Assembly-line polyketide synthases (PKSs) are among the most sophisticated catalysts in nature, responsible for the biosynthesis of many medicinally important natural products including antibiotics, anticancer agents, immunosuppressants and veterinary agents. While conventional methods for polyketide natural product discovery have fallen out of favor, the rapid expansion of microbial genome sequencing has revealed a vast untapped diversity of assembly-line PKSs, most of which can be regarded as “orphans” in that their product identity is unknown. A clear picture of how this sequence diversity reflects the evolutionary history of assembly-line PKSs could enable judicious prioritization of experimental efforts aimed at decoding these orphans. To this end we have used a scalable curation workflow to hand-curate an updated catalogue, PKSClusterDB, that includes 16 633 non-redundant assembly-line PKSs. We have also formulated an automatable “anchor-window” framework based on conserved multimodular segments of assembly-line PKSs to identify and interrogate PKS families of interest. Application of this framework to three different PKS families extracted from PKSClusterDB revealed lineage-dependent patterns of diversification, ranging from broadly diversified families to more compact or sharply bounded lineages. PKSClusterDB has also proven useful in exploring other aspects of PKS diversity, including scaffold architecture, extender-unit specificity, reductive-state programming, stereochemical control and modular organization. In closing, we consider how advances in machine learning could be harnessed to accelerate our understanding of assembly-line PKS evolution, diversity and biosynthetic mechanisms.

Trichoderma is a genus of beneficial fungi widely used in agriculture disease suppression, plant growth promotion, and soil health improvement. Although some studies have shown that Trichoderma spp. can be seed-borne, albeit at low frequency, no specific study has so far focused on the diversity of Trichoderma isolates capable of adapting to this particular environment. Over 2 years of routine seed health analyses, we isolated 107 Trichoderma strains associated with seeds from 32 cultivated plant species, and we investigated the diversity of this specific library using multi-locus analysis. These isolates are distributed among 18 different species, but Trichoderma atroviride is by far the most frequent. Representative strains of each species all have the ability to parasitise damping-off agents, although the intensity of parasitism can vary greatly from one strain to another and depending on the targeted plant pathogens. For two isolates of T. atroviride, we showed that these fungi can be transmitted from fruit to seed and limit the transmission of the plant pathogen Alternaria brassicicola. Furthermore, these strains stimulated seedling growth and provided protection against Globisporangium ultimum. Together, these findings support the benefits of focusing on certain selected seed-borne Trichoderma which seem particularly well suited to improving seed health.

Microbial community assembly is shaped by the nature of available resources, with labile carbon sources like glucose often expected to support low diversity due to rapid growth and competitive exclusion. In contrast, recalcitrant substrates like cellulose are thought to support higher diversity through slower growth and increased niche partitioning. In previous work, we showed that compost-derived microbial communities propagated on cellulose maintained high diversity over nearly a year. To determine whether such diversity is specific to recalcitrant substrates or reflects more general features of assembly, we tracked community dynamics in three environments—cellulose paper, cellulose broth and glucose—using daily 16S rRNA profiling. Communities maintained through four bi-weekly serial transfers, with five replicates per treatment, yielded a high-resolution dataset of over 800 samples. Despite originating from the same inoculum, communities diverged sharply in both taxonomic and functional composition. Cellulose environments yielded stable communities enriched in specialists, while glucose environments exhibited rapid succession and dominance by generalists. Surprisingly, all environments sustained comparably high levels of taxonomic diversity. Functional inference suggested extensive cross-feeding and resource salvaging in both cases. Our results reveal distinct assembly trajectories under simple carbon regimes and provide a foundation for future mechanistic study.

