In nature, viruses frequently evolve overlapping genes (OLG) in alternate reading frames of the same nucleotide sequence despite the drastically reduced protein sequence space resulting from the sharing of codon nucleotides. Their existence leads one to wonder whether amino acid sequences are sufficiently degenerate with respect to protein folding to broadly allow arbitrary pairs of functional proteins to be overlapped. Here, we investigate this question by engineering synthetic OLGs using state-of-the-art generative models. To evaluate the approach, we first design overlapped sequences targeting two different protein families. We then encode distinct highly ordered de novo protein structures and observe surprisingly high in silico and experimental success rates. This demonstrates that the overlap constraints under the structure of the standard genetic code do not significantly restrict simultaneous accommodation of well defined 3D folds in alternative reading frames. Our work suggests that OLG sequences may be frequently accessible in nature and could be readily exploited to compress and constrain synthetic genetic circuits.

Archaeal transcription is a hybrid of eukaryotic and bacterial features: a RNAP II-like polymerase transcribes genes organized in circular chromosomes within cells devoid of a nucleus. Consequently, archaeal genomes are depleted in canonical transcription factors (TFs) found in other domains of life. Here we outline the discovery of a cryptic archaea-specific family of ligand-binding TFs, called AmzR (Archaeal Metabolite-sensing Zipper-like Regulators). We identify AmzR through an evolution-based genetic screen and show that it is a negative regulator of methanogenic growth on methylamines in the model archaeon, Methanosarcina acetivorans. AmzR binds its target promoters as an oligomer using paired basic α-helices akin to eukaryotic leucine zippers. AmzR also binds methylamines, which reduces its DNA-binding affinity, and allows it to function as a one-component system commonly found in prokaryotes, while containing a eukaryotic-like DNA-binding motif. The AmzR family of TFs are widespread in archaea and broadens the scope of innovations at the prokaryote-eukaryote interface.

Increasing drought frequency poses a significant threat to agricultural productivity. A promising strategy to enhance crop resilience against drought is the utilization of root channels left by winter cover crops, which can improve access to subsoil water and nutrients for subsequent cash crops like maize (Zea mays L.). The impact of drought on bacterial communities inhabiting these root channels remains largely unknown. Here, we investigated drought-induced shifts in maize rhizosphere bacterial communities and their functional adaptation in cover crop root channels across three soil types in northern Germany (Luvisol, Podzol, and Phaeozem).  Using a multi-omics approach (16S rRNA gene amplicon sequencing, qPCR, and metaproteomics), we identified significant taxonomic and functional responses to drought. A rise in the abundance of K-strategist bacterial communities indicate a shift towards stress-tolerant populations with drought. Under drought stress, the relative abundances of Acidobacteriota, Actinomycetota, Planctomycetota, and Pseudomonadota increased, while Chloroflexota, Methylomirabilota, Ca. Patescibacteria, and Verrucomicrobiota declined. Functional analyses revealed that drought-stressed aerobic taxa among the Pseudomonadota and Verrucomicrobiota upregulated the glyoxylate cycle, potentially enhancing carbon and energy conservation, and increased antioxidant defences (catalase-glutathione peroxidase and methionine cycle-transsulfuration pathway). These drought-mitigating strategies were especially pronounced in root channels formed by Brassicaceae and Poaceae cover crops in the Luvisol and Podzol soils. These findings demonstrate the functional plasticity of rhizosphere bacterial communities in reused root channels in response to drought, highlighting the potential to leverage microbiome-mediated resilience for agricultural practices.

Plant-associated microorganisms interact with each other and host plants via intricate chemical signals, offering multiple benefits, including plant nutrition. We report such a mechanism through which the rhizosphere microbiome improves plant growth under sulfur (S) deficiency. Disruption of plant S homeostasis resulted in a coordinated shift in composition and S-metabolism of the rhizosphere microbiome. Using this insight, we developed an 18-membered synthetic rhizosphere bacterial community (SynCom) that rescued growth of Arabidopsis and a leafy Brassicaceae vegetable under S-deficiency. This beneficial trait is taxonomically widespread among SynCom members, with bacterial pairs predominantly providing synergistic benefits to host plants. Notably, stronger competitive interactions among SynCom members conferred greater fitness benefits to the host, suggesting a trans-kingdom fitness tradeoff. Finally, guided chemical screening, deletion knockout mutant, and targeted metabolomics identified and validated microbially released glutathione (GSH) as the necessary bioactive signal that orchestrates plant-microbe (trans-kingdom) fitness tradeoff and improves plant growth under sulfur limitation.

