USE OF A NOVEL-APPROACH, TERMED ISLAND PROBING, IDENTIFIES THE SHIGELLA-FLEXNERI SHE PATHOGENICITY ISLAND WHICH ENCODES A HOMOLOG OF THE IMMUNOGLOBULIN-A PROTEASE-LIKE FAMILY OF PROTEINS

The she gene of Shigella flexneri 2a, which also harbors the internal enterotoxin genes set1A and set1B (F. R. Noriega, GenBank accession no . U35656, 1995) encodes a homolog of the virulence-related immunoglobu lin A (IgA) protease-like family of secreted proteins, Tsh, EspC, SepA , and Hap, from an avian pathogenic Escherichia coli, an enteropathoge nic E. coli, S. flexneri 5, and Haemophilus influenzae, respectively. To investigate the possibility that this locus was carried on a larger deletable element, the S. flexneri 2a YSH6000T she gene was insertion ally disrupted by allelic exchange using a Tn10-derived tetAR(B) casse tte. Then, to detect loss of the she locus, the tetracycline-resistant derivative was plated onto fusaric acid medium to select for tetracyc line-sensitive revertants, which were observed to arise at a frequency of 10(-5) to 10(-6). PCR and pulsed-field gel electrophoresis analysi s confirmed loss of the she::tetAR(B) locus in six independent tetracy cline-sensitive isolates. Sample sequencing over a 25-kb region flanki ng she identified four insertion sequence-like elements, the group II intron-like sequence Sf.IntA, and the 3' end of a second IgA protease- like homolog, sigA, lying 3.6 kb downstream and in an orientation inve rted with respect to she. The deletion was mapped to chromosomal NotI fragment A and determined to have a size of 51 kb. Hybridization with flanking probes confirmed that at least 17.7 kb of the 51-kb deletable element was unique to the seven she(+) strains investigated, supporti ng the conclusion that she lay within a large pathogenicity island. Th e method described in this study, termed island probing, provides a us eful tool to further the study of pathogenicity islands in general. Im portantly, this approach could also be of value in constructing safer live attenuated bacterial vaccines.