Could codon composition condition the immediate success and the orientation of horizontal gene transfer? Horizontal gene transfer represents a change in the genome of expression of the transferred gene, and experimental evidence has accumulated indicating that the codon composition of a sequence is an important determinant of its compatibility with the translation machinery of the genome in which it is expressed. This suggests that codon composition influences the phenotype and the fitness conferred by a transferred gene and thus the immediate success of the transfer. To directly test this hypothesis, we characterized the resistance conferred by synonymous variants of a gentamicin resistance gene in three bacterial species: Escherichia coli, Acinetobacter baylyi and Pseudomonas aeruginosa. The strongest determinant of the resistance level conferred was the species in which the resistance gene was transferred, very likely because of important differences in the copy number of the plasmid carrying the gene. Significant differences in resistance were also found between synonymous variants within each of the three species, but more importantly, there was a strong interaction between species and variant: variants conferring high resistance in one species confer low resistance in another. However, the similarity in codon usage between the synonymous variants and the host genome only explained part of the phenotypic differences between variants in one species, P. aeruginosa. Further investigation of alternative explanations did not reveal common universal mechanisms across our three bacterial species. We conclude that codon composition can be a determinant of post-horizontal gene transfer success. However, there are multiple paths leading from synonymous sequence to phenotype, and sensitivity to these different paths is species-specific.

Termination of DNA replication is a surprisingly complex process that contributes critically to genome stability and cell viability. And even though progress was made to establish the consequences that arise if termination is going awry, the precise molecular mechanisms of fork fusion events and the coordination with key factors that ensure that DNA replication is brought to a successful conclusion remain poorly understood. We therefore investigated replication termination in E. coli, focusing specifically on the interplay between replication fork fusions and genomic stability, the Tus–ter replication fork trap, and key DNA-processing enzymes. By utilizing whole genome sequencing, immunoblotting, and recombination reporter assays, we demonstrate that local hyper-recombination is induced wherever forks meet and that the combined loss of factors such as RecG helicase and 3′ exonucleases causes extreme over-replication in the terminus region of the chromosome. Unexpectedly, cells lacking Tus exhibit elevated R-loop levels, revealing an unanticipated connection between the fork trap and R-loop metabolism. These findings underscore the complexity of replication termination and its central role in maintaining bacterial genome stability, while providing mechanistic insights with implications for understanding replication termination in more complex organisms and developing new antimicrobial strategies.

The canonical PAM site TTTV (where V = A, G, or C) is widely used in the design of CRISPR-Cas12a systems for both genome editing and diagnostic applications. Although several non-canonical protospacer-adjacent motifs (PAM) have been identified, they generally exhibit weak Cas12a cleavage activity. In this study, we find that increasing the reaction temperature to 45 °C or higher allows the identification of numerous non-canonical PAMs with trans-cleavage activity comparable to that of canonical PAMs, while displaying only weak cis-cleavage activity. Moreover, we observe that combining these non-canonical PAMs with elevated temperatures significantly enhances the Cas12a system’s ability to discriminate highly similar sequences. Based on these findings, we develop a non-canonical PAM-mediated, poikilothermal, one-pot CRISPR-Cas12a detection platform (POP-CRISPR), which demonstrates substantial improvements in sensitivity, specificity, speed, and target adaptability for nucleic acid detection compared to existing methods. These advantages are validated through the reliable detection of clinical samples, including those of Human papillomavirus (HPV), Mycoplasma pneumoniae (MP), and its drug-resistant strains. Additionally, we show that POP-CRISPR enables rapid, on-site pathogen detection within 20 min, using a fast sample processing protocol and a miniaturized detection device. CRISPR-Cas12a diagnostics are limited by strict PAM requirements. Here, authors show that higher temperatures activate numerous noncanonical PAMs, enabling a versatile one-pot platform that improves sensitivity, specificity, and rapid on-site pathogen detection.

Phylogenetic signal describes the tendency of related organisms to resemble each other in morphology and function. Related organisms tend to also live in similar ecological niches, which is termed niche conservatism. The concepts of both phylogenetic signal and niche conservatism are widely used to understand crucial aspects of evolution and speciation, and they are well established in animals and plants. However, although assumed to be present, the extension of these concepts to microorganisms is challenging to assess. Here we hypothesize that two closely related microbial species should be found in samples with similar community compositions, reflecting their ecological similarity. We propose ‘community conservatism’ to refer to this phenomenon and leverage a database with millions of samples and hundreds of thousands of pairs of microorganisms to assess their relatedness and the similarity of the communities they occupy. Our findings reveal that community conservatism can be observed globally in all environments and phyla tested, over nearly all taxonomic ranks, but to varying extents. Analyzing community conservatism shows promise to advance our understanding of evolution, speciation and the mechanisms governing community assembly in microorganisms. Furthermore, we propose that it can be used to reintegrate ecological parameters into operational taxonomic unit delimitation. This study reveals that closely related microorganisms tend to inhabit similar communities across all major environments and phyla. The authors term this phenomenon ‘community conservatism’, extending the ecological concepts of phylogenetic signal and niche conservatism to the microbial world.

