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Infect Immun. Jan 1992; 60(1): 175–182.
PMCID: PMC257519

Spontaneous insertion of an IS1-like element into the virF gene is responsible for avirulence in opaque colonial variants of Shigella flexneri 2a.


Colonial variation of Shigella flexneri serotype 2a from the translucent (2457T) to the opaque form (2457O) occurs spontaneously once in 10(4) cell divisions, with concomitant loss of ipa gene expression and virulence. The appearance of 2457O was associated with the insertional inactivation of virF, an invasion plasmid-encoded positive regulator of ipa gene expression. Plasmid pWR110, a Tn5-tagged invasion plasmid that restores the invasive phenotype to plasmid-cured Shigella derivatives, was conjugally transferred into 2457O. Synthesis of the invasion-associated IpaB and IpaC polypeptides, normally present on the surface of virulent shigellae, and the invasive phenotype were restored in 2457O(pWR110) transconjugants. Plasmid DNA restriction endonuclease patterns of 2457T and 2457O, along with hybridization analysis, showed that a SalI fragment carrying the virF gene in 2457O had increased in size relative to its counterpart in 2457T. Analysis of virF DNA sequences amplified by the polymerase chain reaction revealed that the virF sequence from 2457O was 780 bp larger than that amplified from 2457T. Moreover, the virF sequence amplified from 2457O hybridized to an IS1 DNA probe whereas the amplified 2457T virF sequence did not. DNA sequence analysis mapped the insertion element, designated IS1SFO, within an A.T-rich region of the virF open reading frame and identified a 9-bp virF target sequence that was duplicated at the insertion site of IS1SFO. The DNA sequence of IS1SFO was greater than 99% homologous to IS1F. Plasmid pWR600, carrying a 1,260-bp HpaII fragment encoding a wild-type virF gene, was able to restore the virulent phenotype and translucent colonial morphology to nine independently isolated 2457O hosts.

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  • Bernardini ML, Mounier J, d'Hauteville H, Coquis-Rondon M, Sansonetti PJ. Identification of icsA, a plasmid locus of Shigella flexneri that governs bacterial intra- and intercellular spread through interaction with F-actin. Proc Natl Acad Sci U S A. 1989 May;86(10):3867–3871. [PMC free article] [PubMed]
  • Birnboim HC. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 1983;100:243–255. [PubMed]
  • Buysse JM, Stover CK, Oaks EV, Venkatesan M, Kopecko DJ. Molecular cloning of invasion plasmid antigen (ipa) genes from Shigella flexneri: analysis of ipa gene products and genetic mapping. J Bacteriol. 1987 Jun;169(6):2561–2569. [PMC free article] [PubMed]
  • Buysse JM, Venkatesan M, Mills JA, Oaks EV. Molecular characterization of a trans-acting, positive effector (ipaR) of invasion plasmid antigen synthesis in Shigella flexneri serotype 5. Microb Pathog. 1990 Mar;8(3):197–211. [PubMed]
  • Chosa H, Makino S, Sasakawa C, Okada N, Yamada M, Komatsu K, Suk JS, Yoshikawa M. Loss of virulence in Shigella strains preserved in culture collections due to molecular alteration of the invasion plasmid. Microb Pathog. 1989 May;6(5):337–342. [PubMed]
  • Daskaleros PA, Payne SM. Cloning the gene for Congo red binding in Shigella flexneri. Infect Immun. 1985 Apr;48(1):165–168. [PMC free article] [PubMed]
  • Daskaleros PA, Payne SM. Characterization of Shigella flexneri sequences encoding congo red binding (crb): conservation of multiple crb sequences and role of IS1 in loss of the Crb+ phenotype. Infect Immun. 1986 Nov;54(2):435–443. [PMC free article] [PubMed]
  • DuPont HL, Hornick RB, Snyder MJ, Libonati JP, Formal SB, Gangarosa EJ. Immunity in shigellosis. I. Response of man to attenuated strains of Shigella. J Infect Dis. 1972 Jan;125(1):5–11. [PubMed]
  • Elsinghorst EA, Baron LS, Kopecko DJ. Penetration of human intestinal epithelial cells by Salmonella: molecular cloning and expression of Salmonella typhi invasion determinants in Escherichia coli. Proc Natl Acad Sci U S A. 1989 Jul;86(13):5173–5177. [PMC free article] [PubMed]
