The ability to construct entirely new synthetic DNA sequences de novo is essential to engineering and studying biology. However, the ability to produce long complex synthetic DNA sequences and libraries currently lags behind the ability to sequence and edit DNA. All existing DNA-assembly technologies rely on DNA sequence information found within the final construct to direct assembly between DNA molecules. As a result of this paradigm, these sequences cannot be extensively optimized specifically for assembly without affecting the final sequence. To fundamentally address this challenge, here we show the development of a new DNA assembly technique named Sidewinder that separates the information that guides assembly from the final assembled sequence using DNA three-way junctions. We demonstrate the transformative nature of the Sidewinder technique with highly robust and accurate construction of a 40-piece multifragment assembly, complex DNA sequences of both high GC content and high repeats, parallel assembly of multiple distinct genes in the same reaction and a combinatorial library with a large number of diversified positions across the entire length of the gene for high coverage of a library of 442,368 variants. This technology enables high-fidelity DNA assembly with a misconnection rate at the three-way junction of approximately 1 in 1,000,000. Sidewinder enables high-fidelity DNA assembly by separating the information that guides assembly from the final assembled sequence.

Aminoacyl-tRNAs, charged by aminoacyl-tRNA synthetases with cognate amino acids, are essential for protein synthesis in primary metabolism. Beyond this canonical role, increasing evidence highlights their involvement in natural product biosynthesis. In this review, we first describe the biosynthesis of the aminoacyl nucleoside sulfamate ascamycin from Streptomyces sp. 80H647, highlighting the discovery of the alanyl-tRNA synthetase-like enzyme AcmF through an AI-driven “Forecasting Biosynthesis” approach. Leveraging recent advances in AlphaFold 3, we constructed complex models of a broadened repertoire of aminoacyl-tRNA-dependent enzymes to provide preliminary structure-function insights. These include the isoleucyl-tRNA synthetase-like enzyme SbzA, Gcn5-related N-acetyltransferase-fold transferases, cyclodipeptide synthase family enzymes, and lantibiotic dehydratase-like peptide aminoacyl-tRNA ligases. The catalytic mechanisms of these aminoacyl-tRNA-dependent enzymes are summarized in detail in this review.

Biofilms are intricately associated with life on Earth, enabling functions essential to human and plant systems, but their susceptibility to spaceflight stressors and functional disruption in space remains incompletely understood. During spaceflight, biofilms have largely been considered as potential infrastructure, life support or infection risks. This review focuses on the prevailing beneficial roles of biofilms in human and plant health, and examines evidence of biofilm adaptability in space environments.

Plant-parasitic nematodes are among the most destructive soil-dwelling pests, posing severe threats to global agriculture. However, the interplay between plant metabolites, rhizosphere microorganisms and their potential role in guiding pathogenic nematodes to their hosts remains poorly understood. Here we explored this gap by investigating the role of benzoxazinoids (BXs), a class of defensive metabolites of maize plants, in influencing the host-seeking behavior of root-knot nematodes (RKNs). Our findings revealed that, surprisingly, BXs secreted by maize roots, particularly 6-methoxy-benzoxazolin-2-one, not only enhance RKN infection but also serve as powerful attractants. Remarkably, BX effects were observed only in the presence of a soil matrix. Further analysis demonstrated that 6-methoxy-benzoxazolin-2-one modulates the abundance and composition of rhizosphere bacteria, which in turn play a crucial role in RKN attraction and infection. We discovered that rhizosphere bacteria of BX-producing plants emit volatile compounds such as methyl ketones and 2-phenylethanol, which are then used by RKNs to locate host plants. RKNs detect these volatiles through chemosensory genes, including Mi-odr-1, Mi-odr-7 and Mi-gpa-6. Our study provides mechanistic insights into how RKNs use secondary-metabolite-shaped plant–microbe interactions to enhance their host-seeking behavior and maximize their performance. Root-knot nematodes locate host plants by sensing microbial volatiles shaped by plant defence metabolites, revealing a surprising ecological link between plant chemistry, soil microbes and parasite behavior in the rhizosphere.

