van de Peppel, A. C. | bioRxiv Science

bioRxiv2026

Experimental evolution of collective β-lactam resistance in Escherichia coli via activation of a dormant outermembrane porin

Understanding the mechanisms that drive antibiotic resistance is relevant for both evolutionary theory and the design of effective drug therapies. A specific challenge are collective resistance mechanisms, where bacterial populations survive drug concentrations that kill individual bacteria. Here, we explore the evolvability of collective resistance mechanisms in bacterial strains expressing antibiotic-degrading {beta}-lactamases with different levels of privatization. Four strains of Escherichia coli, with or without outermembrane porin OmpF to affect drug permeability and expressing either a low or high-activity periplasmic {beta}-lactamase, were subjected to lineage selection in a gradient of the drug cefotaxime. Strains with a low-activity enzyme increased cell-level resistance, while strains with low permeability, and hence a more private {beta}-lactamase function, increased collective resistance. Remarkably, increased collective resistance in the strain with a private high-activity {beta}-lactamase came with decreased cell-level resistance. This tradeoff was primarily caused by the activation of a dormant outermembrane porin, NmpC, via the excision of an insertion sequence. Increased drug permeability through NmpC explained both its lower cell-level resistance and its greater collective resistance through faster drug removal and growth recovery via enhanced filamentation at high cell density. The recovery advantage of the NmpC mutant also explained its initial invasion within the ancestral population, suggesting that drug permeability is a readily evolvable collective-resistance mechanism in bacteria with high-activity {beta}-lactamases. Our findings highlight the role of filamentation and drug permeability in {beta}-lactamase-mediated collective resistance to these widely used drugs.

evolutionary biology↗

bioRxiv2023

Natural gene variation in Cannabis sativa unveils a key region of cannabinoid synthase enzymes

Cannabinoids are well-known specialised metabolites from the plant Cannabis sativa L. (cannabis). They exhibit various therapeutical to intoxicating psychoactive effects and have potential for medicinal applications. Among the enzymes involved in cannabinoid biosynthesis, cannabinoid oxidocyclases such as the tetrahydrocannabinolic acid (THCA) synthase play a key role in determining cannabis chemotype. To improve our understanding of cannabinoid oxidocyclase structure-function relationship, we proposed a new approach to targeted mutagenesis. By reviewing cannabis natural variation, three cannabinoid oxidocyclase mutations (S355N, CONF, G376R) associated to atypical plant chemotypes were selected. In-vitro characterization of THCA synthase mutants demonstrated these mutations significantly impact enzyme activity, correlating with the associated chemotype: S355N nearly inactivated the THCA synthase, CONF impaired CBGA metabolization and altered product specificity, while G376R drastically reduced enzyme activity and altered product specificity. In-silico docking experiments permitted to model the successive steps of THCA synthase substrate metabolization, revealing that the three mutations hamper substrate binding. Collectively, our results demonstrated how plant diversity can be leveraged to guide enzyme targeted mutagenesis, highlighted a key region of cannabinoid oxidocyclases, and permitted the establishment of a new model of the THCA synthase catalytic mechanism. This provides new insights into enzyme function, which can ultimately help developing medicinal cannabis cultivars and cannabinoid biotechnological production.

molecular biology↗