  • Formal SB, Gemski P, Baron LS, Labrec EH. A Chromosomal Locus Which Controls the Ability of Shigella flexneri to Evoke Keratoconjunctivitis. Infect Immun. 1971 Jan;3(1):73–79. [PMC free article] [PubMed]
  • FORMAL SB, DAMMIN GJ, LABREC EH, SCHNEIDER H. Experimental Shigella infections: characteristics of a fatal infection produced in guinea pigs. J Bacteriol. 1958 May;75(5):604–610. [PMC free article] [PubMed]
  • Formal SB, Labrec EH, Palmer A, Falkow S. Protection of Monkeys Against Experimental Shigellosis with Attenuated Vaccines. J Bacteriol. 1965 Jul;90(1):63–68. [PMC free article] [PubMed]
  • Galas DJ, Calos MP, Miller JH. Sequence analysis of Tn9 insertions in the lacZ gene. J Mol Biol. 1980 Nov 25;144(1):19–41. [PubMed]
  • Hale TL. Genetic basis of virulence in Shigella species. Microbiol Rev. 1991 Jun;55(2):206–224. [PMC free article] [PubMed]
  • Hale TL, Oaks EV, Formal SB. Identification and antigenic characterization of virulence-associated, plasmid-coded proteins of Shigella spp. and enteroinvasive Escherichia coli. Infect Immun. 1985 Dec;50(3):620–629. [PMC free article] [PubMed]
  • Jordan E, Saedler H, Starlinger P. O0 and strong-polar mutations in the gal operon are insertions. Mol Gen Genet. 1968;102(4):353–363. [PubMed]
  • Kato J, Ito K, Nakamura A, Watanabe H. Cloning of regions required for contact hemolysis and entry into LLC-MK2 cells from Shigella sonnei form I plasmid: virF is a positive regulator gene for these phenotypes. Infect Immun. 1989 May;57(5):1391–1398. [PMC free article] [PubMed]
  • Kim R, Corwin LM. Mutation in Shigella flexneri resulting in loss of ability to penetrate HeLa cells and loss of glycerol kinase activity. Infect Immun. 1974 May;9(5):916–923. [PMC free article] [PubMed]
  • Kopecko DJ, Holcombe J, Formal SB. Molecular characterization of plasmids from virulent and spontaneously occurring avirulent colonial variants of Shigella flexneri. Infect Immun. 1979 May;24(2):580–582. [PMC free article] [PubMed]
  • Labrec EH, Schneider H, Magnani TJ, Formal SB. EPITHELIAL CELL PENETRATION AS AN ESSENTIAL STEP IN THE PATHOGENESIS OF BACILLARY DYSENTERY. J Bacteriol. 1964 Nov;88(5):1503–1518. [PMC free article] [PubMed]
  • Makino S, Sasakawa C, Kamata K, Kurata T, Yoshikawa M. A genetic determinant required for continuous reinfection of adjacent cells on large plasmid in S. flexneri 2a. Cell. 1986 Aug 15;46(4):551–555. [PubMed]
  • Maurelli AT, Blackmon B, Curtiss R., 3rd Loss of pigmentation in Shigella flexneri 2a is correlated with loss of virulence and virulence-associated plasmid. Infect Immun. 1984 Jan;43(1):397–401. [PMC free article] [PubMed]
  • Maurelli AT, Sansonetti PJ. Identification of a chromosomal gene controlling temperature-regulated expression of Shigella virulence. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2820–2824. [PMC free article] [PubMed]
  • Mills JA, Buysse JM, Oaks EV. Shigella flexneri invasion plasmid antigens B and C: epitope location and characterization with monoclonal antibodies. Infect Immun. 1988 Nov;56(11):2933–2941. [PMC free article] [PubMed]
  • Nyman K, Nakamura K, Ohtsubo H, Ohtsubo E. Distribution of the insertion sequence IS1 in gram-negative bacteria. Nature. 1981 Feb 12;289(5798):609–612. [PubMed]
  • Oaks EV, Wingfield ME, Formal SB. Plaque formation by virulent Shigella flexneri. Infect Immun. 1985 Apr;48(1):124–129. [PMC free article] [PubMed]
  • Oaks EV, Wingfield ME, Formal SB. Plaque formation by virulent Shigella flexneri. Infect Immun. 1985 Apr;48(1):124–129. [PMC free article] [PubMed]
  • Pál T, Newland JW, Tall BD, Formal SB, Hale TL. Intracellular spread of Shigella flexneri associated with the kcpA locus and a 140-kilodalton protein. Infect Immun. 1989 Feb;57(2):477–486. [PMC free article] [PubMed]
  • Payne SM, Finkelstein RA. Detection and differentiation of iron-responsive avirulent mutants on Congo red agar. Infect Immun. 1977 Oct;18(1):94–98. [PMC free article] [PubMed]
  • Peterson BC, Hashimoto H, Rownd RH. Cointegrate formation between homologous plasmids in Escherichia coli. J Bacteriol. 1982 Sep;151(3):1086–1094. [PMC free article] [PubMed]