Extracellular contractile injection systems (eCISs) are bacteriophage tail-derived toxin delivery complexes in prokaryotes. They play roles in microbial interactions with hosts, using tail fiber proteins for target cell binding. Here, we present a comprehensive analysis of eCIS tail fiber genes in bacterial and archaeal genomes, providing insights into their remarkable diversity, target cells, functional adaptations, and evolutionary dynamics. We identified 3445 eCIS tail fiber proteins encoded in 2585 eCIS loci from 1069 microbes. These fibers can be categorized by five new N-terminal domains responsible for tail fiber attachment to eCIS baseplates. We use structure prediction to classify fibers into 276 structural clusters and 1177 domain fold families, which likely mediate glycan and protein binding on the cell surface of eukaryotes or bacterial targets. DNA sequences encoding these rapidly evolving domains may have been acquired from diverse eukaryotes, bacteria, and viruses. Finally, we experimentally show that a candidate tail fiber from a Paenibacillus eCIS can bind and direct effector injection into THP-1 human monocyte-like cells, possibly binding D-mannose on the cell surface. This study reveals the exceptional diversity of eCIS receptor binding domains, suggests new eCIS target cells, and provides thousands of proteins that can adhere to different cell types. Extracellular contractile injection systems (eCISs) are bacteriophage tail-derived toxin delivery complexes that are present in many prokaryotes. Here, the authors present an analysis of eCIS tail fiber genes in bacterial and archaeal genomes, providing insights into their diversity, target cells, functional adaptations, and evolutionary dynamics.

Engineered bacteriophages are emerging as a promising class of precision antimicrobials at a time when gastrointestinal diseases are increasingly linked to microbial dysbiosis, antibiotic resistance, and disruptions in host–microbe interactions. Conventional antibiotics often provide limited benefit in these settings because they lack selectivity and fail to restore microbial ecology. Advances in synthetic biology and nanotechnology have made it possible to redesign phages with enhanced specificity, expanded functionality, and improved stability, positioning them as versatile tools for microbiota-centered therapies. This review summarizes the major engineering approaches, and examines their applications in inflammatory bowel disease (IBD), colorectal cancer (CRC), and infectious enteritis. Key mechanistic insights into pathogen targeting, immune modulation, and barrier protection are highlighted. Remaining challenges, such as ensuring long-term stability, avoiding resistance development, and enabling scalable manufacturing, are discussed together with emerging interdisciplinary strategies that may advance the clinical translation of personalized phage therapies.

The evolutionary arms race between bacteria and phages drives the development of bacterial antiviral defense systems and phage counter-defense strategies. Restriction–modification (RM) systems protect bacteria by methylating ‘self’ DNA and cleaving unmodified phage DNA. Phages like T-even coliphages evade RM systems by substituting cytosine with 5-hydroxymethyl cytosine (5hmC) or 5-glucosylated hmC (5ghmC). Here, we characterize ‌a single-component antiviral defense system featuring a GIY-YIG endonuclease domain. Biochemical and structural analyses demonstrate that this defense system is a type IV modification-dependent restriction endonuclease that specifically degrades 5hmC- or 5ghmC-modified DNA, and we accordingly name it CMoRE (Cytosine Modification ‌R‌estriction Endonuclease). The crystal structures reveal an N-terminal GIY-YIG nuclease domain and a C-terminal modification-sensing domain. Unique features, including a ‘GIYxY-YIG’ motif and an inhibitory negatively charged loop, distinguish CMoRE as an additional member of the GIY-YIG family. This system not only highlights the evolutionary interplay between phages and bacteria but also presents CMoRE as a potential tool for precise genomic detection of 5hmC in mammals, with implications for epigenetics research and disease diagnostics. In this study, the authors identify and structurally characterize a single-component anti-phage defense system named CMoRE (Cytosine Modification Restriction Endonuclease).

In prokaryotes, bidirectional promoters are pseudo-symmetrical DNA sequences that stimulate divergent transcription. Ubiquitous, and far more likely to drive messenger RNA production than directional promoters, nothing is known about their control. For example, symmetry allows bidirectional promoters to engage RNA polymerase in two possible orientations. As one binding event prevents the other, there is potential for regulation at this step. Here, we show that basal transcription, from all five tested bidirectional promoters, is too low for RNA polymerase competition. Hence, synthesis of one RNA does not impact the divergent pair. Conversely, if transcription in one direction is substantially activated, divergent RNA production can be repressed. Often, this results from RNA polymerase competition alone. Unexpectedly, this also impacts population-level gene expression noise. Specifically, if transcription is constrained, by RNA polymerase interference, cell-to-cell variation is reduced. We anticipate that our findings will help to establish rules for understanding bidirectional promoters, which have hardly been studied, in many bacteria.