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MOLECULAR AND CELLULAR PATHOGENESIS

Identification of Virulence Genes ofHelicobacter pylori by Random Insertion Mutagenesis

J. J. E. Bijlsma, C. M. J. E. Vandenbroucke-Grauls, S. H. Phadnis, J. G. Kusters
J. J. E. Bijlsma
Department of Medical Microbiology, Faculty of Medicine, Vrije Universiteit Amsterdam, The Netherlands, and
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C. M. J. E. Vandenbroucke-Grauls
Department of Medical Microbiology, Faculty of Medicine, Vrije Universiteit Amsterdam, The Netherlands, and
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S. H. Phadnis
Department of Pathology, Medical College of Wisconsin, and
Pathology and Laboratory Medicine Service, Department of Veterans Affairs, Clement J. Zablocki Medical Center, Milwaukee, Wisconsin
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J. G. Kusters
Department of Medical Microbiology, Faculty of Medicine, Vrije Universiteit Amsterdam, The Netherlands, and
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DOI: 10.1128/IAI.67.5.2433-2440.1999
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  • Fig. 1.
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    Fig. 1.

    Schematic representation of pBCα3. ColE1, origin of replication (not functional in H. pylori); aphA-3, kanamycin cassette (functional in H. pylori and E. coli); Cm, chloramphenicol cassette (not functional in H. pylori). Only unique restriction sites are indicated. The AluI sites used in the plasmid rescue strategy are not indicated because there are 29 AluI sites in the plasmid.

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    Fig. 2.

    Schematic representation of the integration of pBCα3 in the chromosome. A hypothetical DNA fragment (fragment A) cloned into the plasmid mediates the integration of the plasmid into the H. pylori chromosome via a Campbell-like mechanism. Integration of pBCα3 results in a duplication of fragment A at both sites of insertion and a subsequent disruption of the gene in which fragment A is present. (A) Integration of pBCα3 is in the same direction as the ORF. In this case, point 2 is the actual insertion site. (B) Integration of pBCα3 is in the direction opposite that of the ORF. In this case, point 1 is the actual insertion site. P, hypothetical promoter. The positions of the primers used to sequence the rescued plasmids, the M13 reverse primer (M13-Rev) and aphA3-L, are indicated.

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    Fig. 3.

    Southern blot of 16 randomly selected mutants probed with the kanamycin cassette. Lane 1, plasmid DNA without the kanamycin cassette; lane 2, wild-type chromosomal DNA; lane 3, positive control; lanes 4 to 19, chromosomal DNA of the 16 mutants. Chromosomal DNA was digested with HinfI in all cases. The positions of marker DNA fragments, in kilobase pairs, are given to the left of the autoradiogram.

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    Fig. 4.

    Immunoblot of whole-cell preparations with anti-urease (1:1,000). Lane 1, parental strain 1061; lanes 2 through 4, mutants with disruptions of ureB, ureI, anddeaD, respectively. Approximately 11 μg of protein was loaded in each lane.

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    Fig. 5.

    Negatively stained electron micrographs of H. pylori nonmotile mutants. Representative results are shown for the wild type (a), mutant 3 (b), mutant 4 (c), and mutant 1 (d). Bar in panel a, 500 nm; bars in panels b through d, 1 μm. Arrow in panel c points to the cap-like structure at the site where the flagella protrude from the bacterium.

Tables

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  • Table 1.

    Analysis of genes disrupted in urease-negative mutants, as determined by plasmid rescue and subsequent sequencing

    MutantSite of insertion (bp)aH. pylori gene no.aSp act of urease (U/mg of protein)bHomologue of interrupted ORF
    176,61200720.042 ± 0.001Urease B gene
    275,25800710.074 ± 0.003Urease accessory gene I
    3256,17102470.050 ± 0.002ATP-dependent RNA helicase gene
    • ↵a According to the sequence of Tomb et al. (37).

    • ↵b Mean from three independent experiments ± standard error of the mean. The urease activity of the parental strain was 34.91 ± 0.043 U/mg of protein.

  • Table 2.

    Analysis of genes disrupted in nonmotile mutants, as determined by plasmid rescue and subsequent sequencing

    MutantSite of insertion (bp)aH. pylori gene no.aHomologue of interrupted ORF product
    185,1410080None
    2927,1105′ end of 0876Iron-regulated outer membrane protein
    3954,9710904Phosphotransacetylase
    41,349,3811274Paralyzed flagellar protein
    51,536,7941465ABC transporter
    • ↵a According to the sequence of Tomb et al. (37).

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Identification of Virulence Genes ofHelicobacter pylori by Random Insertion Mutagenesis
J. J. E. Bijlsma, C. M. J. E. Vandenbroucke-Grauls, S. H. Phadnis, J. G. Kusters
Infection and Immunity May 1999, 67 (5) 2433-2440; DOI: 10.1128/IAI.67.5.2433-2440.1999

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Identification of Virulence Genes ofHelicobacter pylori by Random Insertion Mutagenesis
J. J. E. Bijlsma, C. M. J. E. Vandenbroucke-Grauls, S. H. Phadnis, J. G. Kusters
Infection and Immunity May 1999, 67 (5) 2433-2440; DOI: 10.1128/IAI.67.5.2433-2440.1999
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KEYWORDS

Genes, Bacterial
Helicobacter pylori
Mutagenesis, Insertional

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