• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of iaiPermissionsJournals.ASM.orgJournalIAI ArticleJournal InfoAuthorsReviewers
Infect Immun. Nov 1997; 65(11): 4606–4614.
PMCID: PMC175661

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 immunoglobulin A (IgA) protease-like family of secreted proteins, Tsh, EspC, SepA, and Hap, from an avian pathogenic Escherichia coli, an enteropathogenic 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 insertionally disrupted by allelic exchange using a Tn10-derived tetAR(B) cassette. Then, to detect loss of the she locus, the tetracycline-resistant derivative was plated onto fusaric acid medium to select for tetracycline-sensitive revertants, which were observed to arise at a frequency of 10(-5) to 10(-6). PCR and pulsed-field gel electrophoresis analysis confirmed loss of the she::tetAR(B) locus in six independent tetracycline-sensitive isolates. Sample sequencing over a 25-kb region flanking 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 inverted 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, supporting the conclusion that she lay within a large pathogenicity island. The method described in this study, termed island probing, provides a useful tool to further the study of pathogenicity islands in general. Importantly, this approach could also be of value in constructing safer live attenuated bacterial vaccines.

Full Text

The Full Text of this article is available as a PDF (1.1M).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. [PubMed]
  • Benjelloun-Touimi Z, Sansonetti PJ, Parsot C. SepA, the major extracellular protein of Shigella flexneri: autonomous secretion and involvement in tissue invasion. Mol Microbiol. 1995 Jul;17(1):123–135. [PubMed]
  • Bernardini ML, Fontaine A, Sansonetti PJ. The two-component regulatory system ompR-envZ controls the virulence of Shigella flexneri. J Bacteriol. 1990 Nov;172(11):6274–6281. [PMC free article] [PubMed]
  • Bik EM, Bunschoten AE, Gouw RD, Mooi FR. Genesis of the novel epidemic Vibrio cholerae O139 strain: evidence for horizontal transfer of genes involved in polysaccharide synthesis. EMBO J. 1995 Jan 16;14(2):209–216. [PMC free article] [PubMed]
  • Billington SJ, Johnston JL, Rood JI. Virulence regions and virulence factors of the ovine footrot pathogen, Dichelobacter nodosus. FEMS Microbiol Lett. 1996 Dec 1;145(2):147–156. [PubMed]
  • Bloch CA, Rode CK. Pathogenicity island evaluation in Escherichia coli K1 by crossing with laboratory strain K-12. Infect Immun. 1996 Aug;64(8):3218–3223. [PMC free article] [PubMed]
  • Bloch CA, Rode CK, Obreque V, Russell KY. Comparative genome mapping with mobile physical map landmarks. J Bacteriol. 1994 Nov;176(22):7121–7125. [PMC free article] [PubMed]
  • Bloomfield GA, Whittle G, McDonagh MB, Katz ME, Cheetham BF. Analysis of sequences flanking the vap regions of Dichelobacter nodosus: evidence for multiple integration events, a killer system, and a new genetic element. Microbiology. 1997 Feb;143(Pt 2):553–562. [PubMed]
  • Blum G, Ott M, Lischewski A, Ritter A, Imrich H, Tschäpe H, Hacker J. Excision of large DNA regions termed pathogenicity islands from tRNA-specific loci in the chromosome of an Escherichia coli wild-type pathogen. Infect Immun. 1994 Feb;62(2):606–614. [PMC free article] [PubMed]