Healthy plant leaves potentially host both commensal bacteria and opportunistic pathogens, which, under some circumstances, may cause disease. The interactions between commensals and opportunistic pathogens are generally poorly understood, but such understanding is crucial for developing effective biocontrol strategies. In Arabidopsis thaliana, isothiocyanates (ITCs) are defense metabolites that suppress most bacteria; commensals are especially affected as they do not express ITC resistance genes. The ITC hydrolase SaxA detoxifies ITCs, making it an important virulence factor for bacterial and fungal pathogens. To investigate pathogen-commensal interactions based on SaxA-mediated ITC degradation, we used five ITC-sensitive bacterial commensals and the opportunistic pathogen Pseudomonas viridiflava 3D9 (PS). All strains were isolated from healthy A. thaliana leaves. PS degrades 4-methylsulfinylbutyl-ITC (4MSOB-ITC) with SaxA. We examined commensal growth in the presence of 4MSOB-ITC, both in monoculture and in coculture with PS or a saxA-deficient mutant (PSKO). We used the growth data to develop a generalizable consumer-resource mathematical model incorporating ITC toxicity, ITC degradation, and nutrient use. We predicted and confirmed experimentally that the extent to which SaxA benefits the pathogen depends on its effects on commensals. In some contexts, commensal rescue and the resultant nutrient competition limit pathogen growth. In addition, we tested in silico how commensal ITC susceptibility, pathogen ITC degradation rates, and growth parameters affect the trade-off between SaxA-mediated virulence (strong pathogen growth) and commensal rescue (commensal growth). Our findings suggest that the effects of microbial traits—traditionally viewed as either virulence or plant-beneficial factors—are constrained in the microbiome context. This underscores the need to reconsider how such traits are classified in the context of plant-microbiome interactions.

Enhancers activate specific target promoters, but whether intrinsic enhancer-promoter compatibility contributes to this specificity is debated. Recent studies using different reporter assays have reached contradictory conclusions. We compare six reporter assay designs, identify confounders that bias compatibility measurements, and apply improved assays to test 25,000 enhancer-promoter pairs. Promoters differ in their responsiveness to enhancers (>100-fold versus 1.1-fold activation) while enhancers activate all promoters in a similar rank order. Promoter output scales with enhancer activity following a power law, with the exponent varying across promoters. Incorporating this exponent into the Activity-by-Contact model improves prediction of endogenous enhancer effects, explaining why certain active genes are insensitive to distal perturbations and "skipped" by enhancers. Responsiveness is modulated by transcription factor motifs in the core promoter. This work establishes responsiveness as an intrinsic promoter property that enables specific promoters to be highly activated in a landscape of broadly compatible enhancers, while others remain unaffected.

Organisms have continuously evolved in response to environmental conditions. Pathogenic bacteria evolve under host and environmental pressures, reshaping their genomes through insertions, inversions, deletions, and duplications during infection. In clinical settings, phenotypic traits of pathogenic bacteria such as virulence or antimicrobial resistance directly affect disease severity, transmission, and treatment. Conventional genotyping provides insights into genomic relatedness but does not always align with these clinically relevant traits, limiting its utility for phenotype-driven interventions. Here, we develop ABComp (Assembly polishing and Bacterial whole-genome Comparison for multi-group clinical isolates), a modular and Snakemake-based workflow for phenotype-driven comparative genomics. ABComp automates assembly polishing, group-wise pangenome analysis, and enables flexible pathogenic marker discovery through user-defined comparisons. We validated ABComp using a Klebsiella pneumoniae ground truth dataset stratified by yersiniabactin presence and successfully recovered the entire locus as a group-specific core marker. By applying ABComp to another dataset of clinical isolates with experimentally measured virulence, we discovered the ferric citrate (Fec) uptake system as a potential marker specific to a hypervirulent group. These results demonstrate ABComp's utility in uncovering phenotype-linked genomic markers with clinical significance, supporting targeted treatments and rapid diagnosis.

Protein self-assembly is a fundamental biological process of great importance for the design and synthesis of biomaterials. Developing the ability to precisely manipulate protein assembly would greatly expand both our understanding of the process and our biotechnological capabilities. Within bacteria, proteins that self-organize to form bacterial microcompartments (MCPs) offer an excellent model system for studying protein self-assembly and advancing biomaterial design capabilities. MCPs consist of irregular polyhedral shells that encase an enzyme core that acts as enzymatic nanoreactors. In isolation, the abundant shell proteins of the 1,2-propanediol utilization (Pdu) MCP, PduA and PduJ, have a high propensity to self-assemble into tubular structures, analogous in form to carbon nanotubes. Here, we modulate higher-order assembly of PduA and PduJ hexamers by systematically altering their charge through charge inversion and supercharging across multiple platforms including heterologous overexpression and cell-free protein synthesis. Overexpression and cell-free experiments show that increasing the overall negative charge of assembling subunits consistently promotes self-assembly into tubular structures. Using molecular simulations, we determined the preferred bending angle adopted by the hexameric proteins to predict the most probable self-assembled structures, including honeycomb-like sheets and nanotubes. Simulations of closed PduA and PduJ tubes show the interactions responsible for tube stability, chirality, and radius. In vivo, we find that these charge-altered hexamers are assembly competent within the native MCPs in Salmonella enterica LT2. Our results collectively reveal that both electrostatic interactions and fields generated by charges on proteins can be leveraged to control protein-based nanostructures.

