1887

Abstract

A Tn5-generated mutant (strain S2430) of 2a (strain YSH6000) exhibited attenuated virulence and, in addition to the Tn5 insertion in the I K fragment of its virulence plasmid, had a 99-kb deletion within its chromosome. Unlike its wild-type parent, strain S2430 was susceptible to ampicillin, streptomycin, tetracycline and chloramphenicol. An independent multi-antibiotic susceptible variant of strain YSH6000 had a similar deletion. Southern blot analysis of pulsed field electrophoresis gels enabled the sizing of this deletion and its mapping to a region of the chromosome on I fragment D bounded by the homologues of and . Hybridisation experiments with a probe specific to the multi-antibiotic resistance region indicated that this large deletion was responsible for antibiotic susceptibility. Both strain S2430 and a derivative of the antibiotic-susceptible variant, with a Tn5 insertion in its l K fragment, exhibited an equal reduction in contact haemolysis compared with the Tn5-bearing derivative of strain YSH6000. However, strain S2430 alone clearly displayed delayed plaque forming ability in LLC-MK2 monolayers, suggesting that the two examples of this deletion may not be identical.

Loading

Article metrics loading...

/content/journal/jmm/10.1099/00222615-45-1-64
1996-07-01
2022-01-28
Loading full text...

Full text loading...

/deliver/fulltext/jmm/45/1/medmicro-45-1-64.html?itemId=/content/journal/jmm/10.1099/00222615-45-1-64&mimeType=html&fmt=ahah