  • Blum G, Falbo V, Caprioli A, Hacker J. Gene clusters encoding the cytotoxic necrotizing factor type 1, Prs-fimbriae and alpha-hemolysin form the pathogenicity island II of the uropathogenic Escherichia coli strain J96. FEMS Microbiol Lett. 1995 Feb 15;126(2):189–195. [PubMed]
  • Brunder W, Schmidt H, Karch H. EspP, a novel extracellular serine protease of enterohaemorrhagic Escherichia coli O157:H7 cleaves human coagulation factor V. Mol Microbiol. 1997 May;24(4):767–778. [PubMed]
  • Carniel E, Guilvout I, Prentice M. Characterization of a large chromosomal "high-pathogenicity island" in biotype 1B Yersinia enterocolitica. J Bacteriol. 1996 Dec;178(23):6743–6751. [PMC free article] [PubMed]
  • Censini S, Lange C, Xiang Z, Crabtree JE, Ghiara P, Borodovsky M, Rappuoli R, Covacci A. cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14648–14653. [PMC free article] [PubMed]
  • Cheetham BF, Katz ME. A role for bacteriophages in the evolution and transfer of bacterial virulence determinants. Mol Microbiol. 1995 Oct;18(2):201–208. [PubMed]
  • Cherepanov PA, Mikhailova TG, Karimova GA, Zakharova NM, Ershov Iu V, Volkovoi KI. Klonirovanie i detal'noe kartirovanie fra-ymt-oblasti plazmidy pFra Yersinia pestis. Mol Gen Mikrobiol Virusol. 1991 Dec;(12):19–26. [PubMed]
  • Collins CM, Gutman DM. Insertional inactivation of an Escherichia coli urease gene by IS3411. J Bacteriol. 1992 Feb;174(3):883–888. [PMC free article] [PubMed]
  • Cover TL. The vacuolating cytotoxin of Helicobacter pylori. Mol Microbiol. 1996 Apr;20(2):241–246. [PubMed]
  • Dar ME, Bhagwat AS. Mechanism of expression of DNA repair gene vsr, an Escherichia coli gene that overlaps the DNA cytosine methylase gene, dcm. Mol Microbiol. 1993 Aug;9(4):823–833. [PubMed]
  • Egile C, d'Hauteville H, Parsot C, Sansonetti PJ. SopA, the outer membrane protease responsible for polar localization of IcsA in Shigella flexneri. Mol Microbiol. 1997 Mar;23(5):1063–1073. [PubMed]
  • Fasano A, Noriega FR, Maneval DR, Jr, Chanasongcram S, Russell R, Guandalini S, Levine MM. Shigella enterotoxin 1: an enterotoxin of Shigella flexneri 2a active in rabbit small intestine in vivo and in vitro. J Clin Invest. 1995 Jun;95(6):2853–2861. [PMC free article] [PubMed]
  • Ferat JL, Michel F. Group II self-splicing introns in bacteria. Nature. 1993 Jul 22;364(6435):358–361. [PubMed]
  • Fetherston JD, Schuetze P, Perry RD. Loss of the pigmentation phenotype in Yersinia pestis is due to the spontaneous deletion of 102 kb of chromosomal DNA which is flanked by a repetitive element. Mol Microbiol. 1992 Sep;6(18):2693–2704. [PubMed]
  • Freiberg C, Fellay R, Bairoch A, Broughton WJ, Rosenthal A, Perret X. Molecular basis of symbiosis between Rhizobium and legumes. Nature. 1997 May 22;387(6631):394–401. [PubMed]
  • Froehlich BJ, Karakashian A, Melsen LR, Wakefield JC, Scott JR. CooC and CooD are required for assembly of CS1 pili. Mol Microbiol. 1994 May;12(3):387–401. [PubMed]
  • Ghosal D, Sommer H, Saedler H. Nucleotide sequence of the transposable DNA-element IS2. Nucleic Acids Res. 1979 Mar;6(3):1111–1122. [PMC free article] [PubMed]
  • Gouin E, Mengaud J, Cossart P. The virulence gene cluster of Listeria monocytogenes is also present in Listeria ivanovii, an animal pathogen, and Listeria seeligeri, a nonpathogenic species. Infect Immun. 1994 Aug;62(8):3550–3553. [PMC free article] [PubMed]