Temperate bacteriophages are dominant members of the human gut microbiome that can infect and lyse their bacterial hosts or integrate as prophages. During this integrated state, prophages exhibit extensive control over host physiology and lysis via induction. Here, we studied a diverse collection of Bacteroidales isolates, which are amongst the most abundant bacterial orders within the human gut, identifying 902 high-quality prophage genomes present within 305 isolates, 240 of which were poly-lysogens. Despite their prevalence, our understanding of the function and induction triggers of prophages is limited. To predict prophage induction, we employed an iterative profile Hidden Markov Model search across divergent bacterial hosts to identify prophage regulatory components. We found 197 Bacteroidales prophages encoding complete CI-like repressor proteins, which initiate induction upon DNA damage. We selected Bacteroides thetaiotaomicron strain Bt_806 to characterise further as it harbored six diverse prophages, including the prevalent and abundant prophage LoVE, which was the only integrated prophage encoding a complete CI-like repressor. Transcriptomics revealed phage LoVE was routinely induced upon DNA damage, while the five co-habiting prophages remained stably integrated yet exhibited transcriptionally active genes associated with regulation, prophage maintenance, and uncharacterised functions. Finally, we selected an additional eleven Bacteroidales poly-lysogens, confirming that integrated prophages encoding complete CI-like repressors were reliably induced upon DNA damage. Together, we demonstrate that mechanistic understanding of prophage induction linked with identification of regulatory genes enables selective and predictable induction of gut prophage species as a potential tool to modulate the microbiome.

Cell-to-cell communication in microbial systems is known for its vital role in cellular signalling and gene expression. A specific form termed Quorum Sensing (QS) has received considerable attention since its discovery in the marine symbiont Aliivibrio fischeri. QS-controlled microbial functions are associated with bacterial virulence, pathogenicity, host–microbe interactions, and biofilm development. Interference in these signaling systems can modulate microbial virulence and pathogenicity, and microbial infection caused by drug-resistant pathogens. Plant-derived phytochemicals are considered a promising candidate, with coumarins emerging as significant plant-derived signalling molecules shaping microbiome dynamics and pathogen behaviors from a broad spectrum of ecosystems. Here we explored the role of natural and synthetic coumarin compounds in the control of signalling and virulence traits in Pseudomonas aeruginosa and other priority bacterial pathogens, including the fungal opportunist Aspergillus fumigatus. We uncovered an important ‘hydroxylation-bias’ favoring coumarin, umbelliferone (7-OH), and 6-hydroxy-coumarin (6-OH) in the specific competitive inhibition of the Pseudomonas Quinolone Signal (PQS), associated with reduced activity of a PqsR translational fusion and suppression of pyocyanin production. Conversely, while esculetin (6,7-OH) was most effective at Acyl Homoserine Lactone (AHL) QS biosensor inhibition, it did not affect PQS production. Anti-biofilm activity of coumarins against P. aeruginosa was independent of initial attachment but linked to changes in exopolysaccharide production. As the very real threat posed by antimicrobial resistance persists, these data support a role for phytochemicals such as coumarins in delivering an ecological solution to dysbiosis in the host–microbe interaction.

