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Helicobacter2001; 6(1); 15-23; doi: 10.1046/j.1523-5378.2001.00001.x

New approaches for validation of lethal phenotypes and genetic reversion in Helicobacter pylori.

Abstract: Because of limited genetic tools for use in Helicobacter pylori, tests routinely applied in other bacteria for demonstrating a gene's role in viability and other phenotypes have not been applied to this organism. In a mutational study of putative response regulator genes, we aimed to develop such tools for H. pylori. Methods: We attempted to mutate five response regulator genes by allelic exchange insertional mutagenesis. For genes that yielded no viable mutants, a second copy of the gene was inserted into the chromosome via a suicide vector, and it was seen if providing the second copy would permit the gene's disruption. For genes that yielded mutants with selectable phenotypes, a strategy was developed for reversion whereby an intact copy of the gene is introduced to the organism by transformation with PCR products. Following this procedure, revertants were selected by phenotypic tests then tested for genetic reversion. Results: After failure to attain transformants upon attempted mutation of genes HP0166 and HP1365, we inserted a second copy of each gene within the H. pylori chromosome. In each case the second copy relieved the block of transformation. Mutation of genes HP0703 and HP1021 gave non-motile and small-colony phenotypes, respectively. Following transformation with PCR products containing intact copies of the genes, both phenotype and genotype had reverted following phenotypic selections. Conclusions: The methods used in this study provide new approaches for confirming suspected genotype/phenotype associations and should be widely applicable in the study of H. pylori.
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Publication Date: 2001-05-01 PubMed ID: 11328361DOI: 10.1046/j.1523-5378.2001.00001.xGoogle Scholar: Lookup
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  • Journal Article
  • Research Support
  • Non-U.S. Gov't
  • Research Support
  • U.S. Gov't
  • P.H.S.

Summary

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The researchers developed new methods to demonstrate a gene’s role in viability in Helicobacter pylori by experimenting with mutational studies of response regulator genes. These methods have culminated in new ways for confirming genotype/phenotype associations, which could be widely used in the study of this organism.

Research Approach

  • The researchers started by attempting to mutate five response regulator genes through a process known as allelic exchange insertional mutagenesis. This involves replacing one allele with another. The goal was to disrupt the genes and observe the result.
  • When no viable mutants resulted, they took the approach of inserting a second copy of the gene into the chromosome. This was achieved using a suicide vector, which is a type of plasmid or virus that carries material into a cell causing it to change.
  • They wanted to see if providing this second copy would allow the gene’s disruption to take place.
  • For genes where mutants were successfully created with selectable phenotypes, a reversion strategy was developed. This involved introducing an intact copy of the gene to the organism through transformation with PCR (polymerase chain reaction) products.
  • Following this, any potential revertants were selected through phenotypic tests and then tested for genetic reversion.

Research Findings

  • Two genes, HP0166 and HP1365, failed to produce viable mutants. However, when a second copy of each gene was introduced into the H. pylori chromosome, it allowed the block of transformation to be overcome.
  • Two other genes, HP0703 and HP1021, when mutated, yielded non-motile and small-colony phenotypes, respectively.
  • Introduction of PCR products containing intact copies of the mutated genes resulted in the reversal of both phenotype and genotype after phenomenological selections.

Conclusions and Implications

  • The techniques utilised in this study open up new possibilities for confirming suspected genotype/phenotype associations.
  • These methods developed via mutational studies could be widely applicable in the study of Helicobacter pylori, an organism that previously had limited genetic tools available.

Cite This Article

APA
McDaniel TK, Dewalt KC, Salama NR, Falkow S. (2001). New approaches for validation of lethal phenotypes and genetic reversion in Helicobacter pylori. Helicobacter, 6(1), 15-23. https://doi.org/10.1046/j.1523-5378.2001.00001.x

Publication

Helicobacter
ISSN: 1083-4389
NlmUniqueID: 9605411
Country: England
Language: English
Volume: 6
Issue: 1
Pages: 15-23

Researcher Affiliations

McDaniel, T K
  • Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, CA, USA.
Dewalt, K C
    Salama, N R
      Falkow, S

        MeSH Terms

        • Animals
        • Cells, Cultured
        • Chromosomes, Bacterial / genetics
        • Enzyme-Linked Immunosorbent Assay
        • Gastric Mucosa / microbiology
        • Genes, Bacterial
        • Genes, Lethal
        • Genes, Regulator
        • Genetic Vectors
        • Genotype
        • Helicobacter pylori / genetics
        • Helicobacter pylori / growth & development
        • Horses
        • Humans
        • Interleukin-8 / metabolism
        • Mutagenesis, Insertional
        • Mutation
        • Phenotype
        • Polymerase Chain Reaction
        • Transformation, Bacterial

        Grant Funding

        • AI38459 / NIAID NIH HHS

        Citations

        This article has been cited 23 times.
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