Transposons are a convenient vehicle to insert DNA into a bacterial genome, but widely-used transposons such as Tn7 are not able to hit custom targets. Recently, CRISPR RNA-guided integrases have been shown to direct the insertion of a mini transposon to a chosen site using a short guide sequence. We adapted this system for the widespread plant-associated genus Sphingomonas, revealing flexibilities and limitations of the tool. We uniquely tagged five genetically diverse strains, both in neutral sites and in sites with phenotypic consequences, and we demonstrate the utility of the tags for quantitative strain tracking in complex bacterial populations. Although we initially focused on the attTn7 site recognized by Tn7 as an insertion site with minimal fitness effects, we discovered via a genus-wide search that this site disrupts potentially important genes in some strains. Therefore we validated an improved site for benign integration in Sphingomonas and used a construct with guides targeting this site to transform a heterogeneous Sphingomonas population, bypassing prior strain isolation. Using a novel rapid and economical transposon mapping method, we were able to identify correctly-tagged primary transformant colonies with novel genetic content, thus demonstrating a short cut towards the establishment of diverse tagged synthetic communities for the experimental study of bacterial natural variation.

For a rapid and cost-effective evolution of tailor-made enzymes, we established a high-throughput in vitro selection platform named SMART (Single-Molecule Assay on Ribonucleic acid by Translated product), integrating mRNA display, next-generation sequencing, and bioinformatics. SMART represents a versatile system where a module termed an auxiliary unit allows enzyme-specific selection under various experimental conditions. Here, we report on the establishment of SMART for oxidases using a model enzyme, Schizosaccharomyces pombe d-amino acid oxidase (SpDAAO), and ascorbate peroxidase 2 as the auxiliary enzyme to detect hydrogen peroxide produced by the oxidase, and mediate biotinylation of active single-molecule display complexes. As a proof-of-concept, a library including site-saturation mutagenesis at the catalytic residue Y232 of SpDAAO was subjected to a single SMART selection round, yielding enrichment of the active enzyme variant. The results demonstrate the utility of SMART as a fast, robust, and efficient platform with the potential of customization for other enzyme chemistries through appropriate modifications of the auxiliary unit. Using SMART, desired enzyme variants can be selected in just a few hours by a single person without the need for costly equipment or any bias or limitations.

GenomeDepot is an open-source web-based platform for annotation, management, and comparative analysis of microbial genomic sequences and associated data including ortholog families, protein domains, operons, regulatory interactions, strain taxonomy, and sample metadata. GenomeDepot supports rapid creation of websites for user-defined genome collections that include bioinformatic tools for interactive genome browsing, Basic Local Alignment Search Tool (BLAST) search, annotation search, comparative genomic neighborhood visualization, and sequence download. Gene function annotations are generated by a customizable annotation pipeline. The pipeline runs annotation tools in Conda environments and can be easily extended with additional user-specified tools.

Conjugative plasmids drive bacterial evolution and antibiotic resistance spread, yet their gene expression must be silenced to protect the host. A histone-like protein H-NS represses many mobile and sedentary xenogenes but fails to silence the conjugal transfer vir operon of R6K, a prototype IncX plasmid. Instead, R6K encodes its own H-NS homolog, Sfx, to repress the vir operon. Here, we show that, unlike other plasmid silencers that target promoters, Sfx cooperates with Rho factor to arrest transcription elongation. ChIP-seq reveals that despite sharing similar DNA motifs and a preference for negative supercoiling, Sfx and H-NS occupy distinct niches: Sfx binds weakly to the chromosome but is enriched on the R6K vir operon, from which H-NS is excluded. We hypothesize that this selective targeting is mediated by Sfx-vir interactions and phase separation. We show that Sfx binding to vir DNA critically depends on DNA topology but not on the target location. Our results suggest that Sfx phase separates with R6K to ensure its preferential recruitment to the plasmid DNA and forms stable nucleoprotein filaments that are impermeable to competitors. These findings reveal how histone-like proteins can partition the genome into distinct regulatory niches, a strategy likely mirrored across all life. 

Biofilm communities exhibit emergent properties that exceed the sum of contributions from individual members of the community. Here, we describe a multilayered metabolic interaction that drives enhanced biofilm formation among three bacterial species from the plant rhizosphere. Comparative metatranscriptomic and metabolomic analyses reveal that Bacillus velezensis-secreted 5-aminovaleric acid promotes the growth of the other community members, Burkholderia contaminans and Acinetobacter baumannii. In return, B. contaminans supplies branched-chain amino acids for B. velezensis. Branched-chain amino acids and cell-cell signaling acyl-homoserine lactones from B. contaminans induce biosynthesis of the siderophore bacillibactin in B. velezensis, that is further enhanced by A. baumannii. In exchange, the B. velezensis-secreted siderophore promotes the growth of B. contaminans in iron-limited conditions, which benefits the multispecies biofilm community in vitro and promotes plant growth performance in iron-depleted soil. Our study reveals the molecular mechanisms underlying an emergent rhizosphere biofilm community function and demonstrates its importance in plant-microbe interactions.

Mycorrhizal fungi represent one of the oldest and most successful symbioses in plant evolution. Communication among mycorrhizal fungi and plants occurs prior to direct contact among them through different and variable biochemical signals, including microRNAs, hormones, small peptides and volatile organic and inorganic compounds. Volatile organic compounds (VOCs) emerge as key chemical signals that enable the transmission of chemical messages modulating plant and microorganism responses in both below- and above-ground ecosystems. The diversity and concentration of mycorrhizal VOCs will vary depending on the environment and the emitting organism and are usually related to changes in the conformation of root architecture and lateral root formation mediated by auxin and strigolactones. Moreover, the study of the effects of mycorrhizal VOCs in the tolerance to abiotic and biotic stress are still scarce although there are some promising results pointing out to the effect of these VOCs in plant development under osmotic stress conditions, and their properties as antifungal and antibacterial molecules. However, the information regarding the molecular mechanisms involved in mycorrhizal VOCs signaling and their effect on plants remains still elusive. The understanding of VOC-mediated plant-mycorrhizal interactions, together with the technical improvements for their detection and mode of application in the field, will open new avenues for biotechnological crop improvement and management that not only will reduce the dependence on agrochemicals but also fosters soil health and plant resilience.

The plant microbiome plays a central role in regulating plant health and resilience, providing eco-friendly alternatives to agrochemicals. Plant-associated Streptomyces species are prolific producers of structurally diverse natural products with a demonstrated role in promoting plant growth. Coumarins are prevalent plant metabolites that shape the root microbiome, but their impact on microbial natural product biosynthesis is poorly understood. Here, we demonstrate that the coumarins scopoletin and its glucoside scopolin remodel specialized metabolism in the Arabidopsis root endophyte Streptomyces sp. ATMOS53. Multiomics analyses revealed that the coumarins activate the biosynthesis of the pyrrolizidine alkaloids bohemamines and alter the balance in anthracycline biosynthesis, with reduced production of late-stage anthracycline congeners and accumulation of shunt metabolites earlier in the pathway. These metabolic shifts resulted in a marked reduction of the antimicrobial activity of ATMOS53 against plant-associated Bacillus and Paenibacillus species. Notably, coumarin-mediated repression of anthracycline production was also observed in the established producers Streptomyces peucetius and Streptomyces galilaeus, indicating that the regulatory effect on anthracycline biosynthesis is conserved in streptomycetes. Our findings highlight coumarins as modulators of specialized metabolism of Streptomyces and show the significance of plant-derived chemicals for the control of the biosynthetic capacity of plant-associated microbes.