  • Hacker J, Blum-Oehler G, Mühldorfer I, Tschäpe H. Pathogenicity islands of virulent bacteria: structure, function and impact on microbial evolution. Mol Microbiol. 1997 Mar;23(6):1089–1097. [PubMed]
  • Heard PL, Lewis CS, Chaudhuri G. Leishmania mexicana amazonensis: differential display analysis and cloning of mRNAs from attenuated and infective forms. J Eukaryot Microbiol. 1996 Sep-Oct;43(5):409–415. [PubMed]
  • Ishiguro N, Sato G. Nucleotide sequence of insertion sequence IS3411, which flanks the citrate utilization determinant of transposon Tn3411. J Bacteriol. 1988 Apr;170(4):1902–1906. [PMC free article] [PubMed]
  • Jordi BJ, van Vliet AH, Willshaw GA, van der Zeijst BA, Gaastra W. Analysis of the first two genes of the CS1 fimbrial operon in human enterotoxigenic Escherichia coli of serotype 0139:H28. FEMS Microbiol Lett. 1991 May 15;64(2-3):265–270. [PubMed]
  • Knoop V, Brennicke A. Evidence for a group II intron in Escherichia coli inserted into a highly conserved reading frame associated with mobile DNA sequences. Nucleic Acids Res. 1994 Apr 11;22(7):1167–1171. [PMC free article] [PubMed]
  • Kozlov YuV, Kabishev AA, Lukyanov EV, Bayev AA. The primary structure of the operons coding for Shigella dysenteriae toxin and temperature phage H30 shiga-like toxin. Gene. 1988 Jul 30;67(2):213–221. [PubMed]
  • Mahairas GG, Sabo PJ, Hickey MJ, Singh DC, Stover CK. Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J Bacteriol. 1996 Mar;178(5):1274–1282. [PMC free article] [PubMed]
  • Maloy SR, Nunn WD. Selection for loss of tetracycline resistance by Escherichia coli. J Bacteriol. 1981 Feb;145(2):1110–1111. [PMC free article] [PubMed]
  • Matsutani S, Ohtsubo E. Complete sequence of IS629. Nucleic Acids Res. 1990 Apr 11;18(7):1899–1899. [PMC free article] [PubMed]
  • Matsutani S, Ohtsubo H, Maeda Y, Ohtsubo E. Isolation and characterization of IS elements repeated in the bacterial chromosome. J Mol Biol. 1987 Aug 5;196(3):445–455. [PubMed]
  • McDaniel TK, Kaper JB. A cloned pathogenicity island from enteropathogenic Escherichia coli confers the attaching and effacing phenotype on E. coli K-12. Mol Microbiol. 1997 Jan;23(2):399–407. [PubMed]
  • McDaniel TK, Jarvis KG, Donnenberg MS, Kaper JB. A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1664–1668. [PMC free article] [PubMed]
  • Mecsas JJ, Strauss EJ. Molecular mechanisms of bacterial virulence: type III secretion and pathogenicity islands. Emerg Infect Dis. 1996 Oct-Dec;2(4):270–288. [PMC free article] [PubMed]
  • Michel F, Ferat JL. Structure and activities of group II introns. Annu Rev Biochem. 1995;64:435–461. [PubMed]
  • Miller VL, Mekalanos JJ. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. [PMC free article] [PubMed]
  • Mills DA, McKay LL, Dunny GM. Splicing of a group II intron involved in the conjugative transfer of pRS01 in lactococci. J Bacteriol. 1996 Jun;178(12):3531–3538. [PMC free article] [PubMed]
  • Mills DM, Bajaj V, Lee CA. A 40 kb chromosomal fragment encoding Salmonella typhimurium invasion genes is absent from the corresponding region of the Escherichia coli K-12 chromosome. Mol Microbiol. 1995 Feb;15(4):749–759. [PubMed]
  • Mullany P, Pallen M, Wilks M, Stephen JR, Tabaqchali S. A group II intron in a conjugative transposon from the gram-positive bacterium, Clostridium difficile. Gene. 1996 Sep 26;174(1):145–150. [PubMed]