Phages are typically classified as temperate, integrating into host genomes, or lytic, replicating and killing bacteria. Lytic phages are not expected in bacterial genome sequences, yet our analysis of 3.6 million bacterial assemblies from 1,226 species revealed >100,000 complete lytic phage genomes, which we term Bacterial Assembly-associated Phage Sequences (BAPS). This represents a ~five-fold increase in the number of phages with known hosts and fundamentally reshapes our understanding of phage biology. Identifying BAPS has enabled the discovery of entirely novel phage clusters, including clusters distantly related to Salmonella Goslarviruses in E. coli, and Shigella, while significantly expanding known genera such as Seoulvirus (from 16 to >300 members). Notably, close relatives of therapeutic lytic phages were also detected, suggesting clinical isolate sequencing unknowingly archives viable phage candidates. The discovery of complete, lytic phage genomes within bacterial assemblies challenges long-standing assumptions and reveals a vast, untapped reservoir of phages.

Bacillus methanolicus represents a thermophilic methylotroph whose methanol utilization depends on plasmid-encoded genes. It serves as a unique model for deciphering plasmid-dependent methylotrophy and an ideal chassis for low-carbon biomanufacturing using CO2-derived C1 substrates. Despite its evolutionary uniqueness and industrial potential, the lack of synthetic biology tools has hindered both mechanistic understanding and strain engineering. Here, we present a comprehensive synthetic biology platform comprising a high-efficiency electroporation protocol, a CRISPR method enabling robust and multiplex genome editing, diverse neutral loci for gene integration and overexpression, and a cloud-based genome-scale metabolic model iBM822 for user-friendly biodesign. Leveraging this toolkit, we systematically dissected plasmid-dependent methylotrophy, host restriction-modification systems, and functional significance of the chromosomal methylotrophic genes through targeted deletion. To address plasmid loss-induced strain degeneration, we integrated the large endogenous plasmid pBM19 into the chromosome for stable and intact methylotrophic growth. Finally, by integrating metabolic modeling with CRISPR editing, we engineered L-arginine feedback regulation to achieve the first L-arginine biosynthesis from methanol. This study establishes a synthetic biology framework for B. methanolicus, promoting mechanistic exploration of methylotrophy and low-carbon biomanufacturing.

Formate is a single-carbon compound that is challenging to assimilate, including when assimilation involves a CO2 intermediate that can diffuse from the cell. Mutations that overcome such challenges can be identified through adaptive laboratory evolution. We evolved the anoxygenic phototrophic bacterium Rhodopseudomonas palustris to use formate as the sole carbon source. Through gene deletions, we determined that formate is assimilated via oxidation to CO2 by formate dehydrogenase followed by CO2 fixation by the Calvin cycle. However, this pathway had no clear link to three genes that were commonly mutated in evolved isolates: (i) ribB, a flavin synthesis enzyme, (ii) ppsR2, a repressor of light-harvesting genes, and (iii) RPA0893, a regulator of unknown function. A RibB mutation was necessary and sufficient for formate assimilation and improved formate oxidation. PpsR2 mutations occurred early, facilitated formate assimilation, and caused elevated pigmentation. Pigment production generates CO2 and alkaline conditions that, along with intracellular chromatophore membranes, could represent a rudimentary but important CO2-concentrating mechanism. RPA0983 mutations emerged late and facilitated formate assimilation. RPA0983 proximity to a CO2-liberating pigment synthesis gene suggests a similar effect as PpsR2 mutations. Our findings reveal unintuitive pathway intersections that could have broad implications for formate and CO2-utilizing organisms.