Uneven translation rates resulting from mRNA context, tRNA abundance, nascent amino acid sequence or various external factors play a key role in controlling the expression level and folding of the proteome. Inverse toeprinting coupled to next-generation sequencing (iTP-seq) is a scalable in vitro method for characterizing bacterial translation landscapes, complementary to ribosome profiling (Ribo-seq), a widely used method for determining transcriptome-wide protein synthesis rates in vivo. In iTP-seq, ribosome-protected mRNA fragments known as inverse toeprints are generated by using RNase R, a highly processive 3′ to 5′ RNA exonuclease. Deep sequencing of these fragments reveals the position of the leading ribosome on each mRNA with codon resolution, as well as the full upstream coding regions translated by these ribosomes. Consequently, the method requires no a priori knowledge of the translated sequences, enabling work with fully customizable transcript libraries rather than previously sequenced genomes. As a standardized framework for inverse toeprint generation, amplification and sequencing, iTP-seq can be used in combination with different types of libraries, in vitro translation conditions and data-analysis pipelines tailored to address a range of biological questions. Here, we present a robust protocol for iTP-seq and show how it can be integrated into a broader workflow to enable the study of context-dependent translation inhibitors, such as antibiotics. The time required to complete this workflow is ~10 d, and the workflow can be carried out by an experienced molecular biologist, with data analysis also requiring a working knowledge of command-line tools and Python scripts. This protocol describes inverse toeprinting coupled to next-generation sequencing, an in vitro approach to characterize bacterial translation at codon resolution that can accommodate custom synthetic libraries and various translation perturbations.

Nitrogen-fixing microbes are a primary contributor of this important nutrient to the global nitrogen cycle. Biological nitrogen fixation (BNF) through the enzyme nitrogenase requires extensive energy that in whole cells is generally studied during the oxidation of carbohydrates such as sugars. The nitrogen-fixing bacterium Azotobacter vinelandii is a model diazotroph for the study of aerobic BNF. Much is known about metabolism in A. vinelandii when cultured on a simple medium where energy is provided primarily in the form of sucrose or glucose. Outside of the laboratory, this soil bacterium grows on metabolites primarily derived from plant root exudates or from the degradation of dead plant matter. In this work, we expand on previous studies looking at genes that are essential to BNF in A. vinelandii when grown on sucrose medium using transposon sequencing (Tn-seq). We applied Tn-seq to determine the genes essential to growth when the medium was shifted to acetate, succinate or glycerol as the primary carbon and energy source to fuel both growth and BNF. A global overview of the genes of central metabolism and those directing substrates toward central metabolism, along with a selection of unexpected genes that were essential for specific growth substrates, is provided.

Computational analysis of large-scale metagenomics sequencing datasets provides valuable isolate-level taxonomic and functional insights from complex microbial communities. However, the ever-expanding ecosystem of metagenomics-specific methods and file formats makes designing scalable workflows and seamlessly exploring output data increasingly challenging. Although one-click bioinformatics pipelines can help organize these tools into workflows, they face compatibility and maintainability challenges that can prevent replication. To address the gap in easily extensible yet robustly distributable metagenomics workflows, we have developed the Core Analysis Modular Pipeline (CAMP), a module-based metagenomics analysis system written in Snakemake, with a standardized module and directory architecture. Each module can run independently or in sequence to produce target data formats (e.g. short-read preprocessing alone or followed by de novo assembly), and provides output summary statistics reports and Jupyter notebook-based visualizations. We applied CAMP to a set of 10 metagenomics samples, demonstrating how a modular analysis system with built-in data visualization facilitates rich seamless communication between outputs from different analytical purposes. The CAMP ecosystem (module template and analysis modules) can be found at https://github.com/Meta-CAMP.

Life’s development and adaption to fluctuating environments are underpinned by DNA–protein interactions, which have also spurred the development of artificial systems ranging from genetic circuits to nanoarchitectures. Nonetheless, in cellulo there remains an untapped design space for DNA–protein systems that are incompatible with the role of DNA as genetic material. Here we engineer retrons to intracellularly express non-genetic small DNAs embedding specific protein-binding sequences. These DNA species are products of genes and therefore their quantitative, spatial and functional control is decoupled from genetic stability, enabling alternative architectures of DNA–protein systems with unique functions. Using synthetic networks of proteins and the engineered retron-DNA, we demonstrated precise, multiplexed gene regulation and construction of feedback circuits for dynamic responses. Further, we developed DNA-based molecular scaffolds and bridges that enable modular, post-translational and spatial control of multiple proteins within cells. Finally, we transformed an allosteric transcription factor into inducible post-translational switches. Our work suggests that the non-genetic DNA–protein systems represent a promising control layer for creating synthetic cellular behaviours. The design space of synthetic DNA–protein systems in cells has been constrained by DNA’s role as genetic material. Now it has been shown that engineering retron-derived ‘non-genetic’ small DNAs for sequence-specific protein binding enables alternative DNA–protein networks for gene regulation, feedback loops, molecular scaffolds and post-translational switches.