References

  1. Sansonetti P. J. Molecular and cellular biology of Shigella flexneri invasiveness: from cell assay systems to shigellosis. Curr Top Microbiol Immunol 1992; 180:1–19
    [Google Scholar]
  2. Sasakawa C., Buysse J. M., Watanabe H. The large virulence plasmid of Shigella. Curr Top Microbiol Immunol 1992; 180:21–44
    [Google Scholar]
  3. High N., Mounier J., Prévost M. C., Sansonetti P. J. IpaB of Shigella flexneri causes entry into epithelial cells and escape from the phagocytic vacuole. EMBO J 1992; 11:1991–1999
    [Google Scholar]
  4. Sasakawa C., Adler B., Tobe T. Functional organization and nucleotide sequence of virulence Region-2 on the large virulence plasmid in Shigella flexneri 2a. Mol Microbiol 1989; 3:1191–1201
    [Google Scholar]
  5. Clerc P., Sansonetti P. J. Entry of Shigella flexneri into HeLa cells: evidence for directed phagocytosis involving actin polymerization and myosin accumulation. Infect Immun 1987; 55:2681–2688
    [Google Scholar]
  6. Allaoui A., Mounier J., Prévost, M-C., Sansonetti P. J., Parsot C. icsB: a Shigella flexneri virulence gene necessary for the lysis of protrusions during intercellular spread. Mol Microbiol 1992; 6:1605–1616
    [Google Scholar]
  7. Suzuki T., Murai T., Fukuda I., Tobe T., Yoshikawa M., Sasakawa C. Identification and characterization of a chromosomal virulence gene, vacJ, required for intercellular spreading of Shigella flexneri. Mol Microbiol 1994; 11:31–41
    [Google Scholar]
  8. Andrews G. P., Hromockyj A. E., Coker C., Maurelli A. T. Two novel virulence loci, mxiA and mxiB, in Shigella flexneri 2a facilitate excretion of invasion plasmid antigens. Infect Immun 1991; 59:1997–2005
    [Google Scholar]
  9. Sasakawa C., Komatsu K., Tobe T., Suzuki T., Yoshikawa M. Eight genes in region 5 that form an operon are essential for invasion of epithelial cells by Shigella flexneri 2a. J Bacteriol 1993; 175:2334–2346
    [Google Scholar]
  10. Venkatesan M. M., Buysse J. M., Oaks E. V. Surface presentation of Shigella flexneri invasion plasmid antigens requires the products of the spa locus. J Bacteriol 1992; 174:1990–2001
    [Google Scholar]
  11. Bemardini M. L., Mounier J., d’Hauteville H., Coquis-Rondon M., Sansonetti P. J. 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 USA 1989; 86:3867–3871
    [Google Scholar]
  12. Nakata N., Sasakawa C., Okada N. Identification and characterization of virK, a virulence-associated large plasmid gene essential for intercellular spreading of Shigella flexneri. Mol Microbiol 1992; 6:2387–2395
    [Google Scholar]
  13. Adler B., Sasakawa C., Tobe T., Makino S., Komatsu K., Yoshikawa M. A dual transcriptional activation system for the 230 kb plasmid genes coding for virulence-associated antigens of Shigella flexneri. Mol Microbiol 1989; 3:627–635
    [Google Scholar]
  14. 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; 2:589–597
    [Google Scholar]
  15. Hromockyj A. E., Tucker S. C., Maurelli A. T. Temperature regulation of Shigella virulence: identification of the repressor gene virR, an analogue of hns, and partial complementation by tyrosyl transfer RNA (tRNA1 Tyr). Mol Microbiol 1992; 6:2113–2124
    [Google Scholar]
  16. Bemardini M. L., Fontaine A., Sansonetti P. J. The two-component regulatory system OmpR-EnvZ controls the virulence of Shigella flexneri. J Bacteriol 1990; 172:6274–6281
    [Google Scholar]
  17. Lindberg A. A., Kärnell A., Weintraub A. The lipopolysaccharide of Shigella bacteria as a virulence factor. Rev Infect Dis 1991; 13: Suppl 4S279–S284
    [Google Scholar]
  18. Nassif X., Mazert, M-C., Mounier J., Sansonetti P. J. Evaluation with an iuc::Tn10 mutant of the role of aerobactin production in the virulence of Shigella flexneri. Infect Immun 1987; 55:1963–1969
    [Google Scholar]
  19. Franzon V. L., Arondel J., Sansonetti P. J. Contribution of superoxide dismutase and catalase activities to Shigella flexneri pathogenesis. Infect Immun 1990; 58:529–535
    [Google Scholar]
  20. Tobe T., Sasakawa C., Okada N., Honma Y., Yoshikawa M. vacB, a novel chromosomal gene required for expression of virulence genes on the large plasmid of Shigella flexneri. J Bacteriol 1992; 174:6359–6367