Polyhydroxyalkanoates are biopolyesters synthesized and stored in intracellular granules by diverse prokaryotes. Despite intense research efforts and prior evidence of a rather widespread phylogenetic occurrence of the related genetic machinery, reports on extreme thermophilic and hyperthermophilic polyhydroxyalkanoates producers remain scarce. However, thermophilic cell factories for bioplastic production would serve as an excellent example of Next-Generation Industrial Biotechnology. In this study, we aim to address this research gap by establishing a bioinformatics pipeline to mine genomes of extremely and moderately thermophilic microorganisms for signatures of potential polyhydroxyalkanoate PHB production. Based on a collection of verified protein sequences of polyhydroxyalkanoate polymerase PhaC, the key biosynthetic enzyme, carefully curated sets of thermophilic bacterial and archaeal genomes were screened. This revealed that although PhaC-encoding genes are prevalent in diverse moderately thermophilic bacteria, they are absent in the considered extreme thermophilic bacteria. In contrast, a few limited examples of extreme thermophilic archaea were found to encode putative phaC genes embedded within a typical polyhydroxyalkanoate synthesis operon in their genomes, namely within the genera Ferroglobus, Geoglobus and Archaeoglobus, while no hits were found in extreme thermophilic bacteria. The latter included Thermus thermophilus, which was previously reported as a polyhydroxyalkanoates producer. This was refuted in our bioinformatics analysis and moreover, the predicted absence of polyhydroxyalkanoates synthesis in T. thermophilus was experimentally confirmed by employing various extraction and analytical methods. Based on the findings in this study, we conclude that polyhydroxyalkanoate production is very scarce in extreme thermophiles and hyperthermophiles, for reasons that remain to be elucidated.

Prophages are prevalent features of bacterial genomes that can reduce susceptibility to lytic phage infection, yet the mechanisms involved are often elusive. Here, we identify a small RNA (svsR) encoded by the lambdoid prophage NC-SV in adherent-invasive Escherichia coli (AIEC) strain NC101 that confers resistance to lytic coliphages. Comparative genomic analyses revealed that NC-SV-like prophages and svsR homologs are conserved across diverse Enterobacteriaceae. Transcriptional analyses reveal that svsR represses maltodextrin transport genes, including lamB, which encodes the outer membrane maltoporin LamB--a known receptor for multiple phages. Nutrient supplementation experiments show that maltodextrin enhances phage adsorption, while glucose suppresses it, consistent with established effects of these sugars on lamB expression. In vivo, we compared wild-type NC101 and a prophage-deletion strain (NC101deltaNC-SV) in mice to assess the impact of NC-SV on lytic phage susceptibility. Although intestinal E. coli densities remained stable across groups, animals colonized with NC101 exhibited markedly reduced phage burdens in both the intestinal lumen and mucosa compared to mice colonized with NC101deltaNC-SV. This reduced phage pressure was associated with increased dissemination of NC101 to extraintestinal tissues, including the spleen and liver. Together, these findings highlight a nutrient-responsive, prophage-encoded mechanism that protects AIEC from phage predation and may promote bacterial persistence and dissemination in the inflamed gut.

Over the past two decades, metagenomics has greatly expanded our understanding of microbial phylogenetic and metabolic diversity. However, most microbial taxa remain uncultured, hindering research and biotechnological applications. Isolating environmental anaerobes using traditional methods is particularly cumbersome and low throughput. Here, we present a novel, high-throughput approach for the cultivation and isolation of anaerobes, which involves trapping and growing single microbes within selectively permeable hydrogel capsules followed by fluorescence-activated cell sorting to distribute compartmentalized isolates into liquid medium for further growth. We show that diverse anaerobes can grow within capsules and that slower-growing ones (e.g. methanogens) can be enriched with this platform. We also applied this approach to isolate anaerobes from soil, including strains of the sulfate-reducing bacteria Desulfovibrio desulfuricans and Nitratidesulfovibrio vulgaris. Overall, this work introduces a robust, high-throughput alternative to traditional techniques for isolating environmental anaerobes and expands the emerging set of microfluidics-based tools for the cultivation of novel taxa.