Journal: bioRxiv
Article Title: Plasmids weaponize conjugation to eliminate non-permissive recipients
doi: 10.64898/2026.02.10.705089
Figure Lengend Snippet: (A) Model of coercive assimilation. In non-permissive recipients (pink oval), intracellular defense systems (e.g., CRISPR-Cas9, teal) degrade the incoming plasmid, preventing TrbK expression. The unshielded recipient remains susceptible to repetitive, T4SS punctures and uncontrolled DNA delivery (lethal zygosis). (B) Restriction-Modification (defense). Left: Schematic of the RM competition assay. RM+ recipients (non-permissive, pink) and RM-recipients (permissive, yellow) compete in the presence of varying amounts of donor cells carrying RK2 plasmids (blue) that are either methylated (protected from digestion) or unmethylated (susceptible to digestion). Right: Competitive index of RM+ recipients relative to RM-recipients in the presence of donors with methylated (left facet) or unmethylated (right facet) RK2. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. Different letters (a - b) indicate statistically significant differences within and between facets. When donors carried unmethylated RK2, RM+ recipients suffered significant fitness costs compared to the methylated plasmid treatment (p < 0.05) (C) CRISPR-Cas defense. Left: Schematic of the CRISPR competition assay. Recipients with Cas9 and an RK2-targeting gRNA (non-permissive, pink) or a non-targeting gRNA (permissive, yellow) were competed in the presence of varying amounts of RK2 donor. Right: Competitive index of targeting recipients relative to non-targeting recipients under uninduced or induced conditions. Different letters (a - b) indicate statistically significant differences (p < 0.05). Circles represent the competitive index of replicates, bars represent the mean (n = 3).
Article Snippet: CRISPR guide RNAs were designed to target RK2’s oriV using the Benchling guide design tool ( ).
Techniques: CRISPR, Plasmid Preparation, Expressing, Modification, Competitive Binding Assay, Methylation
