dichotomy: lineages capable of free-living nitrogen fixation possess a conserved nif island architecture that consistently includes the oxygen-protective gene glbO, whereas the symbiotic nif-associated regions are highly variable and universally lack glbO. Using both loss-of-function and gain-of-function genetic approaches, we show that glbO contributes significantly to nitrogenase activity under free-living conditions, whereas it is dispensable within the protected nodule environment. This work establishes a new framework for the evolution of symbiosis, identifying free-living ancestors as the source from which nitrogen-fixing symbiosis repeatedly and independently evolved in Bradyrhizobium.

We present CBR-db, a database with detailed chemical properties data for biochemical compounds and reactions. We provide the first chemically consistent analyses and detailed chemical property data for a biologically relevant set of 148,673 reactions and 18,716 small molecules derived from the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the ATLAS of Biochemistry. CBR-db provides detailed atom tracking, reaction similarity classification, compound properties, chiral centers, molecular assembly indices, and thermodynamic properties. CBR-db is designed to be continuously updated, open-source, and reproducible. This report highlights the cheminformatic data, explains the curation and data set, and highlights the data’s utility for researchers working at the intersection of chemical properties and biology. We anticipate a broad range of potential applications for these data, given their position at the intersection of cheminformatics and bioinformatics, including researchers interested in molecular properties and reaction mechanisms found in metabolism, the chemical underpinnings of synthetic biochemical design and engineering, and how the origins and evolution of biochemistry have selected among properties of molecules and reactions within chemical space.

Multiplexed assays of variant effects (MAVEs) make it possible to measure the functional impact of all possible single amino acid residue substitutions in a protein in a single experiment. Combination of variant effect data from several such experiments provides the opportunity to conduct large-scale analyses of variant effect scores measured across proteins, but can be complicated by variations in the phenotypes that are probed across experiments. Thus, using variant effect datasets obtained with similar MAVE techniques can help reveal general rules governing the effects of amino acid variation for a single molecular phenotype. In this work, we accordingly combined data from six individual variant abundance by massively parallel sequencing (VAMP-seq) experiments and analysed a total of 31,614 variant effect scores reporting solely on the impact of single amino acid residue substitutions on the cellular abundance of proteins. Using our combined variant effect dataset, we derived and analysed a collection of amino acid substitution matrices describing the average impact on cellular abundance of all residue substitution types in different structural environments. We found that the substitution matrices predict the cellular abundance of protein variants with surprisingly high accuracy when given structural information only in the form of whether a residue is buried or exposed. We thus propose our substitution matrix-based predictions as strong baselines for future abundance model development.

Integral membrane proteins comprise at least a quarter of every proteome. To fold and function properly, these proteins must insert into the correct membrane with the proper topology and orientation. Although their transmembrane helices are chemically compatible with the bilayer, insertion inside cells is not a simple spontaneous event. It must occur in a crowded environment, at the correct membrane, and it often involves challenges such as transferring hydrophilic segments across the bilayer or accommodating helices that are only marginally compatible with it. Cells therefore rely on dedicated systems that direct membrane proteins to the membrane, mediate their insertion, and monitor the process to ensure that it occurs correctly. This review outlines the principles that govern membrane protein insertion in cells and explains how transmembrane sequence features interact with the machineries that mediate their entry into the bilayer. We highlight the major insertion systems of bacteria and eukaryotes and the auxiliary factors that support them, and describe how these pathways accommodate the broad range of membrane protein architectures found in cells, from single-pass proteins to complex multispanning transporters. We also discuss how cells maintain accuracy when insertion fails, through mechanisms that detect and resolve misinsertion. Together, these concepts present membrane protein insertion as a coordinated, adaptable, and safeguarded process, shaped by the interplay between sequence properties, membrane environments, and the machinery responsible for building the membrane proteome.

Transposon-insertion sequencing (TIS) is a high-throughput approach that uses randomly generated transposon mutant libraries to assess bacterial gene essentiality and their contribution to fitness under defined conditions. The method integrates classical transposon mutagenesis with next-generation sequencing. However, conventional TIS workflows generally require at least one active growth step to enrich for replicating or fit mutants. The outcome of these experiments can be biased by the recovery of non-replicating cells or by the loss of slow-growing mutants. To overcome this limitation, we modified the traditional TIS workflow by incorporating propidium monoazide (PMA). PMA is membrane-impermeant and excluded from viable cells but can enter dead cells when the membrane has lost its integrity. PMA covalently crosslinks to DNA upon exposure to UV light. Such binding inhibits PCR amplification, an essential step in TIS library preparation. This property enables selective exclusion of DNA originating from dead cells and reduces the signal to noise ratio in TIS experiments. We refer to this modified approach as Viability-TIS (Vi-TIS). Applying Vi-TIS to our ultra-dense Escherichia coli K-12 BW25113 TIS library enhanced the accuracy of essential gene identification. We further exposed the library to subinhibitory concentrations of carbenicillin and found that Vi-TIS produced results consistent with those obtained using culture-based methodologies. In addition, Vi-TIS revealed previously unrecognized carbenicillin-susceptible mutants. Finally, we demonstrate that the mutations are linked to perturbations in peptidoglycan biosynthesis.

Ribonucleases (RNases) play a key role in modulating gene expression at the post-transcriptional level, enabling bacteria to rapidly adapt to their environment. The B. subtilis genome encodes more than 20 RNases and understanding the role of each of these enzymes is crucial for a comprehensive knowledge of bacterial post-transcriptional adaptation mechanisms. Among these, RNase J1 and J2, belonging to the β-lactamase enzyme family, form a heterodimer. While the primary function of RNase J1 as a 5′-exoribonuclease in B. subtilis has been known for some time, the role of RNase J2, encoded by the rnjB gene, has remained unclear. Indeed, it has been considered a minor degradation factor due its very weak 5′-exoribonuclease in vitro, the lack of a growth phenotype and the limited number of mRNAs with altered equilibrium levels in rnjB mutants. In this study, we demonstrate that the absence of RNase J2 influences pellicle and macro-colony biofilm formation, as well as swarming in B. subtilis. We show that RNase J2 plays a far more global role in governing mRNA half-lives than originally thought, by stimulating RNase J1-mediated degradation of a subset of RNase J1′s mRNA substrates. An inter-subunit contact between RNase J1 and J2 is crucial for RNase J2 action. Altogether, our results suggest that RNase J2 acts primarily as a specificity co-factor for RNase J1 rather than as a ribonuclease per se.

A major challenge in the microbial production of valuable compounds is the trade-off between cell growth and biosynthesis. Orthogonal translation systems, which utilize orthogonal ribosomes derived from engineered rRNAs to translate specific mRNAs, offer a means to reallocate translational resources from cellular growth to biosynthesis. However, the in vivo orthogonality and quantitative effectiveness of orthogonal translation systems have not been fully evaluated. Here, we systematically tested orthogonal rRNA–mRNA pairs in E. coli using sfGFP as a reporter and identified a pair that exhibits robust two-sided orthogonality and high translation efficiency. We then applied this orthogonal translation system to the violacein biosynthetic pathway. The orthogonal translation system increased violacein production by 6.30- to 7.77-fold compared with the endogenous translation system and sustained production after entry into the stationary phase. These results demonstrate that a well-characterized two-sided orthogonal translation system successfully improved the productivity of a multienzyme pathway by reallocating translational capacity in vivo.