ICEEc2, a new integrative and conjugative element belonging to the pKLC102/PAGI-2 family, identified in Escherichia coli strain BEN374

Abstract

The diversity of the Escherichia coli species is in part due to the large number of mobile genetic elements that are exchanged between strains. We report here the identification of a new integrative and conjugative element (ICE) of the pKLC102/PAGI-2 family located downstream of the tRNA gene pheU in the E. coli strain BEN374. Indeed, this new region, which we called ICEEc2, can be transferred by conjugation from strain BEN374 to the E. coli strain C600. We were also able to transfer this region into a Salmonella enterica serovar Typhimurium strain and into a Yersinia pseudotuberculosis strain. This transfer was then followed by the integration of ICEEc2 into the host chromosome downstream of a phe tRNA gene. Our data indicated that this transfer involved a set of three genes encoding DNA mobility enzymes and a type IV pilus encoded by genes present on ICEEc2. Given the wide distribution of members of this family, these mobile genetic elements are likely to play an important role in the diversification of bacteria.

FIG. 1.

Schematic representation of the ICEEc2 genomic island. The upper part represents the percent G+C content of ICEEc2. The different genes present in ICEEc2 are depicted. The 50-bp direct repeats (DR) flanking ICEEc2 are represented. Regions of interest mentioned in the text are indicated below the graph. The bottom part of the graphic is a scale in kb.

FIG. 2.

tBLASTx comparisons between ICEEc2 and other elements of the pKLC102/PAGI-2 family. tBLASTx comparisons were performed using DoubleAct (http://www.hpa-bioinfotools.org.uk/pise/double_act.html) and analyzed using the Artemis Comparison Tool (ACT). For each genetic element analyzed, the upper line indicates ORFs encoded on the direct strand, and the bottom line indicates ORFs encoded on the complementary strand. The threshold for ACT visualization was set at 200.

FIG. 3.

Phylogenetic analysis of the pKLC102/PAGI-2 element integrases. Sequences homologous to ORF1 from pKLC102-PAGI-2 elements and integrases of the IntP and IntG family were aligned using MUSCLE. The alignment was then manually curated, taking into account the different boxes in integrases described by Esposito et al. (13). The region upstream of the genes encoding integrases of pKLC102/PAGI-2 elements were analyzed; when a tRNA gene was found immediately upstream of this gene, its name was in included on the figure. The absence of a tRNA or the fact that it was not annotated is also indicated (no tRNA found). Analysis was then performed by neighbor joining, maximum likelihood, and parsimony. The tree represented in this figure is the one generated by Consense and is rooted using IntP_Q77WA5_BPHK0 and IntP_P27077_VINT_BPP21 as outgroups. Nodes relevant for our analysis and found in all three analyses are indicated by filled circles while those supported by only two analyses are indicated by open circles. The arrow indicates the position of ORF1.

FIG. 4.

Formation of a circular intermediate is dependent upon the ORF1-ORF3 region. Formation of a circular intermediate was demonstrated by PCR using primers pheU374-circF and pheU374-circR and genomic DNA from strains BEN374, BEN374 ΔORF1-ORF3, and BEN374 ΔORF14.