The high-pathogenicity island of Yersinia enterocolitica Ye8081 undergoes low-frequency deletion but not precise excision, suggesting recent stabilization in the genome

Abstract

Highly pathogenic strains of Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica are characterized by the possession of a pathogenicity island designated the high-pathogenicity island (HPI). This 35- to 45-kb island carries an iron uptake system named the yersiniabactin locus. While the HPIs of Y. pestis and Y. pseudotuberculosis are subject to high-frequency spontaneous deletion from the chromosome, we were initially unable to obtain HPI-deleted Y. enterocolitica 1B isolates. In the present study, using a positive selection strategy, we identified three HPI-deleted mutants of Y. enterocolitica strain Ye8081. In these three independent clones, the chromosomal deletion was not limited to the HPI but encompassed a larger DNA fragment of approximately 140 kb. Loss of this fragment, which occurred at a frequency of approximately 5 x 10(-7), resulted in the disappearance of several phenotypic traits, such as growth in a minimal medium, hydrolysis of o-nitrophenyl-beta-D-thiogalactopyranoside, Tween esterase activity, and motility, and in a decreased virulence for mice. However, no precise excision of the Ye8081 HPI was observed. To gain more insight into the molecular basis for this phenomenon, the putative machinery of HPI excision in Y. enterocolitica was analyzed and compared to that in Y. pseudotuberculosis. We show that the probable reasons for failure of precise excision of the HPI of Y. enterocolitica Ye8081 are (i) the interruption of the P4-like integrase gene located close to its right-hand boundary by a premature stop codon and (ii) lack of conservation of 17-bp att-like sequences at both extremities of the HPI. These mutations may represent a process of HPI stabilization in the species Y. enterocolitica.

FIG. 1

Genetic organization of the HPI of Y. enterocolitica Ye8081. The HPI is represented by the gray line, and the identified genes are represented by arrows. Dashed lines below the diagram represent probes, named on the basis of their size in kilobases and on the restriction sites used to generate them. E, EcoRI; Bg, BglII; Ba, BamHI; X, XhoI; Cl, ClaI; H, HindIII.

FIG. 2

Comparison of genomic restriction profiles of the parental strain Ye8081 and of the three deletion derivatives (Ye8081H-). (A) Pulsed-field gel electrophoresis of SrfI-digested genomic DNA. M, molecular weight markers, whose sizes (in kilobases; values have been rounded up) are indicated on the right. The arrows point to fragments that differed between the wild-type strain and the three deletion clones. (B) Southern hybridization of the SrfI-digested DNA of the four strains with either the BaH12.5 or the E18 probe. The arrow indicates the size of the hybridizing fragment.

FIG. 3

Motility of the parental strain Ye8081 and of the deletion derivatives (Ye8081H-). (A) Swarm plates inoculated with the parental strain and one isogenic deletion mutant and incubated at 37 or 25°C. (B) Flagellum staining of the parental strain and of the isogenic mutant upon incubation at 25°C.

FIG. 4

(A) Schematic representation of the 707-bp BglII-XhoI region encompassing the left-hand boundary of the Ye8081 HPI and nucleotide sequence of the region where the degenerate att-like site was identified. (B) Hybridization of the EcoRI-digested genomic DNA of the three strains listed with BG1 and BG2 probes.