Recent research has indicated the presence of highly protein occupied, transcriptionally silent regions of bacterial genomes which show functional parallels to eukaryotic heterochromatin. We utilized an integrative approach to track chromatin structure and transcription in Escherichia coli K-12 across a wide range of nutrient conditions. In the process, we identified multiple loci which act similarly to facultative heterochromatin in eukaryotes, normally silenced but permitting expression of genes under specific conditions. We also found a strong enrichment of small regulatory RNAs (sRNAs) among the set of differentially expressed transcripts during nutrient stress. Using a newly developed bioinformatic pipeline, the transcription factors (TFs) regulating sRNA expression were bioinformatically predicted, with experimental follow-up revealing novel relationships for 45 sRNA–TF candidates. Direct regulation of sRNA expression was confirmed by mutational analysis for five sRNAs of metabolic interest: IsrB (also known as AzuCR), CsrB and CsrC, GcvB, and GadY. Our integrative analysis thus reveals additional layers of complexity in the nutrient stress response in E. coli and provides a framework for revealing similar poorly understood regulatory logic in other organisms.

CRISPR–Cas12a enzymes are versatile RNA-guided genome-editing tools with applications encompassing viral diagnosis, agriculture, and human therapeutics. However, their dependence on a 5′-TTTV-3′ protospacer adjacent motif (PAM) next to DNA target sequences restricts Cas12a’s gene targeting capability to only ∼1% of a typical genome. To mitigate this constraint, we used a bacterial-based directed evolution assay combined with rational engineering to identify variants of Lachnospiraceae bacterium Cas12a with expanded PAM recognition. The resulting Cas12a variants use a range of noncanonical PAMs while retaining recognition of the canonical 5′-TTTV-3′ PAM. In particular, biochemical and cell-based assays show that the variant Flex-Cas12a utilizes 5′-NYHV-3′ PAMs that expand DNA recognition sites to ∼25% of the human genome. With enhanced targeting versatility, Flex-Cas12a unlocks access to previously inaccessible genomic loci, providing new opportunities for both therapeutic and agricultural genome engineering.

Bifidobacteria are beneficial saccharolytic microbes that are widely used as probiotics or in synbiotic formulations, yet individual responses to supplementation can vary with strain type, microbiota composition, diet and lifestyle, underscoring the need for strain-level insights into glycan metabolism. Here we reconstructed 68 pathways for the utilization of mono-, di-, oligo- and polysaccharides by analysing the distribution of 589 curated metabolic gene functions (catabolic enzymes, transporters and transcriptional regulators) across 3,083 non-redundant Bifidobacterium genomes of human origin. Thirty-eight predicted phenotypes were validated in vitro for 30 geographically diverse strains, supporting genomics-based predictions. Our analysis uncovered extensive inter- and intraspecies functional heterogeneity, including a distinct clade within Bifidobacterium longum that metabolizes α-glucans and Bangladeshi isolates carrying unique gene clusters for xyloglucan and human milk oligosaccharide utilization. This large-scale genomic compendium advances our understanding of bifidobacterial carbohydrate metabolism and can inform the rational design of probiotic and synbiotic formulations tailored to strain-specific nutrient preferences. A comprehensive genomic analysis reveals species- and strain-level heterogeneity in carbohydrate utilization potential across bifidobacteria of human origin.

Bacterial toxin–antitoxin (TA) pairs transcriptionally autoregulate their expression via a repression/derepression mechanism in response to changing environmental conditions. The structural diversity of TA systems influences the mechanisms of transcriptional regulation. Here, we define the molecular mechanism for the plasmid-encoded HigB–HigA TA pair originally identified in a post-operative infection with antibiotic-resistant Proteus vulgaris. We determine DNA binding and promoter activity by the HigB–HigA complex supported by structural biology and molecular dynamics simulations of an elusive DNA operator–TA repressor complex. To define the optimal oligomeric TA repressor–DNA operator complex required for derepression, we engineered a dedicated trimeric HigB–HigA2 complex that represses transcription more than 26-fold as compared to the tetrameric HigB2–HigA2. These results expand the known diversity of how the HigB–HigA TA family is autoregulated.