Horizontal gene transfer (HGT) shapes bacterial evolution and microbial ecosystems, yet detecting HGT within microbiomes remains a challenge due to fragmented metagenomic assemblies, reference bias, reliance on gene boundaries, and limited ability to model structural mosaicism and patterns across genomes. We present Kente, a novel pangenome graph-based framework designed for HGT detection that aligns metagenomic assembly contigs to a curated database of >600 genus-level bacterial pangenome graphs constructed using minigraph. Kente infers local taxonomic composition along contigs using alignment evidence and classifies candidate transfers using structured clade-transition topologies (e.g., A-B-A sandwich, open tips, and mosaic patterns). A complementary intra-genus module detects inter-species transfers within a single genus graph using segment-level clade annotations. Across simulated intra- and inter-genus transfer scenarios, Kente achieves higher precision and comparable recall relative to existing gene-centric microbiome HGT detection approaches while reducing false positives from fragmented assemblies. Application to real human gut metagenomes (HMP2, n = 26) demonstrates Kente's ability to detect candidate cross-lineage transfer regions in complex microbial communities. Runtime profiling shows near-linear scaling with input size, enabling efficient analysis of large metagenomic assemblies. Availability and Implementation: https://github.com/treangenlab/Kente

Bacteria in natural environments frequently encounter nutrient limitation leading to growth arrest and must balance the potential benefits of continuing to respond to the environment by making new proteins against the costs of depleting limited resources. We previously showed that the RNA polymerase-binding regulator SutA enhances transcription of hundreds of genes during nutrient limitation in Pseudomonas aeruginosa, suggesting that it might be part of a regulatory network facilitating limited new protein synthesis. Here, we sought to expand our understanding of this network by identifying transcriptional regulators influencing sutA expression. Using northern blotting, western blotting, and reporter assays, we found that the sigma factors FliA and RpoS, and the DNA-binding regulator Lrp, impact expression from a proximal sutA promoter during the transition to stationary phase. This constellation of regulators and the dynamics of SutA expression lead us to propose that SutA is part of a regulatory network that facilitates scavenging. Scavenging includes motility toward possible nutrient sources and uptake mechanisms for these nutrients, activities which require an investment of resources but can yield important benefits during starvation. In vitro transcription experiments, proteomic analysis and reporter assays suggest that SutA directly supports new protein synthesis driven by RpoS and indirectly supports flagellar motility, perhaps by helping maintain protein biosynthetic capacity against the metabolic costs of motility. SutA expression is controlled by multiple regulatory inputs, including negative autoregulation, and the protein appears to be short-lived. These properties are consistent with a role in supporting short, controlled bursts of gene expression during nutrient limitation.

Genetic diversification serves as the foundation for in vivo molecular evolution. The fusion of a cytidine deaminase with T7 RNA polymerase (MutaT7) enables efficient diversification of DNA sequences downstream of the T7 promoter in model microbial hosts E. coli and S. cerevisiae. Vibrio natriegens, currently the fastest-growing nonpathogenic bacterium with a doubling time half that of E. coli, emerged as a promising alternative chassis for synthetic biology. Here, we report the establishment of MutaT7 in V. natriegens (dubbed VnMutaT7), achieving rapid evolution of the model protein TEM-1 to confer high-level ceftazidime resistance in just 36 h. This work establishes the potential of V. natriegens as an accelerated platform for protein engineering.

Symbiotic nitrogen fixation reduces reliance on synthetic fertilizers and is central to agriculture and ecosystem functioning. In legumes, this process occurs in root nodules where rhizobia differentiate into bacteroids that either remain viable or undergo terminal differentiation, a strategy that can enhance nitrogen fixation but limits nodule life span. This irreversible program suits annual legumes and has evolved convergently across multiple lineages, including the inverted repeat-lacking clade (IRLC), where it is enforced by nodule-specific cysteine-rich (NCR) peptides. By contrast, perennial legumes with indeterminate nodules must sustain symbiosis over extended periods, which is incompatible with terminally differentiated bacteroids. Here, we identify a family of nodule-specific proline-glycine-rich peptides (NPGs) in Robinia that are induced upon rhizobial infection. NPGs are highly expressed in nodules, encode intrinsically disordered peptides, and accumulate in infected cells within the fixation zone. Exposure of Mesorhizobium robiniae to recombinant NPGs induces transcriptional changes associated with a fixation-related physiological state while preserving bacterial viability. These findings identify NPGs as candidate host effectors at the plant-microbe interface and point to an alternative mode of symbiont modulation in perennial legumes.