Horizontal transfer of ICEclc-like elements in Pseudomonas aeruginosa clinical isolates

Integrative and conjugative elements (ICEs) are widespread autonomous mobile DNA within bacterial chromosomes. ICEs contain the genes necessary for excision from the chromosome, conjugative transfer to a new recipient cell, and chromosomal reintegration. They can also carry accessory genes that, while not essential for transfer, confer adaptive phenotypes to the host, contributing to host survival under stressful or changing conditions. Genome studies have indicated that Pseudomonas aeruginosa clinical isolates carry a wide range of related ICEs with adaptive genes enriched for heavy metal resistance and efflux systems, however, their mobility has remained understudied. Here, we studied the activation and transfer mechanisms of a representative subset of ICEclc-type elements. We found that ICE excision could be induced in P. aeruginosa by ectopic expression of BisDC, the known master regulator of ICEclc activation, pointing to a similar regulatory cascade. A number of elements could be transferred to P. putida, where they conferred increased tolerance to specific heavy metals. We also assessed ICE excision rates in response to different classes of stressors using qPCR-based quantification. Sub-lethal copper exposure significantly increased ICE excision rates in several P. aeruginosa strains, although this response was strongly strain-dependent and absent in isolates with enhanced copper tolerance, highlighting the importance of host background. Despite elevated excision, copper did not stimulate ICE transfer or induce conjugation gene expression, indicating that ICE excision and conjugation can be uncoupled processes. Transcriptomic analyses revealed strain-specific regulatory responses to copper stress, including differential activation of metal-responsive regulators, oxidative stress pathways, and virulence-associated systems. IMPORTANCEIntegrative and conjugative elements (ICEs) play a major role in bacterial adaptation by mediating horizontal gene transfer, yet the environmental cues governing their activation remain poorly understood. Here, we demonstrate that ICEclc-type elements in Pseudomonas aeruginosa are transferable at low frequencies and that their excision can be selectively induced by specific stress conditions, notably copper exposure and hypoosmotic stress. Our findings reveal that ICE excision and conjugative transfer can be uncoupled and are strongly influenced by host genetic background, underscoring the complexity of ICE regulation. This work aimed to explore whether clinical conditions or antimicrobial treatment could inadvertently promote ICE-mediated gene transfer, with implications for understanding the evolution of antibiotic resistance and virulence.