RNA processing is essential for proper cellular function, contributing to protein and cell state diversity, and is often dysregulated in diseased states. A key subset of RNA regulators is the double-stranded RNA-specific adenosine deaminase (ADAR) protein family, which hydrolytically deaminates double-stranded RNA, causing an adenosine-to-inosine edit (A-to-I). Active ubiquitously throughout the body, this pleiotropic protein family plays critical roles in embryonic patterning, neurological function, and immune regulation. Their aberrant activity has in turn been implicated in a spectrum of disorders, including cancer, metabolic diseases, and autoimmune conditions. By instead purposefully modulating their activity, ADARs have been leveraged to create a versatile toolset for transcriptome engineering. This includes enabling programmable RNA editing, controlled RNA splicing, reversibly modulating protein interactions, and altering cellular inflammation. Here, we review the pleiotropic functions and versatile applications of ADARs, as well as outline areas for growth and potential new avenues in both therapeutics and research.

Most people in the USA manage their health by taking at least one prescription drug, and drugs classified as non-antibiotics can adversely affect the gut microbiome and disrupt intestinal homeostasis. Here we identify medications that are associated with an increased risk of gastrointestinal infections across a population cohort of more than one million individuals monitored over 15 years. Notably, the cardiac glycoside digoxin and other drugs identified in this epidemiological study are sufficient to alter the composition of the microbiome and the risk of infection with Salmonella enterica subsp. enterica serovar Typhimurium (S. Tm) in mice. The effect of digoxin treatment on S. Tm infection is transmissible through the microbiome, and characterization of this interaction highlights a digoxin-responsive β-defensin that alters the microbiome composition and consequent immune surveillance of the invading pathogen. Combining epidemiological and experimental approaches thus provides an opportunity to uncover drug–host–microbiome–pathogen interactions that increase the risk of infections in humans. An analysis of prescription medications shows that several non-antibiotic drugs, such as the heart medication digoxin, can reduce the immune response to pathogens and increase the risk of gastrointestinal infections by altering the composition of the microbiome.

a, Mastrangelo et al. show that the molecule imidazole propionate (ImP), which is made by bacteria in the gut, enters the bloodstream and interacts with the imidazoline 1 receptor (I1R). I1R is expressed in a class of blood cell known as myeloid cells. Activation of I1R stimulates the protein complex mTORC1, which leads to the proliferation of pro-inflammatory immune cells. b, This results in immune cells being recruited to fatty plaques on the walls of blood vessels such as the aorta, the body’s largest artery. The build-up of cholesterol and immune cells is known as atherosclerosis, and can result in obstruction of blood vessels and further cardiovascular problems. Inhibiting I1R using a compound called AGN (shown in a) reverses this pro-atherosclerotic effect.

Remodelling plant immune receptors has become a vital strategy for creating new disease resistance traits to combat the growing threat of plant pathogens to global food security and environmental sustainability. However, current methods are constrained by the rapid evolution of plant pathogens and often lack broad-spectrum and durable protection. Here we report an innovative strategy to engineer broad-spectrum, durable and complete disease resistance in plants through expression of a chimeric protein containing a flexible polypeptide coupled with a single or dual conserved pathogen-originated protease cleavage site fused in frame to the N terminus of an autoactive nucleotide-binding and leucine-rich-repeat immune receptor (NLR) containing a coiled-coil or RESISTANCE TO POWDERY MILDEW 8-like coiled-coil domain. Following invasion, pathogen-originated specific proteases cleave the inactive chimeric protein to form free autoactive NLR, triggering broad-spectrum plant disease resistance. We demonstrate that a single engineered NLR can confer broad-spectrum and complete resistance against multiple potyviruses. Given that many pathogenic organisms across kingdoms encode proteases, this strategy has the potential to be exploited to control viruses, bacteria, oomycetes, fungi, nematodes and pests in plants. Cleavage by pathogen-derived proteases of an engineered chimeric protein activates its plant immune receptor component, enabling broad-spectrum resistance to pathogens in plants.