RMH

The E. coli chromosomal origin, oriC, contains a duplex-unwinding element (DUE) flanked by two clusters of the initiator DnaA-binding sites (DnaA boxes). ATP-DnaA oligomerizes to construct Left- and Right–DnaA subcomplexes on these clusters. These complexes coordinately promote DUE unwinding and DnaB helicase loading onto the unwound region for replication initiation. However, despite the strong conservation of DnaA and DnaB across bacterial species, oriC architectures vary largely. Remarkably, bipartite and bilateral configurations contain the two DnaA box-clusters separated by an insertion of the dnaA gene or positioned on both sides of the DUE, respectively. Here, we demonstrate that E. coli oriC variants mimicking these configurations remain fundamentally functional. Notably, DnaB loading activity of these variants showed increased dependence on single-stranded DUE binding by the relocated DnaA subcomplex and on the AT cluster upstream of the DUE, reflecting reduced stability of DUE unwinding. Consistent with these in vitro results, these variants supported cellular replication initiation with only slow cell growth rates. Together, these findings reveal remarkable architectural flexibility, mechanistic robustness, and a conserved mechanism in oriC. Also, those highlight unwinding stability as a key determinant of oriC adaptation to rapid cell growth, providing insight into the evolution of oriC architecture.

The rational engineering of non-model microorganisms is often constrained by the scarcity of high-performance genetic regulatory tools. To overcome this limitation, we developed ARCANE – a reprogrammable circuit architecture for non-model engineering – featuring a cascaded design in which an inducer controls orthogonal bacterial transcription factors (bTFs), which in turn drive both activation and repression of endogenous promoters. This architecture provides strong insulation from host regulatory networks, minimizes leakage, and enables a broad dynamic range. As a proof-of-concept, we implemented ARCANE in the industrially significant non-model yeast, Yarrowia lipolytica. The resulting genetic switch exhibited exceptionally precise and tunable control over both single reporter genes and a multi-gene carotenoid biosynthesis pathway. Beyond providing a powerful regulatory tool for Y. lipolytica, ARCANE offers a versatile and transferable modular foundation for programmable gene control in emerging microbial platforms, paving the way for scalable synthetic biology in non-model systems.

We present a simple, low-cost, and efficient protocol for fluorescent staining of Bacillus spp. endospores using the thioflavin T (ThT) dye. The article provides step-by-step procedures for culturing Bacillus paranthracis, Bacillus thuringiensis (2 strains), and Bacillus subtilis, purifying endospores from pure cultures, and staining them with ThT. ThT-stained endospores retain fluorescence for at least 2 years when stored at 4°C in the dark and remain fluorescent following formaldehyde fixation. Importantly, ThT staining does not impair spore viability, and stained endospores germinate normally. In contrast, vegetative Bacillus cells exhibit minimal to no ThT fluorescence. Overall, this robust and straightforward staining protocol is well suited for applications requiring fluorescently labeled endospores, including fluorescence microscopy, and provides a convenient plate-based readout for germinant screening and related assays. methods

The instability of nitroaromatic compounds or nitroarenes in the presence of microbial cells has hindered the ability to biomanufacture these industrially and pharmaceutically relevant chemicals. Although nitroreductase (NTR) gene deletions have been linked to nitroarene stability, the extent of this effect is unclear. Here we perform a comprehensive analysis of bacterial NTR activity in cells by engineering strains of E. coli that contain up to 15 knockouts of known and candidate NTR genes. We evaluate the stability of over 20 exogenously supplemented nitroaromatic compounds to wild-type and engineered strains. For several chemistries, such as di-nitro compounds and nitro-aldehydes, our engineered nitroaromatic reductase knockout strains enable retention of compounds. We leverage these tools and insights to improve or enable nitroaromatic compound biosynthesis, including the biocatalytic transformation of an amine precursor to a nitro compound, the biosynthesis of nitrobenzaldehydes and the combined semi-synthesis and site-specific incorporation of nitrophenylalanines from supplemented nitrobenzaldehydes. This work advances access to nitro functional group chemistry in cell-based biocatalysis, metabolic engineering and synthetic biology. Nitroarenes are unstable in microbial cultures, limiting the biomanufacturing of these industrially and pharmaceutically important chemicals. Genome engineering of nitroreductase-deficient E. coli strains has now been shown to improve nitroarene retention during fermentation, expanding access to nitro functional group chemistry in whole-cell biocatalysis, metabolic engineering and synthetic biology.

