Role of attP in integrase-mediated integration of the Shigella resistance locus pathogenicity island of Shigella flexneri

Abstract

The Shigella resistance locus (SRL) pathogenicity island (PAI) in Shigella spp. mediates resistance to streptomycin, ampicillin, chloramphenicol, and tetracycline. It can be excised from the chromosome via site-specific recombination mediated by the P4-related int gene. Here, we show that SRL PAI attP is capable of RecA-independent, site-specific, int-mediated integration into two bacterial tRNA attB sites.

FIG. 1.

Model of the site-specific deletion of the SRL PAI YSH6000. The integrated SRL PAI is deleted via site-specific recombination to form a reconstituted attB site and an extrachromosomal circular form of the SRL PAI. Thin lines represent chromosomal DNA, and a thick line represents the SRL PAI. The SRL is represented by a filled box, and attL (attBOP′), attR (attPOB′), attP (attPOP′), and attB (attBOB′) sites are represented as open boxes. Integrase (int) and serX genes are represented as large arrows, and the position and orientation of primers used in amplifications are indicated with small arrows. The diagram is not drawn to scale.

FIG. 2.

Schematic representation of the SRL PAI attP (POP′) structure. The 14-bp O core is shown as a filled box. The numbers correspond to the positions of the DRs, with the center of the core region being considered position 0. Short horizontal arrows indicate DR1 and DR2, with sequences being given below the diagram. Sequences showing similarity to IHF binding sites are indicated with open boxes, alignments with the E. coli IHF binding site are shown below the diagram, and mismatched bases are shown in lowercase letters.

FIG. 3.

Schematic representation of the integration of the SRL PAI attP (carried on plasmid pAL226) into the chromosomal attB sites. Chromosomal DNA is represented by thin lines, and the plasmid pAL226 is represented by thick lines. att sites are represented by black or white boxes, with the common O core site being represented by shaded boxes. Genes are represented as large arrows, and the positions and orientations of primers used in amplifications are indicated with small arrows. The diagram is not drawn to scale.