Complete DNA sequence and analysis of the large virulence plasmid of Shigella flexneri

Abstract

The complete sequence analysis of the 210-kb Shigella flexneri 5a virulence plasmid was determined. Shigella spp. cause dysentery and diarrhea by invasion and spread through the colonic mucosa. Most of the known Shigella virulence determinants are encoded on a large plasmid that is unique to virulent strains of Shigella and enteroinvasive Escherichia coli; these known genes account for approximately 30 to 35% of the virulence plasmid. In the complete sequence of the virulence plasmid, 286 open reading frames (ORFs) were identified. An astonishing 153 (53%) of these were related to known and putative insertion sequence (IS) elements; no known bacterial plasmid has previously been described with such a high proportion of IS elements. Four new IS elements were identified. Fifty putative proteins show no significant homology to proteins of known function; of these, 18 have a G+C content of less than 40%, typical of known virulence genes on the plasmid. These 18 constitute potentially unknown virulence genes. Two alleles of shet2 and five alleles of ipaH were also identified on the plasmid. Thus, the plasmid sequence suggests a remarkable history of IS-mediated acquisition of DNA across bacterial species. The complete sequence will permit targeted characterization of potential new Shigella virulence determinants.

FIG. 1

Circular map of the plasmid. Outer ring depicts ORFs and their orientations, color coded according to functional category: 1, identical or essentially identical to known virulence-associated proteins (red); 2, homologous to known pathogenesis-associated proteins (pink); 3, highly homologous to IS elements or transposases (blue); 4, weakly homologous to IS elements or transposases (light blue); 5, homologous to proteins involved in replication, plasmid maintenance, or other DNA metabolic functions (yellow); 6, no significant similarity to any protein or ORF in the database (brown); 7, homologous or identical to conserved hypothetical ORFs, i.e., proteins of unknown function (orange); and 8, Tn501 insertion-associated genes (green). The second ring shows complete IS elements. The third ring graphs G+C content, calculated for each ORF and plotted around the mean value for all ORFs, with each value color coded for the corresponding ORF. Scale is in base pairs. The figure was generated by Genescene (DNASTAR).

FIG. 2

Schematic representation of IS elements flanking virulence-associated genes on pWR501. (A) The 32-kb ipa-mxi-spa region; (B) the virA-virG region; (C) the virF region. Sequence coordinates are indicated above each map. frag, fragment; put tnp, putative transposase; seq, sequence; URF, unknown ORF; inc, incomplete. (D) The region downstream of virF (left side, as shown in panel C) shown at the base pair level to demonstrate that the nucleotide marking the end of one IS element or IS fragment is the start of another. The relevant bases are indicated in bold underline.

FIG. 3

Plot of G+C content (y axis) of each ORF on pWR501 (x axis). Unknown ORFs and selected known ORFs with G+C content of less than 40% are labeled.

FIG. 4

Alignment of ShET2 alleles on pWR501. The ShET2 toxin (S0097) is compared with the second copy of ShET2 (S0012) and a smaller ORF (S0030) with which it bears similarity. The similarities are indicated as for BLAST searches.

FIG. 5

The five IpaH alleles of pWR501. (A) Alignment of the amino-terminal halves of the five IpaH alleles, using the Clustal method with PAM250 residue weight table (MEGALIGN algorithm; DNASTAR software). Beginning with the sequence LADAV (residues 307 to 311 of IpaH1.4), the sequences are conserved among all five alleles; the carboxy termini are not shown. Asterisks, approximate extent of the leucine-rich repeat sequence; arrow, beginning of region conserved in all five alleles. (B) Sequence identity and divergence among the five IpaH alleles. Percent divergence is calculated by comparing sequence pairs in relation to the phylogeny reconstructed by MEGALIGN, whereas percent identity is determined for individual pairs without regard to phylogenetic relationship. (C) G+C content distribution of IpaH7.8. Scale is in base pairs.

FIG. 6

Replicon of pWR501. (A) Replicon region and flanking DNA sequences (sequence coordinates 187081 to 214607), including the insertion site of Tn501. (B) Expanded view of the replicon shown in panel A (sequence coordinates 207312 to 214912), indicating the position of inc RNA, ori, and the G site. Distances are indicated in base pairs below each map.