    [Google Scholar]
  21. Adeleye I. A. Conjugal transferability of multiple antibiotic resistance in the three genera of Enterobacteriaceae in Nigeria. J Diarrhoeal Dis Res 1992; 10:93–96
    [Google Scholar]
  22. Ling J. M., Shaw P. C., Kam K. M., Cheng A. F., French G. L. Molecular studies of plasmids of multiply-resistant Shigella spp. in Hong Kong. Epidemiol Infect 1993; 110:437–446
    [Google Scholar]
  23. Gebre-Yohannes A., Drasar B. S. Plasmid profiles of antibiotic-resistant Shigella dysenteriae types 2, 3, 4, 6 and 7 isolated in Ethiopia during 1976–85. Epidemiol Infect 1990; 105:65–72
    [Google Scholar]
  24. Okada N., Sasakawa C., Tobe T. Virulence-associated chromosomal loci of Shigella flexneri identified by random Tn5 insertion mutagenesis. Mol Microbiol 1991; 5:187–195
    [Google Scholar]
  25. Sasakawa C., Kamata K., Sakai T., Murayama S. Y., 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; 51:470–475
    [Google Scholar]
  26. Ausubel F. M., Brent R., Kingston R. E. (eds) Current protocols in molecular biology. New York: Greene Publishing Associates and Wiley-Interscience; 1991
    [Google Scholar]
  27. Morelle G. A plasmid extraction procedure on a miniprep scale. Focus 1989; 11:7–8
    [Google Scholar]
  28. Rajakumar K., Jost B. H., Sasakawa C., Okada N., Yoshikawa M., Adler B. Nucleotide sequence of the rhamnose biosynthetic operon of Shigella flexneri 2a and role of lipopolysaccharide in virulence. J Bacteriol 1994; 176:2362–2373
    [Google Scholar]
  29. Makino S., Sasakawa C., Yoshikawa M. Genetic relatedness of the basic replicon of the virulence plasmid in shigellae and enteroinvasive Escherichia coli. Microb Pathog 1988; 5:267–274
    [Google Scholar]
  30. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 1977; 74:5463–5467
    [Google Scholar]
  31. Smith C. L., Cantor C. R. Purification, specific fragmentation, and separation of large DNA molecules. Methods Enzymol 1987; 155:449–467
    [Google Scholar]
  32. Freudl R. Insertion of peptides into cell-surface-exposed areas of the Escherichia coli OmpA protein does not interfere with export and membrane assembly. Gene 1989; 82:229–236
    [Google Scholar]
  33. Inokuchi K., Mutoh N., Matsuyama S., Mizushima S. Primary structure of the ompF gene that codes for a major outer membrane protein of Escherichia coli K-12. Nucleic Acids Res 1982; 10:6957–6968
    [Google Scholar]
  34. Wilson H. R., Chan P. T., Tumbough C. L. Nucleotide sequence and expression of the pyrC gene of Escherichia coli K-12. J Bacteriol 1987; 169:3051–3058
    [Google Scholar]
  35. Sansonetti P. J., Ryter A., Clerc P., Maurelli A. T., Mounier J. Multiplication of Shigella flexneri within HeLa cells: lysis of the phagocytic vacuole and plasmid-mediated contact hemolysis. Infect Immun 1986; 51:461–469
    [Google Scholar]
  36. Sasakawa C., Kamata K., Sakai T. Virulence-associated genetic regions comprising 31 kilobases of the 230-kilobase plasmid in Shigella flexneri 2a. J Bacteriol 1988; 170:2480–2484
    [Google Scholar]
  37. Okada N., Sasakawa C., Tobe T., Talukder K. A., 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; 5:2171–2180
    [Google Scholar]
  38. Ott M. Dynamics of the bacterial genome: deletions and integrations as mechanisms of bacterial virulence modulation. Int J Med Microbiol Virol Parasitol Infect Dis 1993; 278:457–468
    [Google Scholar]
  39. Bachmann B. J. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev 1990; 54:130–197
    [Google Scholar]
  40. Médigue C., Hénaut A., Danchin A. Escherichia coli molecular genetic map (1000 kbp): update 1. Mol Microbiol 1990; 4:1443–1454
    [Google Scholar]
  41. Campbell A. M. Chromosomal insertion sites for phages and plasmids. J Bacteriol 1992; 174:7495–7499
    [Google Scholar]
  42. Bar-Nir D., Cohen A., Goedeke M. E. tDNAser sequences are involved in the excision of Streptomyces griseus plasmid pSG1. Gene 1992; 122:71–76
    [Google Scholar]
  43. Ka J. O., Tiedje J. M. Integration and excision of a 2, 4-dichlorophenoxyacetic acid-degradative plasmid in Alcaligenes paradoxus and evidence of its natural intergeneric transfer. J Bacteriol 1994; 176:5284–5289
    [Google Scholar]
