Hernandez Viera, A. J. | Search bioRxiv

bioRxiv2026

RNA Polymerase Backtracking Promotes Type III-A CRISPR DNA Targeting

Type III CRISPR-Cas systems provide transcription-dependent immunity against mobile genetic elements, yet the mechanism underlying their DNA targeting remains unclear. Although the type III effector Cas10-Csm functions as a single-stranded DNase in vitro, type III systems eliminate double-stranded DNA targets in vivo, suggesting that transcription generates critical DNA substrates. Here, we show that conserved RNA polymerase anti-backtracking factors, such as GreA, selectively inhibit DNA-targeting of type III immunity. GreA overexpression abolishes protection against both phages and conjugative plasmids, whereas mutations that disrupt GreA anti-backtracking activity eliminate this effect. In contrast, immunity mediated by the type II CRISPR-Cas system or the auxiliary type III RNase Csm6 remain unaffected, demonstrating that GreA specifically suppresses Cas10-dependent DNA interference. These findings identify RNA polymerase backtracking as a key determinant of Cas10-Csm activity and support a model in which arrested transcription complexes generate substrates for DNA degradation, opening up new anti-CRISPR strategies against type III.

microbiology↗

bioRxiv2026

Staphylococcus epidermidis Genomic Plasticity Modulates Horizontal Gene Transfer

Horizontal gene transfer (HGT) is an important driver of evolution, enabling organisms to rapidly adapt to environmental stressors. In bacteria, anti-mobile genetic element (MGE) defense systems are believed to limit HGT, raising the question of how bacterial populations balance defense with the acquisition of genetic novelty. Most insights into anti-MGE defense come from heterologous experimental setups, where candidate systems are artificially expressed in a host and observed over only a few generations. Such approaches can obscure the long-term evolutionary and ecological dynamics of native anti-MGE systems. Here, we show that laboratory-evolved Staphylococcus epidermidis populations rapidly delete a large, flexible genomic region containing anti-MGE defense systems and other accessory genes. This process generates stable, heterogeneous populations composed of cells that either retain or lack these defenses. Loss of this region is associated with a marked increase in HGT. Notably, the balance between anti-MGE defense and HGT is strongly influenced by environmental conditions. In nutrient-rich liquid media, defense systems and adjacent accessory genes are frequently lost, whereas under stress, such as antibiotic exposure, these defenses are maintained, leading to reduced HGT rates. This genetic and phenotypic heterogeneity enhances adaptive potential by enabling the uptake of beneficial MGEs while preserving protection under adverse conditions. Overall, the rapid gain and loss of anti-MGE defense systems highlight how environmental factors, independent of MGEs themselves, can shape HGT dynamics.

microbiology↗