Excision of the Shigella resistance locus pathogenicity island in Shigella flexneri is stimulated by a member of a new subgroup of recombination directionality factors

Abstract

Pathogenicity islands are capable of excision and insertion within bacterial chromosomes. We describe a protein, Rox, that stimulates excision of the Shigella resistance locus pathogenicity island in Shigella flexneri. Sequence analysis suggests that Rox belongs to a new subfamily of recombination directionality factors, which includes proteins from P4, enterohemorrhagic Escherichia coli, and Yersinia pestis.

FIG. 1.

Multiple alignment of Rox homologues. Amino acids that are identical or similar in at least two-thirds of the sequences are rendered as white characters on a black or grey background, respectively. Z1124 and Z1563 are encoded by EHEC EDL933 O islands 43 and 48, respectively. YPO1904, Rox_she, Vis, and AlpA are proteins encoded by the HPIs of Y. pestis, the S. flexneri she PAI, bacteriophage P4, and cryptic prophage CP4-57, respectively. The proposed helix-turn-helix (HTH) motif is indicated by a bar. Alignment was performed by using Clustal W (24).

FIG. 2.

Phylogenetic tree representing subgroups of RDFs. Members of each subgroup were chosen based on the Lewis and Hatfull definition of RDF subgroups (12). The proposed new subgroup is boxed. Mapping was performed by using Clustal W (24). B.melitensis, Brucella melitensis; M.loti, Mesorhizobium loti; S.melioti, Sinorhizobium meliloti; M.leprae, Mycobacterium leprae.

FIG. 3.

The effect of Rox_SRL and ORF41 on SRL PAI excision. (i) The shaded box denotes the SRL PAI, and the lines denote YSH6000-flanking chromosomal DNA. The primers used to determine if the PAI has excised are shown by arrowheads. (ii) Lane λ, lambda HindIII markers. Other lanes contain 10-fold serial dilutions of genomic DNA. AL66, AL67, and AL75 are S. flexneri 2a strains overexpressing rox, overexpressing orf41, and carrying an empty plasmid, respectively. The SRL PAI excisions in AL67 and the plasmid control can be seen as PCR products and are indicated by the arrowheads.

FIG. 4.

The effect of Int and Rox_SRL on SRL PAI excision. Lane λ, lambda HindIII markers. The various strains used as templates for PCR were as follows: AL296 (int mutant, pUC19-Tp) (lane 1), AL295 (int mutant, pUC19-Tp/rox) (lane 2), SBA1363 (SRL PAI⁻) (lane 3), AL325 (wild-type int, pUC19-Tp/rox) (lane 4).