  44. 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; 59:792–799
    [Google Scholar]
  45. Komine Y., Adachi T., Inokuchi H., Ozeki H. Genomic organization and physical mapping of the transfer RNA genes in Escherichia coli K12. J Mol Biol 1990; 212:579–598
    [Google Scholar]
  46. Reiter W.-D., Palm P., Yeats S. Transfer RNA genes frequently serve as integration sites for prokaryotic genetic elements. Nucleic Acids Res 1989; 17:1907–1914
    [Google Scholar]
  47. Blum G., Ott M., Lischewski A. 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; 62:606–614
    [Google Scholar]
  48. Leong J. M., Nunes-Duby S. E., Landy A. Generation of single base-pair deletions, insertions, and substitutions by a site-specific recombination system. Proc Natl Acad Sci USA 1985; 82:6990–6994
    [Google Scholar]
  49. Durand J. M., Okada N., Tobe T. vacC, a virulence-associated chromosomal locus of Shigella flexneri, is homologous to tgt, a gene encoding tRNA-guanine transglycosylase (Tgt) of Escherichia coli K-12. J Bacteriol 1994; 176:4627–4634
    [Google Scholar]
  50. Maurelli A. T., Blackmon B., Curtiss R. Loss of pigmentation in Shigella flexneri 2a is correlated with loss of virulence and virulence-associated plasmid. Infect Immun 1984; 43:397–401
    [Google Scholar]
  51. Monack D. M., Arico B., Rappuoli R., Falkow S. Phase variants of Bordetella bronchiseptica arise by spontaneous deletions in the vir locus. Mol Microbiol 1989; 3:1719–1728
    [Google Scholar]
  52. Fetherston J. D., Schuetze P., Perry R. D. 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; 6:2693–2704
    [Google Scholar]
  53. Lucier T. S., Brubaker R. R. Determination of genome size, macrorestriction pattern polymorphism, and nonpigmentation-specific deletion in Yersinia pestis by pulsed-field gel electrophoresis. J Bacteriol 1992; 174:2078–2086
    [Google Scholar]
  54. Fetherston J. D., Perry R. D. The pigmentation locus of Yersinia pestis KIM6+ is flanked by an insertion sequence and includes the structural genes for pesticin sensitivity and HMWP2. Mol Microbiol 1994; 13:697–708
    [Google Scholar]
  55. Lawlor K. M., Payne S. M. Aerobactin genes in Shigella spp. J Bacteriol 1984; 160:266–272
    [Google Scholar]
  56. Griffiths E., Stevenson P., Hale T. L., Formal S. B. Synthesis of aerobactin and a 76,000-dalton iron-regulated outer membrane protein by Escherichia coli K-12–Shigella flexneri hybrids and by enteroinvasive Escherichia coli. Infect Immun 1985; 49:67–71
    [Google Scholar]
  57. Cohen S. P., Hächler H., Levy S. B. Genetic and functional analysis of the multiple antibiotic resistance (mar) locus in Escherichia coli. J Bacterial 1993; 175:1484–1492
    [Google Scholar]
  58. Hächler H., Cohen S. P., Levy S. B. marA, a regulated locus which controls expression of chromosomal multiple antibiotic resistance in Escherichia coli. J Bacteriol 1991; 173:5532–5538
    [Google Scholar]
  59. Poole K., Krebes K., McNally C., Neshat S. Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon. J Bacteriol 1993; 175:7363–7372
    [Google Scholar]
  60. Cohen S. P., Yan W., Levy S. B. A multidrug resistance regulatory chromosomal locus is widespread among enteric bacteria. J Infect Dis 1993; 168:484–488
    [Google Scholar]
  61. Ramirez-Santos J., Alvarez G., Cisneros E., Gomez-Eichelmann M. C. Distribution of insertion sequence ISI in multjple-antibiotic resistant clinical Enterobacteriaceae strains. FEMS Microbiol Lett 1992; 93:189–194
    [Google Scholar]
  62. Womble D. D., Rownd R. H. Genetic and physical map of plasmid NR1: comparison with other IncFII antibiotic resistance plasmids. Microbiol Rev 1988; 52:433–451
    [Google Scholar]
  63. Inglis B., El-Adhami W., Stewart P. R. Methicillin-sensitive and - resistant homologues of Staphylococcus aureus occur together among clinical isolates. J Infect Dis 1993; 167:323–328
    [Google Scholar]
  64. Silva R. M., Saadi S., Maas W. K. 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; 56:836–842
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/jmm/10.1099/00222615-45-1-64
Loading
/content/journal/jmm/10.1099/00222615-45-1-64
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error