Some microorganisms present in the cultivation environment serve as biocontrol agents and contribute to enhanced mushroom production. However, the native bacteria naturally associated with commercial cultivation bags of Pleurotus ostreatus, as well as their growth-promoting roles and underlying mechanisms, remain poorly understood. This study aimed to identify native bacteria that promote Pleurotus ostreatus development and to investigate the underlying mechanisms. Four native bacteria, including Brevibacterium epidermidis (P6), Acinetobacter soli (A7), Pseudomonas parafulva (A8), and Pseudomonas hunanensis (A12), were isolated based on their ability to promote mycelial growth of P. ostreatus. B. epidermidis P6 shortened complete mycelial colonization time from ~30 d to 14 d in dual cultivation bags. All four strains increased fresh mushroom yield, with B. epidermidis P6, A. soli A7, and P. parafulva A8 increasing the number of basidiomata, while P. hunanensis A12 enhanced their size. These strains produced exopolysaccharides that enhanced mycelial growth. Additionally, B. epidermidis P6, A. soli A7, and P. parafulva A8 also secreted extracellular crude proteins that also promoted mycelial growth. Bi-plates and further gas chromatography–mass spectrometry analysis demonstrated that volatile organic compounds from P. hunanensis A12, including acetone, 2-butanone, benzaldehyde, and 1-undecene, enhanced fungal mycelial growth. The mycelial growth rates of Ganoderma lucidum and Pleurotus pulmonarius were also enhanced by these four strains. These results reveal that four native bacterial strains promote mushroom development through complex mechanisms.

Domain insertion is an established method to engineer ligand-mediated control of activity in protein scaffolds. Whether this strategy can be systematically applied to large, structured RNAs remains unclear. In this study, we investigated the feasibility of engineering ligand-activated splicing ribozymes (LASRs) from group I catalytic introns. Using domain-insertion profiling coupled with high-throughput screening, we mapped the nucleotide-resolution landscape of aptamer insertion across the ribozyme and identified sites that support robust ligand-dependent control. We showed LASRs function across multiple kingdoms of life, including diverse species of bacteria and even fungi, and can be used to regulate various genetic outputs. Finally, we integrated LASRs with a genetic recorder that writes information into ribosomal RNA, enabling sequencing-based recovery of intracellular chemical signals from microbial consortia. This work establishes LASRs as an RNA-based inducible control platform for sensing diverse chemical inputs, regulating the expression of diverse genes of interest, and recording intracellular information.

Genes for CO2 fixation occur in soil microorganisms, but little is known about the pathways that are most common across ecosystem types, the organisms with these genes, where different CO2 fixation pathways are most prevalent, and the energy sources that support autotrophy across ecosystems. Here, we investigated microbial capacity for autotrophy in soils using 853 metagenomes and 201 metatranscriptomes from a wide range of terrestrial ecosystems (agricultural soils, wetlands, weathering rock). Autotrophy-associated RuBisCO (Form I and II) is widely encoded across all soils and occurs in bacteria from numerous lineages (38 phyla). RuBisCO Form IE is consistently more phylogenetically diverse in soils than in marine ecosystems, suggesting that it may have evolved to function in soil-like environments. A newly discovered deeply branching Form I RuBisCO, Form I triple prime, supports the hypothesis that Form I RuBisCO originated in anaerobic environments. Further, saturated soils harbor more, and more distinct, autotrophic microbes, many of which may use the Calvin-Benson-Bassham cycle or Wood-Ljungdahl pathway for CO2 fixation. Overall, the results indicate that autotrophy is a particularly important metabolism in deep, saturated soils and weathering rock.

The three-dimensional organization of the bacterial chromosome is critical for gene regulation. Chromosome conformation capture (Hi-C) has enabled genome-wide mapping of chromosomal folding, yet ensemble- averaged contact maps entangle biologically specific-folding interactions (SFIs) with nonspecific polymer compaction and mask single-cell heterogeneity. Here, we developed a polymer-based simulation framework in E. coli to address these limitations. We found that a null model of 300,000 random polymer configurations recapitulated the global chromosomal organization observed in Hi-C data, establishing that most Hi-C signals reflect generic polymer behavior in a confined volume. Contrasting null-model predictions with experimental Hi-C data isolated a small subset of SFIs (< 7%) and generated a specific-fold ensemble of 20,000 single-cell conformations that reproduced chromosomal interaction domains and single-cell heterogeneity. SFIs were enriched in the ter region, reduced nucleoid accessibility, colocalized with cryptic prophages, and depleted in positively supercoiled regions. H-NS and MatP emerged as major chromosome-wide and local determinants of SFIs, respectively. Furthermore, high-SFI regions correlated with stress-adaptive genes, whereas low-SFI regions harbored housekeeping genes. Together, our results established that a small number of biologically encoded SFIs superimposed on a polymer background shape the E. coli chromosome and gene expression, providing a quantitative framework for dissecting chromosome architecture and function.