  • Sakai T, Sasakawa C, Makino S, Kamata K, Yoshikawa M. Molecular cloning of a genetic determinant for Congo red binding ability which is essential for the virulence of Shigella flexneri. Infect Immun. 1986 Feb;51(2):476–482. [PMC free article] [PubMed]
  • Sakai T, Sasakawa C, Makino S, Yoshikawa M. DNA sequence and product analysis of the virF locus responsible for congo red binding and cell invasion in Shigella flexneri 2a. Infect Immun. 1986 Nov;54(2):395–402. [PMC free article] [PubMed]
  • Sakai T, Sasakawa C, Yoshikawa M. Expression of four virulence antigens of Shigella flexneri is positively regulated at the transcriptional level by the 30 kiloDalton virF protein. Mol Microbiol. 1988 Sep;2(5):589–597. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Sansonetti PJ, Hale TL, Dammin GJ, Kapfer C, Collins HH, Jr, Formal SB. Alterations in the pathogenicity of Escherichia coli K-12 after transfer of plasmid and chromosomal genes from Shigella flexneri. Infect Immun. 1983 Mar;39(3):1392–1402. [PMC free article] [PubMed]
  • Sansonetti PJ, Kopecko DJ, Formal SB. Involvement of a plasmid in the invasive ability of Shigella flexneri. Infect Immun. 1982 Mar;35(3):852–860. [PMC free article] [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, Adler B, Tobe T, Okada N, Nagai S, Komatsu K, Yoshikawa M. Functional organization and nucleotide sequence of virulence Region-2 on the large virulence plasmid in Shigella flexneri 2a. Mol Microbiol. 1989 Sep;3(9):1191–1201. [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]
  • Sasakawa C, Makino S, Kamata K, Yoshikawa M. Isolation, characterization, and mapping of Tn5 insertions into the 140-megadalton invasion plasmid defective in the mouse Sereny test in Shigella flexneri 2a. Infect Immun. 1986 Oct;54(1):32–36. [PMC free article] [PubMed]
  • SERENY B. Experimental shigella keratoconjunctivitis; a preliminary report. Acta Microbiol Acad Sci Hung. 1955;2(3):293–296. [PubMed]
  • Shapiro JA. Mutations caused by the insertion of genetic material into the galactose operon of Escherichia coli. J Mol Biol. 1969 Feb 28;40(1):93–105. [PubMed]
  • Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. [PubMed]
  • Stadler J, Adelberg EA. Temperature dependence of sex-factor maintenance in Escherichia coli K-12. J Bacteriol. 1972 Jan;109(1):447–449. [PMC free article] [PubMed]
  • Tobe T, Nagai S, Okada N, Adler B, Yoshikawa M, Sasakawa C. Temperature-regulated expression of invasion genes in Shigella flexneri is controlled through the transcriptional activation of the virB gene on the large plasmid. Mol Microbiol. 1991 Apr;5(4):887–893. [PubMed]
  • Tomoeda M, Inuzuka M, Kubo N, Nakamura S. Effective elimination of drug resistance and sex factors in Escherichia coli by sodium dodecyl sulfate. J Bacteriol. 1968 Mar;95(3):1078–1089. [PMC free article] [PubMed]
  • Umeda M, Ohtsubo E. Four types of IS1 with differences in nucleotide sequence reside in the Escherichia coli K-12 chromosome. Gene. 1991 Feb 1;98(1):1–5. [PubMed]
  • Venkatesan M, Buysse JM, Vandendries E, Kopecko DJ. Development and testing of invasion-associated DNA probes for detection of Shigella spp. and enteroinvasive Escherichia coli. J Clin Microbiol. 1988 Feb;26(2):261–266. [PMC free article] [PubMed]
  • Venkatesan MM, Buysse JM, Kopecko DJ. Characterization of invasion plasmid antigen genes (ipaBCD) from Shigella flexneri. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9317–9321. [PMC free article] [PubMed]
  • WATKINS HM. Some attributes of virulence in Shigella. Ann N Y Acad Sci. 1960 Nov 21;88:1167–1186. [PubMed]
  • Yamada M, Sasakawa C, Okada N, Makino SI, Yoshikawa M. Molecular cloning and characterization of chromosomal virulence region kcpA of Shigella flexneri. Mol Microbiol. 1989 Feb;3(2):207–213. [PubMed]
  • Zerbib D, Gamas P, Chandler M, Prentki P, Bass S, Galas D. Specificity of insertion of IS1. J Mol Biol. 1985 Oct 5;185(3):517–524. [PubMed]

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