The SPI-3 pathogenicity island of Salmonella enterica

Abstract

Pathogenicity islands are chromosomal clusters of pathogen-specific virulence genes often found at tRNA loci. We have determined the molecular genetic structure of SPI-3, a 17-kb pathogenicity island located at the selC tRNA locus of Salmonella enterica serovar Typhimurium. The G+C content of SPI-3 (47.5%) differs from that of the Salmonella genome (52%), consistent with the notion that these sequences have been horizontally acquired. SPI-3 harbors 10 open reading frames organized in six transcriptional units, which include the previously described mgtCB operon encoding the macrophage survival protein MgtC and the Mg2+ transporter MgtB. Among the newly identified open reading frames, one exhibits sequence similarity to the ToxR regulatory protein of Vibrio cholerae and one is similar to the AIDA-I adhesin of enteropathogenic Escherichia coli. The distribution of SPI-3 sequences varies among the salmonellae: the right end of the island, which harbors the virulence gene mgtC, is present in all eight subspecies of Salmonella; however, a four-gene cluster at the center of SPI-3 is found in only some of the subspecies and is bracketed by remnants of insertion sequences, suggesting a multistep process in the evolution of SPI-3 sequences.

FIG. 1

Physical and genetic maps of the SPI-3 pathogenicity island. (Top) G+C content of the SPI-3 island. The graph was created by using the program Cricket Graph with data generated by the program Windows (GCG) (window, 100 bp; sliding increment, 100 bp). The line at 52% indicates the overall G+C content estimated for the S. enterica serovar Typhimurium chromosome. (Bottom) Positions and orientations of ORFs encoding products larger than 120 amino acids and containing potential Shine-Dalgarno sequences. DNA sequences reported in Table 2 are indicated by numbers (IS-like sequences are represented by gray squares). The map positions of MudJ insertions in the SPI-3 region are indicated by triangles. RSA refers to a family of protected sequences present in the genomes of members of the family Enterobacteriaceae.

FIG. 2

(A) Alignment of the C-terminal domains of the S. enterica MisL protein, the plasmid-encoded AIDA-1 protein from enteropathogenic E. coli, and the VirG protein of S. flexneri. An 18-amino-acid duplicated region rich in Pro, Asp, and Val within MisL is indicated by a horizontal line. Amino acids that are identical between the MisL and AIDA-1 proteins or between the AIDA-1 and VirG proteins are linked by vertical lines. Amino acids that are identical between the MisL and VirG proteins are indicated by a short underline in the VirG residue. (B) Alignment of the N-terminal domains of the MarT protein, ORF269 (O269) of E. coli, and the ToxR regulator from V. cholerae. Highly conserved residues among OmpR homologs are indicated by dots (30). Amino acids that have been shown to be important for ToxR function (41) are marked by asterisks. Amino acids that are identical between the MarT and ORF269 proteins or between the ORF269 and ToxR proteins are linked by vertical lines. Amino acids that are identical between the MarT and ToxR proteins are indicated by a short underline in the ToxR residue. Alignments were performed by using the PILEUP program (GCG).

FIG. 3

Organization of the rmbA, misL, fidL, and marT genes in S. enterica serovar Typhimurium SPI-3 in comparison to the orf230 (o230), orf269 (o269), and orf164 (o164) genes of E. coli K-12. The deduced amino acid sequences of rmbA, fidL, and marT are about 40% identical to the deduced proteins encoded by o230, o164, and o269, respectively (Table 2), suggesting that these proteins are orthologues rather than homologues.

FIG. 4

Phylogenetic distribution of SPI-3 sequences among salmonellae based on Southern blot experiments carried out as described in Materials and Methods. Positions of the remnants of ISs are indicated. +, presence of a positive hybridization signal with the designated strain. Evolutionary relationships of Salmonella Reference Collection C strains are based on variation in the nucleotide sequences of five housekeeping genes (6). The roman numerals indicate the eight Salmonella subspecific groups.