Characterization of an atypical but widespread type IV secretion system for transfer of the integrative and conjugative element (ICEclc) inPseudomonas putida

Abstract

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.

Diversity and Evolution of an Abundant ICEclc-Family of Integrative and Conjugative Elements inPseudomonas aeruginosa

“…Highly similar ICEs were also found in strains isolated from hospitals in different geographical regions, suggesting that both exchange of strains and of their ICEs can occur and contribute to P. aeruginosa diversification and adaptation. As far as we could analyze, the ICEclcfamily members appear to encode all genes necessary for their horizontal gene transfer, in comparison to a recent functional study of ICEclc conjugative transfer genes 37 . This includes all genes for a complete type IV conjugative system, an integrase, and a relaxase gene.…”

“…First, we tried to obtain a broad overview of the presence of ICEs related to ICEclc. For this, we focused on forty ICEclc genes, which we expected might be conserved: the intB13 integrase, the traI relaxase, the DNA topoisomerase topB, thirteen ORFs whose products are involved in regulation and activation of the element 33 , and twenty-four ORFs whose products are implicated in the type IV ICE conjugative system 37 . BLASTN searches with these queries against the 181 P. aeruginosa draft genomes returned hits to intB13 for 91% of the genomes (74% to 91% nucleotide identity over 63% to 94% of the gene length, Fig.…”

“…A ∼50 kb region on ICEclc, with high sequence similarity to other (suspected) ICEs 25,28,33 , contains the genes for ICE activation 33,34 , excision and integration 35 , DNA processing 36 and conjugative transfer 37 . A wide variety of ICEclc family elements has been identified in different bacterial hosts, and more recent surveys point to several instances of relatives carrying antibiotic resistance determinants in clinical isolates 26,[38][39][40][41][42][43] 42 showed ICEs related to ICEclc encoding carbapenem resistance in P. aeruginosa.…”

Diversity and evolution of an abundant ICE clc family of integrative and conjugative elements in Pseudomonas aeruginosa

“…First, we tried to obtain a broad overview of the presence of ICEs related to ICE clc . For this, we focused on 40 ICE clc genes, which we expected might be conserved: the intB13 integrase, the traI relaxase, the DNA topoisomerase topB , 13 open reading frames (ORFs) whose products are involved in regulation and activation of the element ( 33 ), and 24 ORFs whose products are implicated in the type IV ICE conjugative system ( 37 ). BLASTN searches with these queries and a 70% sequence identity cut-off against the 181 P .…”

“…In addition to the clc genes themselves, ICE clc further carries genes for 2-aminophenol degradation ( 25 ), for a multidrug efflux pump ( 32 ), as well as for additional proteins of unknown function. A 50-kb region on ICE clc , with high sequence similarity to other (suspected) ICEs ( 25 , 28 , 33 ), contains the genes for ICE activation ( 33 , 34 ), excision and integration ( 35 ), DNA processing ( 36 ) and conjugative transfer ( 37 ). A wide variety of ICE clc family elements have been identified in different bacterial hosts, and more recent surveys point to several instances of relatives carrying antibiotic-resistance determinants in clinical isolates ( 26 , 38 – 43 ).…”