Rapid, specific quantification of erythritol in complex matrices remains challenging due to its structural similarity to sugars like sucrose. To overcome this, we developed a survival-fluorescence cascade screening circuit (uFAST) to engineer a selective, σ54-dependent BmoR-based biosensor. This circuit integrated SacB-mediated negative and mCherry-mediated positive selection to rapidly isolate erythritol-specific mutants. Guided by structural analysis, we optimized BmoR, achieving a 6.03-fold increase in GFP output and a 2.09-fold improvement in apparent affinity (Km). Coupled with a VioABCE-based visual module, the biosensor enabled accurate erythritol quantification in complex matrices, matching HPLC results. Integrated with an erythritol production module and fluorescence-activated droplet sorting (FADS), this platform screened an ARTP-mutagenized library, identifying a strain with 2.88-fold higher titer, yielding 3.51 g/L erythritol in fermentation. The uFAST circuit provides a generalizable framework for developing highly specific biosensors, offering an efficient tool for erythritol detection and strain improvement.

Bacteriocins are ribosomally synthesized antimicrobial peptides with promising applications in biotechnology, particularly in food preservation and animal and human health. Circular bacteriocins are especially attractive due to their head-to-tail cyclized structure, which confers enhanced stability and antimicrobial potency relative to linear peptides. Here, we report an in vitro cell-free protein synthesis system coupled with an enhanced Split Intein-Mediated Ligation platform (IV-CFPS/SIML) for the efficient synthesis of circular bacteriocins through systematic evaluation of cyclization sites and alternative split inteins. Using enterocin AS-48 as a model, we systematically evaluated multiple serine-based cyclization sites in combination with three split inteins, NpuDnaE, Gp41-1, and SspGyrB, to identify configurations supporting efficient splicing and high antimicrobial activity. Gp41-1 emerged as the most effective intein and was subsequently applied to the production of garvicin ML, amylocyclicin, and 27 naturally occurring sequence variants, demonstrating that cyclization site selection, intein identity, and minor sequence variations strongly influence antimicrobial potency and target range. Finally, SIML expression cassettes encoded in pUC-derived vectors enabled in vivo production and functional expression of selected circular bacteriocins in recombinant E. coli. Collectively, these results establish SIML as a versatile platform for in vitro and in vivo production, screening, and functional characterization of known and putative circular bacteriocins.

Strain-resolved metagenomics characterizes microbial communities at nucleotide-level resolution, enabling researchers to differentiate identical from closely related organisms and characterize population structure and gene content variation. Here we introduce ZipStrain, a program that performs highly accurate strain-resolved metagenomics over 500 times faster than available methods while offering superior RAM management. Applied to a dataset of 2,754 samples spanning human populations, we identify a strain-sharing gradient across social relationships, reveal striking variation in clonal structure across bacteria and bacteriophage, and pinpoint genes whose nucleotide identity deviates from genome-wide expectations. ZipStrain is distributed as an open-source Python package and accompanying Nextflow pipeline at https://github.com/OlmLab/ZipStrain.

Flagella are large transenvelope nanomachines but how they transit the peptidoglycan in Gram positive bacteria is poorly understood. A recent model suggested that flagellar basal bodies diffuse in the membrane and become captured at locations in the peptidoglycan with a pore diameter that could accommodate the axle-like flagellar rod. Mutation of penicillin binding protein 1 (PBP1/PonA), a cell wall repair protein thought to decrease peptidoglycan pore frequency and/or size, resulted in a severe growth defect and cell lysis in the ancestral strain of Bacillus subtilis that was dependent on flagellar synthesis. Genetic analysis indicated that toxicity was due to completion of the flagellar hook, which activated the flagellar sigma factor SigD. SigD, in turn, activated a suite of peptidoglycan hydrolases that caused cellular lysis when PBP1 was absent. In addition, mutations that resulted in high levels of the stress response factor Spx could lessen the toxicity, while PBPX, a putative teichoic acid D-alanylase, was required for autolysis. In sum our results indicate that flagellar synthesis, not normally associated with cell viability, causes cell wall stress and under some conditions, cell death. Moreover, our work indicates that cost of envelope integrity by flagellar synthesis may be underappreciated due to strain domestication, and suggests that specialized systems may compensate for the cost of assembly of transenvelope machines in general.