Nested deletions of the SRL pathogenicity island of Shigella flexneri 2a

Abstract

In this study, we determined the boundaries of a 99-kb deletable element of Shigella flexneri 2a strain YSH6000. The element, designated the multiple-antibiotic resistance deletable element (MRDE), had recently been found to contain a 66-kb pathogenicity island (PAI)-like element (designated the SRL PAI) which carries the Shigella resistance locus (SRL), encoding resistance determinants to streptomycin, ampicillin, chloramphenicol, and tetracycline. The YSH6000 MRDE was found to be flanked by two identical IS91 elements present at the S. flexneri homologs of the Escherichia coli genes putA and mdoA on NotI fragment D. Sequence data from two YSH6000-derived MRDE deletants, YSH6000T and S2430, revealed that deletion of the MRDE occurred between the two flanking IS91 elements, resulting in a single IS91 element spanning the two original IS91 loci. Selection for the loss of tetracycline resistance confirmed that the MRDE deletion occurred reproducibly from the same chromosomal site and also showed that the SRL PAI and the SRL itself were capable of independent deletion from the chromosome, thus revealing a unique set of nested deletions. The excision frequency of the SRL PAI was estimated to be 10(-5) per cell in the wild type, and mutation of a P4-like integrase gene (int) at the left end of the SRL PAI revealed that int mediates precise deletion of the PAI.

FIG. 1

Map of the right flank of the YSH6000 MRDE. (A) Schematic representation of the YSH6000 mdoA-pyrC region. (B) The 2.4-kb EcoRI/EcoRV fragment within pSBA348 bearing the right deletion endpoint of the MRDE. Chromosomal DNA is represented as a thin black line, and genes are represented as arrows. The presence (+) or absence (−) of regions as tested by Southern hybridization in MRDE deletants YSH6000T and S2430 is also shown. Open arrowheads indicate the positions of primers BAP499 and BAP531, which were used to amplify across the MRDE deletion region in YSH6000T by inverse PCR of SalI-digested and religated genomic DNA. Abbreviations for restriction sites: E, EcoRI; EV, EcoRV; S, SalI.

FIG. 2

Deletion of the YSH6000 MRDE. (A) Schematic representation of the genetic organization of the wild-type YSH6000 MRDE. The boundaries of the MRDE are defined by the two IS91 elements, indicated by arrows. The SRL PAI is represented as an open rectangle. Genes and IS elements are represented as arrows, with truncations indicated by a capital delta. The fec, csg, and SRL loci are shown as thin black lines. The tetramers immediately downstream of the IS91 elements at the left and right flanks of the MRDE are shown boxed below each element. Shaded boxes indicate the positions of probes used to determine the presence (+) or absence (−) of these regions within six streptomycin-, ampicillin-, chloramphenicol-, and tetracycline-sensitive strains, YSH6000T, and YSH6000. Open arrowheads represent primers BAP649 and BAP530/BAP470, which were used to amplify across the deletion endpoint. The physical distance between putA and csg is based on PCR and/or sequence analysis, while the distance between csg and mdoA was calculated based on the original sizing of the MRDE at 99 kb (42). Regions designated the MRDE left and right flanks, common to both the wild type and MRDE deletants, are indicated above the diagram. (B) Schematic representation of the resultant structure across the deletion point following loss of the MRDE element in YSH6000T, S2430, SBA1365, and SBA1369. (C) Detection of the MRDE deletion point. Shown are PCR products amplified using primers BAP649 and BAP470 (panels A and B). Lanes: 0, HindIII-digested λ DNA size markers; 1, YSH6000; 2, YSH6000T; 3, SBA1365; 4, SBA1369.

FIG. 3

Deletion of the SRL. (A) Diagram of the SRL in YSH6000. (B) Schematic representation of the structure across the deletion point following loss of the SRL element in SBA1366 and SBA1368. IS elements are represented as arrows, and chromosomal DNA is represented by thin black lines. Determinants encoding resistance to streptomycin, ampicillin, chloramphenicol, and tetracycline are represented as open boxes labeled Sm^r, Ap^r, Cm^r, and Tc^r, respectively. Left and right flanking regions common to both the wild type and SRL deletants are indicated by shaded boxes, with the solid part of each box corresponding to the identical 487 bp of each IS1.

FIG. 4

Deletion of the SRL PAI. (A) Diagram of the genetic organization of the left and right SRL PAI flanking regions in YSH6000. (B) Schematic representation of the structure across the deletion point following loss of the SRL PAI in SBA1363 and SBA1367. Thin black line, chromosomal DNA; open rectangle, SRL PAI; arrows, genes; solid boxes, 14-bp DRs. Also shown are the locations of primers BAP1157 and BAP679, used to amplify across the SRL PAI deletion point.

FIG. 5

Complementation of the int mutation. PCR products spanning the SRL PAI excision site were obtained by amplification using primers BAP1157 and BAP679 (Fig. 4). From 100-μl PCR mixtures, all was loaded for lanes 1 and 2, 75 μl was loaded for lane 3, and 25 μl was loaded for lane 4. Lanes: 1, AL108 (YSH6000/pBRTp^rΔ); 2, AL109 (AL11/pBRTp^rΔ); 3, AL110 (AL11/pAL66); 4, SBA1363 (YSH6000-derived spontaneous PAI deletant).