Conjugation of DNA relies on multicomponent protein complexes bridging two bacterial cytoplasmic compartments. Whereas plasmid conjugation systems have been well documented, those of integrative and conjugative elements (ICEs) have remained poorly studied. We characterize here the conjugation system of the ICEclc element in Pseudomonas putida UWC1 that is a model for a widely distributed family of ICEs. By in frame deletion and complementation, we show the importance on ICE transfer of 22 genes in a 20-kb conserved ICE region. Protein comparisons recognized seven homologs to plasmid type IV secretion system components, another six homologs to frequent accessory proteins, and the rest without detectable counterparts. Stationary phase imaging of P. putida ICEclc with in-frame fluorescent protein fusions to predicted type IV components showed transfer-competent cell subpopulations with multiple fluorescent foci, largely overlapping in dual-labeled subcomponents, which is suggestive for multiple conjugation complexes per cell. Cross-dependencies between subcomponents in ICE-type IV secretion system assembly were revealed by quantitative foci image analysis in a variety of ICEclc mutant backgrounds. In conclusion, the ICEclc family presents an evolutionary distinct type IV conjugative system with transfer competent cells specialized in efficient transfer.

Integrative and conjugative elements (ICEs) are widespread autonomous mobile DNA, containing the genes necessary for their excision, conjugative transfer, and insertion into a new host cell. ICEs can carry additional genes that are non-essential for their transfer, but that can confer adaptive phenotypes to the host. Our aim here was to better characterize the presence, distribution and evolution of ICEs related to the well-described ICEclcamongPseudomonas aeruginosaclinical isolates, and to understand their potential role in spreading genes with adaptive benefit. We examined a total of 181P. aeruginosagenome sequences obtained from patient or hospital environment isolates. More than 90% of the isolates carried one or more ICEclc-like elements, with different degrees of conservation to the known ICEclc-lifestyle and transfer genes. ICE clones closely matched their host clonal phylogeny, but not exclusively, indicating that both clonal evolution and ICE-horizontal transfer are occurring in the hospital environment. Variable gene regions among the clinicalP. aeruginosaICEclc-type elements were notably enriched for heavy metal resistance genes, toxin-antitoxin systems, potential efflux systems and multidrug resistance proteins, a metalloprotease and for a variety of regulatory systems, but not for specific recognizable antibiotic resistance cassettes. Clonal persistence suggests adaptive benefits of these functional categories; and micro-patterns of gene gain and loss indicate ongoing ICE evolution within theP. aeruginosahosts.