  • Nataro JP, Seriwatana J, Fasano A, Maneval DR, Guers LD, Noriega F, Dubovsky F, Levine MM, Morris JG., Jr Identification and cloning of a novel plasmid-encoded enterotoxin of enteroinvasive Escherichia coli and Shigella strains. Infect Immun. 1995 Dec;63(12):4721–4728. [PMC free article] [PubMed]
  • Noriega FR, Liao FM, Formal SB, Fasano A, Levine MM. Prevalence of Shigella enterotoxin 1 among Shigella clinical isolates of diverse serotypes. J Infect Dis. 1995 Nov;172(5):1408–1410. [PubMed]
  • Noriega FR, Losonsky G, Lauderbaugh C, Liao FM, Wang JY, Levine MM. Engineered deltaguaB-A deltavirG Shigella flexneri 2a strain CVD 1205: construction, safety, immunogenicity, and potential efficacy as a mucosal vaccine. Infect Immun. 1996 Aug;64(8):3055–3061. [PMC free article] [PubMed]
  • Okada N, Sasakawa C, Tobe T, Talukder KA, Komatsu K, Yoshikawa M. Construction of a physical map of the chromosome of Shigella flexneri 2a and the direct assignment of nine virulence-associated loci identified by Tn5 insertions. Mol Microbiol. 1991 Sep;5(9):2171–2180. [PubMed]
  • Okada N, Sasakawa C, Tobe T, Yamada M, Nagai S, Talukder KA, Komatsu K, Kanegasaki S, Yoshikawa M. Virulence-associated chromosomal loci of Shigella flexneri identified by random Tn5 insertion mutagenesis. Mol Microbiol. 1991 Jan;5(1):187–195. [PubMed]
  • Ott M. Dynamics of the bacterial genome: deletions and integrations as mechanisms of bacterial virulence modulation. Zentralbl Bakteriol. 1993 Jun;278(4):457–468. [PubMed]
  • Paton AW, Paton JC, Goldwater PN, Heuzenroeder MW, Manning PA. Sequence of a variant Shiga-like toxin type-I operon of Escherichia coli O111:H-. Gene. 1993 Jul 15;129(1):87–92. [PubMed]
  • Provence DL, Curtiss R., 3rd Isolation and characterization of a gene involved in hemagglutination by an avian pathogenic Escherichia coli strain. Infect Immun. 1994 Apr;62(4):1369–1380. [PMC free article] [PubMed]
  • Rajakumar K, Sasakawa C, Adler B. A spontaneous 99-kb chromosomal deletion results in multi-antibiotic susceptibility and an attenuation of contact haemolysis in Shigella flexneri 2a. J Med Microbiol. 1996 Jul;45(1):64–75. [PubMed]
  • Rajakumar K, Bulach D, Davies J, Ambrose L, Sasakawa C, Adler B. Identification of a chromosomal Shigella flexneri multi-antibiotic resistance locus which shares sequence and organizational similarity with the resistance region of the plasmid NR1. Plasmid. 1997;37(3):159–168. [PubMed]
  • Ried JL, Collmer A. An nptI-sacB-sacR cartridge for constructing directed, unmarked mutations in gram-negative bacteria by marker exchange-eviction mutagenesis. Gene. 1987;57(2-3):239–246. [PubMed]
  • Sansonetti PJ, Ryter A, Clerc P, Maurelli AT, Mounier J. Multiplication of Shigella flexneri within HeLa cells: lysis of the phagocytic vacuole and plasmid-mediated contact hemolysis. Infect Immun. 1986 Feb;51(2):461–469. [PMC free article] [PubMed]
  • Sasakawa C, Kamata K, Sakai T, Murayama SY, Makino S, Yoshikawa M. Molecular alteration of the 140-megadalton plasmid associated with loss of virulence and Congo red binding activity in Shigella flexneri. Infect Immun. 1986 Feb;51(2):470–475. [PMC free article] [PubMed]
  • Shea JE, Hensel M, Gleeson C, Holden DW. Identification of a virulence locus encoding a second type III secretion system in Salmonella typhimurium. Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2593–2597. [PMC free article] [PubMed]
