Genetic structure and distribution of the colibactin genomic island among members of the family Enterobacteriaceae

Abstract

A genomic island encoding the biosynthesis and secretion pathway of putative hybrid nonribosomal peptide-polyketide colibactin has been recently described in Escherichia coli. Colibactin acts as a cyclomodulin and blocks the eukaryotic cell cycle. The origin and prevalence of the colibactin island among enterobacteria are unknown. We therefore screened 1,565 isolates of different genera and species related to the Enterobacteriaceae by PCR for the presence of this DNA element. The island was detected not only in E. coli but also in Klebsiella pneumoniae, Enterobacter aerogenes, and Citrobacter koseri isolates. It was highly conserved among these species and was always associated with the yersiniabactin determinant. Structural variations between individual strains were only observed in an intergenic region containing variable numbers of tandem repeats. In E. coli, the colibactin island was usually restricted to isolates of phylogenetic group B2 and inserted at the asnW tRNA locus. Interestingly, in K. pneumoniae, E. aerogenes, C. koseri, and three E. coli strains of phylogenetic group B1, the functional colibactin determinant was associated with a genetic element similar to the integrative and conjugative elements ICEEc1 and ICEKp1 and to several enterobacterial plasmids. Different asn tRNA genes served as chromosomal insertion sites of the ICE-associated colibactin determinant: asnU in the three E. coli strains of ECOR group B1, and different asn tRNA loci in K. pneumoniae. The detection of the colibactin genes associated with an ICE-like element in several enterobacteria provides new insights into the spread of this gene cluster and its putative mode of transfer. Our results shed light on the mechanisms of genetic exchange between members of the family Enterobacteriaceae.

FIG. 1.

Genetic structure of the colibactin island of E. coli strain IHE3034. This genomic island is flanked by direct repeats (DR) and is inserted at asnW into the bacterial chromosome. The colibactin biosynthesis gene cluster is indicated by gray arrows. DNA regions used for PCR screening are indicated by black lines above the gene cluster. The VNTR located between clbB and clbR in different E. coli isolates differs in size. The number of repeats, the ST of the host strain, and the colibactin phenotype is given. EcN, E. coli strain Nissle 1917.

FIG. 2.

Phenotypic analysis of colibactin expression in different enterobacteria. HeLa cells were infected for 4 h with C. koseri, K. pneumoniae, E. aerogenes, and ECOR-B1 E. coli representative isolates or with DH10B pBACpks as a positive control (23). In the left panels, histone H2AX Ser139 phosphorylation (γH2AX) indicative of DNA double-strand breaks was assayed by confocal immunofluorescence at 4 h after infection. DNA and γH2AX are pseudocolored in blue and red, respectively. Bars, 20 μm. In the middle panels, the cell morphology was observed after Giemsa staining at 72 h after infection. Bars, 50 μm. In the right panels, G2 cell cycle arrest and increased sub-G1 cell populations (cell death) after DNA damage were assayed by flow cytometry at 72 h after infection. Similar results were obtained with all other colibactin island-positive C. koseri, K. pneumoniae, E. aerogenes, and ECOR-B1 E. coli isolates (data not shown).

FIG. 3.

Association of an ICE-like DNA region with the colibactin determinant in different members of the family Enterobacteriaceae. The tRNA-proximal sequence context of the integrative element comprising the colibactin gene cluster in C. koseri ATCC strain BAA-895 was screened by overlapping PCR in different colibactin-positive enterobacterial strains. The results of the PCR screening are summarized in the box below the genetic map. Homologous DNA regions are indicated in light gray. Regions with minor nucleotide sequence differences are boxed. Variable DNA stretches representing insertions or deletions are indicated in dark gray, and DNA regions that could not be amplified by PCR are shown in white.

FIG. 4.

Genetic structure of the sequence context of the colibactin determinant in C. koseri strain ATCC BAA 895 and its comparison to other corresponding enterobacterial genome regions. Nucleotide sequence homology between different DNA regions is indicated by red areas between the corresponding genetic maps. Homologous regions are indicated by different colors according to their function: the yersiniabactin gene cluster is represented in blue, the DNA transfer and mobilization region is represented in yellow, the colibactin determinant is represented in green, and pLVK-related DNA regions are represented in pink. Direct repeat regions are indicated in orange.