Antibiotic heteroresistance, characterized by rare resistant subpopulations of bacteria within a susceptible main population, is associated with treatment failure and often caused by tandem amplification of resistance genes. Here, we investigated how the distribution of tandem amplifications affects heteroresistance using an approach combining genetic engineering and ultra-deep Nanopore sequencing to accurately quantify the distribution of tandem amplification copy numbers on plasmids down to frequencies of 10-5. Using an Escherichia coli isolate, we describe the direct relation between the distribution of tandem amplifications increasing the copy number of a blaSHV gene and a heteroresistance phenotype to piperacillin-tazobactam, and reveal how this distribution expands under antibiotic pressure and partially reverts upon its removal. Mathematical modeling indicates that indirect resistance and fitness cost of amplifications influence the dynamic distribution of tandem amplifications. These findings provide insights into amplification-mediated phenotypes and enhance possibilities for the development of improved therapeutic and diagnostics strategies for heteroresistance. Antibiotic heteroresistence is commonly caused by repeated amplification of resistance genes. Jonsson et al., reveals the relation between these two events, and how antibiotic pressure, fitness cost and indirect resistance affect the distribution of tandem repeats.

The design of large genetic circuits requires genetic regulatory devices capable of performing complex logic operations that place no excessive metabolic burden on the host cell. Hybrid riboswitches, synthetically enhanced compact RNA elements (<100 nucleotides) that form a tertiary structure with the ability to specifically bind two different target molecules, can be used to design genetic regulators that emulate Boolean logic. When inserted into the 5′ UTR of a messenger RNA, these devices can regulate translation initiation upon specific binding of one or both ligands without the need for additional auxiliary factors. The goal of this study is to design hybrid riboswitches that emulate Boolean NAND logic in yeast. We propose a novel machine learning-based design framework combining high-throughput in vivo screening and deep Bayesian optimization. Through an initial screening, we discovered a hybrid riboswitch with NAND behavior. Using batch Bayesian optimization with an ensemble neural network as surrogate, we improved the NAND functionality of our hybrid riboswitch with respect to a performance score, thereby achieving near-digital NAND behavior. With its focus on model-based and score-driven design, our proposed method can complement experiment-driven approaches by allowing fine grained adaptation of functionality, including constructs sensitive to single nucleotide changes.

The advent of nano-technology has revolutionized approaches to identifying and combating bacterial infections. This review highlights the latest applications of nanoparticles (NPs) for bacterial detection and treatment, with a focus on their translational potential in clinical settings. We discuss advanced nanotechnology-enabled biosensing platforms that offer ultra-sensitive, rapid and precise diagnostics capabilities crucial for addressing antibiotic-resistant pathogens. In addition to detection, various nanoparticles demonstrate multiple antibacterial mechanisms and function as targeted drug-delivery vehicles. The review also examines current clinical trials involving nanoparticle-based therapeutics, underscoring their promise for overcoming antimicrobial resistance. 

1,5-Pentanediol (1,5-PDO) is a high-value chemical with broad uses in polymer, cosmetic, and pharmaceutical industries. Although diverse biosynthetic pathways have been constructed, current recombinant strains typically rely on plasmid-based overexpression, which necessitates antibiotics and hinders industrial-scale production.  We developed a robust, plasmid-free E. coli platform for de novo 1,5-PDO synthesis by integrating pathway genes (davB, davA, gabT, yahK, car, sfp and yqhD) into the chromosome of a lysine-hyperproducing strain via CRISPR/Cas9. Screening of carboxylic acid reductases identified Nocardia iowensis CAR-Ni as the most effective, yielding a base strain (D13) that produced 0.672 g/L 1,5-PDO. Integrated analysis confirmed the alcohol dehydrogenase (ADH)-mediated reduction of 5-hydroxypentanal (5-HP) as an underappreciated bottleneck. We subsequently screened ten endogenous ADHs and selected YjgB for computational optimization. Docking-guided saturation mutagenesis at position E205 yielded the variant YjgB(E205C), which exhibited a 3.34-fold increase in in vitro activity, reduced 5-HP accumulation, and elevated the titer to 0.935 g/L. Enhancing NADPH supply by integrating pntAB further raised the shake-flask titer to 1.5 g/L. In a 5-L fed-batch bioreactor, the final strain (D91) achieved 12.1 g/L 1,5-PDO (yield of 0.225 mol/mol glucose) without antibiotics or inducers. To our knowledge, this is the highest reported 1,5-PDO titer in E. coli. This study establishes a scalable, sustainable biosynthetic platform through synergistic metabolic engineering and computational enzyme optimization.