  • Shearman C, Godon JJ, Gasson M. Splicing of a group II intron in a functional transfer gene of Lactococcus lactis. Mol Microbiol. 1996 Jul;21(1):45–53. [PubMed]
  • Silva RM, Saadi S, Maas WK. A basic replicon of virulence-associated plasmids of Shigella spp. and enteroinvasive Escherichia coli is homologous with a basic replicon in plasmids of IncF groups. Infect Immun. 1988 Apr;56(4):836–842. [PMC free article] [PubMed]
  • Smith CL, Cantor CR. Purification, specific fragmentation, and separation of large DNA molecules. Methods Enzymol. 1987;155:449–467. [PubMed]
  • Stein MA, Leung KY, Zwick M, Garcia-del Portillo F, Finlay BB. Identification of a Salmonella virulence gene required for formation of filamentous structures containing lysosomal membrane glycoproteins within epithelial cells. Mol Microbiol. 1996 Apr;20(1):151–164. [PubMed]
  • Stein M, Kenny B, Stein MA, Finlay BB. Characterization of EspC, a 110-kilodalton protein secreted by enteropathogenic Escherichia coli which is homologous to members of the immunoglobulin A protease-like family of secreted proteins. J Bacteriol. 1996 Nov;178(22):6546–6554. [PMC free article] [PubMed]
  • St Geme JW, 3rd, de la Morena ML, Falkow S. A Haemophilus influenzae IgA protease-like protein promotes intimate interaction with human epithelial cells. Mol Microbiol. 1994 Oct;14(2):217–233. [PubMed]
  • Swenson DL, Bukanov NO, Berg DE, Welch RA. Two pathogenicity islands in uropathogenic Escherichia coli J96: cosmid cloning and sample sequencing. Infect Immun. 1996 Sep;64(9):3736–3743. [PMC free article] [PubMed]
  • Uchiya K, Tobe T, Komatsu K, Suzuki T, Watarai M, Fukuda I, Yoshikawa M, Sasakawa C. Identification of a novel virulence gene, virA, on the large plasmid of Shigella, involved in invasion and intercellular spreading. Mol Microbiol. 1995 Jul;17(2):241–250. [PubMed]
  • Venkatesan MM, Buysse JM, Hartman AB. Sequence variation in two ipaH genes of Shigella flexneri 5 and homology to the LRG-like family of proteins. Mol Microbiol. 1991 Oct;5(10):2435–2445. [PubMed]
  • von Heijne G. How signal sequences maintain cleavage specificity. J Mol Biol. 1984 Feb 25;173(2):243–251. [PubMed]
  • Wang RF, Kushner SR. Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in Escherichia coli. Gene. 1991 Apr;100:195–199. [PubMed]
  • Womble DD, Rownd RH. Genetic and physical map of plasmid NR1: comparison with other IncFII antibiotic resistance plasmids. Microbiol Rev. 1988 Dec;52(4):433–451. [PMC free article] [PubMed]
  • Wu SS, Kaiser D. Markerless deletions of pil genes in Myxococcus xanthus generated by counterselection with the Bacillus subtilis sacB gene. J Bacteriol. 1996 Oct;178(19):5817–5821. [PMC free article] [PubMed]
  • Zagaglia C, Casalino M, Colonna B, Conti C, Calconi A, Nicoletti M. Virulence plasmids of enteroinvasive Escherichia coli and Shigella flexneri integrate into a specific site on the host chromosome: integration greatly reduces expression of plasmid-carried virulence genes. Infect Immun. 1991 Mar;59(3):792–799. [PMC free article] [PubMed]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Gene
    Gene links
  • GSS
    Published GSS sequences
  • MedGen
    Related information in MedGen
  • Nucleotide
    Published Nucleotide sequences
  • Protein
    Published protein sequences
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links
  • Taxonomy
    Related taxonomy entry
  • Taxonomy Tree
    Taxonomy Tree