The hybridization chain reaction (HCR) is an amplification method recognized for detecting analytes present in trace quantities owing to its high specificity, sensitivity, and straightforward approach. Simultaneously, G-quadruplex DNAzymes augment HCR-based biosensors, serving as transducers due to their elevated catalytic activity and nonenzymatic methodology. The current review aims to provide readers with a critical overview of significant aspects of biosensors that utilize HCR for analyte detection amplification and G-quadruplex as transducers, focusing on the latest activities of global researchers. A bibliometric analysis was conducted to identify key research topics in HCR and G-quadruplex-based sensors. Further, the review aims to elucidate the advantages and disadvantages of each strategy employed for detection using this sort of sensor. Specific sections have been included to critically evaluate the potential of these biosensors across many scientific domains. In conclusion, the limitations of the existing sensor that restrict its applicability in real-time scenarios have been identified for subsequent enhancement. Plausible strategies that could enhance these sensors and emerging scientific fields that could benefit from HCR and G-quadruplex DNAzyme-based sensors have been elucidated.

Maximizing the nitrogen fixation occurring in rhizobia-legume associations represents an opportunity to sustainably reduce nitrogen fertilizer inputs in agriculture. High-throughput measurement of symbiotic traits has the potential to accelerate the identification of elite rhizobium/legume associations and enable novel research approaches. Plasmid-ID technology, recently deployed in Rhizobium leguminosarum, facilitates the concurrent assessment of rhizobium nitrogen-fixing effectiveness and competitiveness for root nodulation. This study adapts Plasmid-ID technology to function in Sinorhizobium species that are central models for studying rhizobium-legume associations and form economically important symbioses with alfalfa. New Sino-Plasmid-IDs were developed and tested for stability and their ability to measure competitiveness for root nodulation and nitrogen-fixing effectiveness. Rhizobial competitiveness is measured by identifying strain-specific nucleotide barcodes using next-generation sequencing, whereas effectiveness is measured by GFP fluorescence driven by the synthetic nifH promoter. Sino-Plasmid-IDs allow researchers to efficiently study competitiveness and effectiveness in a multitude of Sinorhizobium strains simultaneously.

Interactions between microbes and their mobile genetic elements (MGEs), including viruses and plasmids, are critical drivers of microbiome structures and processes. CRISPR-Cas systems are known to be important regulators of these host-MGE interactions, but a global understanding of CRISPR-Cas diversity, activity, and roles across Earth's biomes is still lacking. Here, we use an optimized computational approach to search short-read data and collect ~800 million CRISPR spacers across ~450,000 public metagenomes. Comparing spacers across samples and taxa revealed a high population diversity for CRISPR loci overall, with typically only a small subset of spacers detected as prevalent and conserved within a microbial population. From this extensive CRISPR spacer dataset, we identified 1.18 billion hits between 41 million spacers and 2.5 million viruses and plasmids. Prevalent and conserved spacers were over-represented in these MGE-matching spacers, and CRISPR spacers frequently matched multiple MGEs, consistent with a positive selection pressure associated with MGE targeting. Focusing on the role of CRISPR as antiphage defense, we observed surprising cases of viruses targeted by microbes not expected to be viable hosts. These were more frequent for viruses encoding a diversification mechanism (DGR) for their host attachment proteins, and associated with a reduced rate of escape mutations. This suggests that broad spacer targeting may derive from the recurring entry of a virus genome into a non-host microbial cell, leading to some viruses being targeted by taxonomically diverse microbes well outside of their actual host range. Taken together, this petabase-scale exploration of CRISPR arrays in nature outlines the extensive diversity of CRISPR array loci across microbiomes. It highlights several key genomic and ecological parameters driving the activity of CRISPR arrays that are likely influencing strain-level diversification and selection processes within microbial populations.

3-prime end sequencing (3′-seq) is a high-throughput sequencing technique that is used to specifically quantify the changes in 3′-end formation of transcripts in bacterial cells, which is increasingly being utilized to address fundamental questions regarding transcription termination and pausing across a range of different bacterial species. However, the growing number of 3′-seq studies is accompanied by an increase in study-specific 3′-seq data analysis approaches. Thus, differences in a number of factors including: experimental design, data collection approaches, analysis methodologies, and interpretation decisions, make it challenging to confidently compare results derived from different studies, even those that were performed on the same organism. To assess the potential severity of these discrepancies, we used PIPETS, a statistically robust and genome-annotation agnostic 3′-seq analysis package, to study Escherichia coli 3′-seq data sets from three different groups collected under similar conditions. By using a consistent analysis and results interpretation approach, we identified large disparities in the characteristics of the raw 3′-seq data between each of the studies, despite all three studies using the same strain and very similar reported experimental conditions. Additionally, we found strand-specific inconsistencies, with some data sets having reference strand 3′-seq read coverage distributions that differed greatly from the complement strand within the same replicate. Finally, when the 3′-seq distribution profiles of the three E. coli studies are compared to studies from four additional bacteria, we identified 3′-seq results clustering patterns that are not explained by phylogenetic similarity between organisms. With the large differences seen between data sets from the same organism as well as the inconsistencies seen between replicates from the same data sets, we urge the field to reconsider the assumptions around 3′-seq data homogeneity and move towards consistent analysis approaches, and cautious interpretation of the data.