Experimental evolution is a powerful method that has been instrumental for revealing core mechanisms of adaptation and coevolution. It has mostly been used in very simple settings of one or two species. Yet, it is now increasingly being employed in more complex community settings that include indirect effects, higher-order interactions, and multidimensional selection typical of natural communities. Here we synthesize the emerging field of experimental evolution in communities and show how community context reshapes selection and evolutionary trajectories, beyond what single-species or pairwise designs predict. We conducted a systematic literature survey targeting multi-species, multi-generation evolution, identifying 100 such studies with the number increasing recently. Despite this progress, most experiments are biased toward microbial systems and competitive interactions, leaving major gaps for predicting evolution in realistic communities. We discuss community ecology concepts in the light of experimental evolution, together with designs that address these concepts. We emphasize three main research areas: indirect and higher-order interactions that make selection multidimensional, eco-evolutionary feedbacks linking trait change to community dynamics, and genetic constraints that shape responses across interaction networks. We then discuss routes to increase ecological realism with field experiments and conclude by outlining key research fronts for experimental evolution in communities.

Nitrite-oxidizing bacteria (NOB) use either periplasmic (pNXR) or cytoplasmic (cNXR) nitrite oxidoreductase to oxidize nitrite, and this distinction influences nitrite affinity and energy yield. cNXR-containing NOB have historically been considered low-affinity, copiotrophic nitrifiers adapted to high nitrite and neutral pH. Here, we report a previously uncharacterized pH- and substrate-dependent modulation of nitrite affinity in cNXR NOB that is not observed in pNXR NOB and is not a universal microbial trait. Nitrobacter winogradskyi Nb-255, grown at low nitrite (1 mM), had a high apparent affinity (Km(app) = 25.9 μM; specific affinity ao = 440.5 l g cells−1 h−1) comparable to oligotrophic pNXR NOB. However, when grown at high nitrite (10 mM), these cells showed a low affinity at pH 7.5 (Km(app) = 388.0 μM) but exhibited a rapid increase in affinity upon immediate exposure to pH 5.5 (Km(app) = 19.2 μM) without prior acid adaptation. In contrast, pNXR NOB exhibited consistent kinetic behavior across different pH conditions, underscoring that this kinetic plasticity is unique to cNXR NOB. Kinetic inhibition assays revealed that this plasticity is mechanistically underpinned by a shift from a low-affinity nitrite/nitrate antiporter (NarK) to a high-affinity nitrite channel (NirC), coupled with enhanced HNO2 diffusion at low pH, together increasing intracellular nitrite availability. These findings establish that cNXR NOB can dynamically tune nitrite affinity via transporter-level regulation in response to nitrite concentration and pH. This novel mechanism provides a mechanistic explanation for the unexpected prevalence of Nitrobacter in acidic, low-nitrite environments, highlighting its ecological relevance.

The emergence of CRISPR–Cas systems has transformed nucleic acid detection and manipulation. Cas13, a type VI CRISPR effector, targets RNA with high sensitivity through both cis (target RNA) and trans (collateral RNA) cleavage. This property enables the use of fluorescent reporters for sensitive diagnostics. However, Cas13’s heightened sensitivity also leads to reduced specificity due to its susceptibility to single-nucleotide mismatches, potentially causing off-target effects. To overcome this limitation, we developed the first Dual-guide RNA system for Cas13 that improves mismatch discrimination and enhances target specificity. This system employs two distinct RNAs—dcrRNA and dtracrRNA—which cooperatively recognize the target and reduce off-target activity. In vitro experiments demonstrated robust cis- and trans-RNase activity, indicating efficient and specific cleavage. The system accurately detected SARS-CoV-2 RNA, distinguished KRAS G12D and G12C mutations, and differentiated mucocutaneous from cutaneous Leishmania sequences in analytical assays, with clinical validation confirming accurate detection of positive and negative samples. These results highlight the Dual-guide Cas13 platform’s potential for precise, rapid, and reliable RNA detection. Overall, this approach represents a substantial advance over conventional Cas13 systems, offering improved specificity while maintaining clinically relevant sensitivity, and provides a generalizable tool for next-generation molecular diagnostics and precision RNA targeting and regulation.