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. 1997 May;65(5):1949–1952. doi: 10.1128/iai.65.5.1949-1952.1997

Introduction of unmarked mutations in the Helicobacter pylori vacA gene with a sucrose sensitivity marker.

M Copass 1, G Grandi 1, R Rappuoli 1
PMCID: PMC175249  PMID: 9125586

Abstract

Research on Helicobacter pylori has been hindered by the lack of useful genetic tools. Using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows introduction of unmarked mutations in H. pylori. A kan-sacB cassette, consisting of the sacB gene expressed from the H. pylori flagellin promoter and the kanamycin resistance module, was introduced by homologous recombination into a target H. pylori gene. The resultant strains were sucrose sensitive and kanamycin resistant. Following transformation with a mutated allele, growth in sucrose-containing medium allowed the selection of strains that had lost the kan-sacB module and had integrated the unmarked allele. We have used this cassette to perform a site-directed modification of two histidine residues encoded by the vacA gene in a two-step procedure. This system should prove useful in the site-directed mutagenesis of H. pylori genes.

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Selected References

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  1. Blaser M. J., Perez-Perez G. I., Kleanthous H., Cover T. L., Peek R. M., Chyou P. H., Stemmermann G. N., Nomura A. Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach. Cancer Res. 1995 May 15;55(10):2111–2115. [PubMed] [Google Scholar]
  2. Blaser M. J. The role of Helicobacter pylori in gastritis and its progression to peptic ulcer disease. Aliment Pharmacol Ther. 1995;9 (Suppl 1):27–30. doi: 10.1111/j.1365-2036.1995.tb00780.x. [DOI] [PubMed] [Google Scholar]
  3. Blomfield I. C., Vaughn V., Rest R. F., Eisenstein B. I. Allelic exchange in Escherichia coli using the Bacillus subtilis sacB gene and a temperature-sensitive pSC101 replicon. Mol Microbiol. 1991 Jun;5(6):1447–1457. doi: 10.1111/j.1365-2958.1991.tb00791.x. [DOI] [PubMed] [Google Scholar]
  4. Covacci A., Censini S., Bugnoli M., Petracca R., Burroni D., Macchia G., Massone A., Papini E., Xiang Z., Figura N. Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5791–5795. doi: 10.1073/pnas.90.12.5791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ferrero R. L., Cussac V., Courcoux P., Labigne A. Construction of isogenic urease-negative mutants of Helicobacter pylori by allelic exchange. J Bacteriol. 1992 Jul;174(13):4212–4217. doi: 10.1128/jb.174.13.4212-4217.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gay P., Le Coq D., Steinmetz M., Berkelman T., Kado C. I. Positive selection procedure for entrapment of insertion sequence elements in gram-negative bacteria. J Bacteriol. 1985 Nov;164(2):918–921. doi: 10.1128/jb.164.2.918-921.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Haas R., Meyer T. F., van Putten J. P. Aflagellated mutants of Helicobacter pylori generated by genetic transformation of naturally competent strains using transposon shuttle mutagenesis. Mol Microbiol. 1993 May;8(4):753–760. doi: 10.1111/j.1365-2958.1993.tb01618.x. [DOI] [PubMed] [Google Scholar]
  8. Leying H., Suerbaum S., Geis G., Haas R. Cloning and genetic characterization of a Helicobacter pylori flagellin gene. Mol Microbiol. 1992 Oct;6(19):2863–2874. doi: 10.1111/j.1365-2958.1992.tb01466.x. [DOI] [PubMed] [Google Scholar]
  9. Rappuoli R., Covacci A., Ghiara P., Telford J. Pathogenesis of Helicobacter pylori and perspectives of vaccine development against an emerging pathogen. Behring Inst Mitt. 1994 Dec;(95):42–48. [PubMed] [Google Scholar]
  10. Ried J. L., 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. doi: 10.1016/0378-1119(87)90127-2. [DOI] [PubMed] [Google Scholar]
  11. Steinmetz M., Le Coq D., Djemia H. B., Gay P. Analyse génétique de sacB, gène de structure d'une enzyme secrétée, la lévane-saccharase de Bacillus subtilis Marburg. Mol Gen Genet. 1983;191(1):138–144. doi: 10.1007/BF00330901. [DOI] [PubMed] [Google Scholar]
  12. Telford J. L., Ghiara P., Dell'Orco M., Comanducci M., Burroni D., Bugnoli M., Tecce M. F., Censini S., Covacci A., Xiang Z. Gene structure of the Helicobacter pylori cytotoxin and evidence of its key role in gastric disease. J Exp Med. 1994 May 1;179(5):1653–1658. doi: 10.1084/jem.179.5.1653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Tompkins L. S., Falkow S. The new path to preventing ulcers. Science. 1995 Mar 17;267(5204):1621–1622. doi: 10.1126/science.7886448. [DOI] [PubMed] [Google Scholar]
  14. Trieu-Cuot P., Gerbaud G., Lambert T., Courvalin P. In vivo transfer of genetic information between gram-positive and gram-negative bacteria. EMBO J. 1985 Dec 16;4(13A):3583–3587. doi: 10.1002/j.1460-2075.1985.tb04120.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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