Article Figures & Data
Figures
- 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.
- 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.
- 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.
- 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.
- 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
- Table 1.
Analysis of genes disrupted in urease-negative mutants, as determined by plasmid rescue and subsequent sequencing
Mutant Site of insertion (bp)a H. pylori gene no.a Sp act of urease (U/mg of protein)b Homologue of interrupted ORF 1 76,612 0072 0.042 ± 0.001 Urease B gene 2 75,258 0071 0.074 ± 0.003 Urease accessory gene I 3 256,171 0247 0.050 ± 0.002 ATP-dependent RNA helicase gene - Table 2.
Analysis of genes disrupted in nonmotile mutants, as determined by plasmid rescue and subsequent sequencing
Mutant Site of insertion (bp)a H. pylori gene no.a Homologue of interrupted ORF product 1 85,141 0080 None 2 927,110 5′ end of 0876 Iron-regulated outer membrane protein 3 954,971 0904 Phosphotransacetylase 4 1,349,381 1274 Paralyzed flagellar protein 5 1,536,794 1465 ABC transporter ↵a According to the sequence of Tomb et al. (37).