Nitrogen (N) fertilizers are key drivers of high yields in rice (Oryza sativa L.), yet increasing N inputs often deliver diminishing improvements in N use efficiency (NUE) and can exacerbate N losses via volatilization, leaching, and runoffs, with associated environmental impacts. The rice phyllosphere microbiome, which consists of microbes inhabiting leaf surfaces and internal tissues, has the potential to influence foliar nutrient turnover, N metabolism, and stress responses and may therefore influence internal NUE (IEN) and, under reduced fertilizer-N input, potentially affect agronomic efficiency of applied N (AEN). Here, we summarize the major steps of rice N metabolism (uptake, assimilation, transport, redistribution, and remobilization) and discuss microbial pathways that could modulate N use, including foliar N transformations, microbially mediated N inputs, and indirect effects through hormonal regulation, stress buffering, and microbe–microbe interactions. Key knowledge gaps remain in quantification of underlying mechanistic processes, field robustness across seasons and sites, and genotype/microbiome matching under reduced-N management.

Dense bacterial populations often navigate complex and heterogeneous environments using collective behaviors including group motility. Whether local interactions within a population can regulate collective order is unclear. Here we show using live imaging, single-cell tracking and simulations that Pseudomonas aeruginosa regulates collective order by mechanosensing cell–cell collisions, enabling adaptive surface exploration. Collision-induced reversals suppress collective cell alignment in dense bacterial populations. While a non-reversing mutant remains locked in cohesive near-linear motion regardless of density, wild-type cells adapt to local crowding, transitioning from isotropic motion in crowds to unidirectional exploration at the colony front. With increasing density, collisions generate information that feeds back on single-cell motility to regulate collective order, providing a navigational advantage. This advantage extends to spatially structured environments, such as micromazes, where collision-induced reversals prevent cells from becoming trapped at physical boundaries. These findings reveal that bacterial collective organization is not merely an emergent consequence of physical interactions, but a feedback-regulated state. Density-dependent regulation of collective order through collision sensing and collision-induced reversals provides a navigational advantage to Pseudomonas aeruginosa during surface colonization and navigation through micromazes.

Current genetic engineering technology that based on complementary base pairing for target recognition is a powerful tool to control protein expression at DNA or RNA levels. Nonetheless, limited research has utilized genetic strategies to directly manipulate proteins. To address this gap, we have developed a method of genetically engineered RNA inducers of proximity (GENRIP), one strategy that leverages RNA structure-dependent protein recognition to precisely regulate protein activity. GENRIP is a modular and bifunctional RNA comprising two kinds of motifs: a conserved RNA motif that recruits endogenous RNA-binding enzymes for protein modification, and a variable motif responsible for recognizing different proteins of interest (POIs). By inducing proximity between effector enzymes and POIs, GENRIP enables precise post-translational modification of POIs. Using the RNA-binding E3 ligase MEX3A and RNA-binding kinase CamK as model effectors, we have demonstrated its capability to achieve ubiquitination-mediated degradation and phosphorylation-induced activation of POI, respectively. Furthermore, GENRIP offers superior advantages in modularity, programmability, precise subcellular localization targeting, and cell-type selectivity. This approach represents a paradigm shift in genetic engineering, holding profound implications in the field of post-translational protein regulation. Current genetic engineering techniques enable protein modulation at the DNA/RNA levels. Here, the authors design an RNA platform to bring target protein and effector enzyme into proximity, realizing direct protein regulation.