TNT (2,4,6-trinitrotoluene) from unexploded ordinances is a common environmental pollutant near munitions factories, military training sites, and areas of armed conflict. As physical and chemical approaches for TNT remediation are costly and difficult to scale, in situ bioremediation is an attractive alternative. TNT is a phytoaccumulative pollutant. Herbivores engineered to detoxify TNT are a potentially cost-effective platform to bioremediate large areas of contaminated sites while grazing or browsing. As a first step towards engineering herbivores with metabolic capabilities for TNT degradation, we engineered the animal genetic model, Drosophila melanogaster, to screen a library of microbial nitroreductases that can catalyse the initial degradation steps required for TNT degradation. We find strong, cofactor-dependent activity in fly lysates engineered to express NsfA from Escherichia coli, and NsfI from Enterobacter cloacae. bioremediation

Sialyloligosaccharides, critical components of human milk oligosaccharides, are pivotal for infant immunity and development. Recent advances have identified sialidases with trans-sialylation activity as promising biocatalysts for cost-effective sialyloligosaccharide synthesis using economical substrates. Herein, we report a novel sialidase (KsSiase1) from Kribbella sp. ALI-6-A with enhanced catalytic properties and broad substrate specificity, showing preferential hydrolysis: α2,3 > α2,6 > α2,8. The enzyme demonstrated robust trans-sialidase activity, achieving 3.40 g/L sialyloligosaccharide production using 0.5 U/mL KsSiase1, 6% lactose, and 8% cGMP. Implementing a direct-fed enzymatic strategy in raw milk with 1 U/mL KsSiase1 and 6% cGMP, we achieved in situ sialyloligosaccharide enrichment (2.49 g/L) while concomitantly reducing lactose. This biocatalytic system establishes a sustainable, scalable platform for functional food production, merging lactose valorization with sialyloligosaccharides biosynthesis through enzyme–substrate adaptability, addressing both industrial demands and nutritional needs in infant formula development.

Conjugative plasmids drive bacterial evolution and niche adaptation. Their carriage often corresponds with changes to the host transcriptome, but it is unknown during which stage of the conjugation process these transcriptional changes are initiated, and how gene transfer is modulated as they occur. This is because they are normally studied after their full acquisition by conjugation has already occurred, not during the process. Here, active RP4 conjugation revealed that the transcriptional response to plasmid acquisition is almost immediate and is host and surface factor dependent. Responses included activation of non-SOS stress pathways, motility, exopolysaccharide production, anaerobic respiration, and metabolic adaptation. These responses were not a barrier to conjugation, indicating that their significance relates to host homeostasis rather than gene transfer. The recipient could prevent these responses by blocking conjugation through capsule expression. RP4 expression was also inhibited by recipient capsule, since capsule physically blocked donor-recipient contact. Unexpectedly, within a population, RP4 was found to exploit single cell variation in recipient capsule thickness and to successfully conjugate with recipient bacteria with reduced capsule. These results show a novel interplay between plasmid and surface architecture across diverse species that controls the conjugation transcriptional landscape, with important ramifications for plasmid dissemination and evolution.

During CRISPR-Cas adaptation, prokaryotic cells become immunized by the insertion of foreign DNA fragments, termed spacers, into the host genome to serve as templates for RNA-guided immunity. Spacer acquisition relies on the Cas1-Cas2 integrase and accessory proteins like Cas4, which select DNA sequences flanked by the protospacer adjacent motif (PAM) and insert them into the CRISPR array. It has been shown that in type II-A systems selection of PAM-proximal prespacers is mediated by the effector nuclease Cas9, which forms a 'supercomplex' with the Cas1-Cas2 integrase and the Csn2 protein. However, the supercomplex structure and the role of the ring-like Csn2 protein remain unknown. Here, we present cryo-electron microscopy structures of the type II-A prespacer selection supercomplex in the DNA-scanning and two different PAM-bound configurations. Our study uncovers the mechanism of Cas9-mediated prespacer selection in type II-A CRISPR-Cas systems, and reveals the role of the accessory protein Csn2, which serves as a platform for the assembly of Cas9 and Cas1-Cas2 integrase on prespacer DNA, reminiscent of the sliding clamp in DNA replication. Repurposing of Cas9 by the CRISPR adaptation machinery for prespacer selection characterized here demonstrates Cas9 plasticity and expands our knowledge of the Cas9 biology.

The three main types of live bacterial therapies – probiotics, fecal/microbiome transplants, and engineered bacterial therapies – hold immense potential to revolutionize medicine. While offering targeted and personalized treatments for various diseases, these therapies also carry risks such as adverse immune reactions, antibiotic resistance, and the potential for unintended consequences. Therefore, developing and deploying these therapies necessitates a robust regulatory framework to protect public health while fostering innovation. In this paper, we propose a novel conceptual tool – the Ladder of Regulatory Stringency and Balance—which can assist in the design of robust regulatory regimes which encompass medicine practices based not only on definitive Randomized Controlled Trials (RCTs), but also on meta-analyses, observational studies, and clinicians experience. Regulatory stringency refers to the strictness of regulations, while regulatory balance concerns the degree of alignment between the regulatory framework governing a technology and the actual risks posed by specific products within that technology. Focusing on the US regulatory environment, we subsequently position the three types of live bacterial therapies on the Ladder. The insight gained from this exercise demonstrates that probiotics are generally positioned at the bottom of the Ladder, corresponding to low-stringency regulation, with a proportionate regulatory balance. However, probiotics intended for high-risk populations are currently subject to low-stringency regulations, resulting in under-regulation. Our analysis also supports the conclusion that fecal microbiota transplants (FMT) for recurrent Clostridium difficile infection should be positioned close to but below the threshold for under regulation by the U.S. Food and Drug Administration (FDA), and we recommend improved donor screening procedures, preservation and processing, storage, and distribution. Our framework can serve as a scale to assess regulatory gaps for live bacterial therapies and to identify potential solutions where such gaps exist.