In this study, the sigA gene situated on the she pathogenicity island of Shigella flexneri 2a was cloned and characterized. Sequence analysis showed that sigA encodes a 139.6-kDa protein which belongs to the SPATE (serine protease autotransporters of Enterobacteriaceae) subfamily of autotransporter proteins. The demonstration that SigA is autonomously secreted from the cell to yield a 103-kDa processed form and possesses a conserved C-terminal domain for export from the cell were consistent with the autotransporter pathway of secretion. Functional analysis showed that SigA is a secreted temperature-regulated serine protease capable of degrading casein. SigA was cytopathic for HEp-2 cells, suggesting that it may be a cell-altering toxin with a role in the pathogenesis of Shigella infections. SigA was at least partly responsible for the ability of S. flexneri to stimulate fluid accumulation in ligated rabbit ileal loops.

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Iron uptake systems which are critical for bacterial survival and which may play important roles in bacterial virulence are often carried on mobile elements, such as plasmids and pathogenicity islands (PAIs). In the present study, we identified and characterized a ferric dicitrate uptake system (Fec) in Shigella flexneri serotype 2a that is encoded by a novel PAI termed the Shigella resistance locus (SRL) PAI. The fec genes are transcribed in S. flexneri, and complementation of a fec deletion in Escherichia coli demonstrated that they are functional. However, insertional inactivation of fecI, leading to a loss in fec gene expression, did not impair the growth of the parent strain of S. flexneri in iron-limited culture media, suggesting that S. flexneri carries additional iron uptake systems capable of compensating for the loss of Fec-mediated iron uptake. DNA sequence analysis showed that the fec genes are linked to a cluster of multiple antibiotic resistance determinants, designated the SRL, on the chromosome of S. flexneri 2a. Both the SRL and fec loci are carried on the 66,257-bp SRL PAI, which has integrated into the serX tRNA gene and which carries at least 22 prophage-related open reading frames, including one for a P4-like integrase. This is the first example of a PAI that carries genes encoding antibiotic resistance and the first report of a ferric dicitrate uptake system in Shigella.Pathogenicity islands (PAIs) are increasingly recognized as playing a vital role in bacterial virulence. PAIs are distinct virulence cassettes that often integrate into tRNA genes and encode bacteriophage-like integrases. Such islands may occupy large regions of the chromosome and often carry mobile elements, such as insertion sequences and transposons (25). PAIs have been found in many bacterial species, including Yersinia spp. (9, 13), enteropathogenic, enterohemorrhagic, and uropathogenic Escherichia coli (24,38,51), Salmonella enterica serovar Typhimurium (61), Vibrio cholerae (32), Helicobacter pylori (14), and Shigella flexneri (2,42,57,71). Some strains of uropathogenic E. coli and S. enterica serovar Typhimurium may harbor at least five PAIs (19,74). A variety of virulence determinants may be carried on PAIs, including genes encoding fimbriae, hemolysins (31, 64), type III secretion systems (15, 27), and iron uptake systems (13,42,71,75). Various Shigella spp. produce the siderophores enterobactin and/or aerobactin, which are involved in iron uptake (34, 50). The aerobactin locus in S. flexneri was recently shown to be carried on the SHI-2 PAI (42, 71). This was the first report of an iron transport system being carried on a PAI in Shigella.A number of PAI-like elements in Shigella spp. have been described. These include the SHI-2 PAI and a family of structurally related elements (42, 71) and the she PAI, which also belongs to a larger family of structurally related elements (2, 57). One of the characteristics of PAIs is their tendency to excise spontaneously from their sites of integration in the chromosome (26). In...

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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.