Article Figures & Data
Figures
- FIG. 1.
PCR analysis of motB mutants.(A) Schematic representations of wild-type motB (i), motB::cat (ii), and ΔmotB (iii). Arrows represent primers used for PCR. Primer 1 is motB1, primer 2 is motB3, and primer 3 is colicat1 (sequences are in the text). Numbers at the top of panel i indicate nucleotide numbers for the motB open reading frame, while numbers at the bottom represent the genomic sequence numbers from the TIGR sequence (42). The location of the BclI site used for inserting the cat/aphA3-sacB is marked. (B) Agarose gel analysis of PCR amplification products from genomic DNA of SS1, SS1 ΔmotB and SS1 motB::cat, and G27 and G27 motB::cat. The source of genomic DNA is shown at the top of the figure; the primer set (as in panel A) is shown at the bottom. The arrows indicate full-length motB, and the diamonds indicate altered motB (either motB::cat or ΔmotB). Kilobase markers are shown on the left side. Although in some lanes multiple bands can be seen, in all cases the dominant band is the band of interest. Similar results were obtained with G27ΔmotB.
- FIG. 2.
H. pylori strains lacking motB do not spread in soft agar. Photograph of a brucella broth-5% FBS soft agar plate after 4 days of incubation. The strains were stabbed into the middle of each growth area on day 0. The numerals 1, 2, and 3 indicate wild-type SS1, SS1 ΔmotB, and SS1 motB::cat, respectively. Similar results were obtained with G27.
- FIG. 3.
H. pylori SS1 lacking motB is flagellated. Electron micrographs of SS1, stained with phosphotungstate. (A) Wild-type SS1; (B) SS1 ΔmotB; (C) SS1 motB::cat. Flagella are visible in all samples; one filament in each panel is marked with an arrowhead.
- FIG. 4.
motB mutants do not colonize mouse stomachs as well as the wild-type strain. Symbols representing numbers of CFU per gram of stomach in mice infected for 2 weeks with wild-type SS1 (filled circles) or SS1 ΔmotB (filled triangles) are shown. Mice infected by wild-type SS1 were dosed with 9 × 107 CFU, while mice receiving SS1 ΔmotB received 1.9 × 108 CFU. Each point represents one mouse. Three of the ΔmotB mice had no detectable H. pylori; because the limit of detection is estimated to be 250 CFU/gram of stomach, we placed these mice at this level. This detection limit is marked by a dotted line.
Tables
- TABLE 1.
Strains and plasmids used in this study
Strain Genotype or description Antibiotic resistance Reference or source H. pylori G27 Wild type 6-Nina Salama SS1 Wild type 23-Janie O'Rourke SS1 motB::cat SS1 motB::cat1 Cm This study SS1 motB::km-sac SS1 motB::aphA3-sacB1 Km This study SS1 ΔmotB SS1 ΔmotB2 This study G27 motB::cat SS1 motB::cat1 Cm This study G27 motB::km-sac G27 motB::aphA3-sacB1 Km This study G27 ΔmotB G27 ΔmotB2 This study Plasmid pBluescript KS (pBS) Cloning plasmid Ap Stratagene pT7blue Cloning plasmid Ap Novagen pBS-cat pBS with C. coli cat gene Ap, Cm Nina Salama pKSFII pBS with aphA3 and sacB gene Km 9 pKO114 pBS with 1.2-kb motB gene from G27 Ap This study pKO114i pKO114 with aphA3-sacB insert Ap, Km This study pACL14 pKO114 with cat insert Ap, Cm This study pKO124 pT7blue with 2.9-kb region of SS1 genome encompassing motB Ap This study pKO124d pKO124 with in-frame deletion of motB Ap This study - TABLE 2.
In vitro characteristics of H. pylori motB mutantsa
Strain Motile by microscopy Flagellated Swarm rate (mm/day) SS1 Yes Yes 5.6 ± 0.1 SS1 motB::cat No Yes 0.36 ± 0.38 SS1 ΔmotB No Yes 0.48 ± 0.08 G27 Yes Yes 7.4 ± 0.9 G27 motB::cat No n.d. 1.24 ± 0.22 G27 ΔmotB No Yes 1.26 ± 0.16 ↵ a Flagellation was determined by electron microscopy. Swarm rate was determined in brucella broth with 5% FBS soft agar. n.d., not determined.
- TABLE 3.
Infectious doses of SS1 ΔmotB and SS1 in FVB/N micea
Strain Infecting dose No. of mice infected/ no. inoculated CFU (106) SS1 1.3 × 106 4/4 2.7 ± 1.0 1.3 × 105 4/4 1.9 ± 0.28 1.3 × 104 2/2 3.1 ± 1.5 1.3 × 103 4/4 1.6 ± 0.67 1.3 × 102 4/4 0.58 ± 0.15 SS1 ΔmotB 5 × 107 2/5 0.14 ± 0.07 5 × 106 3/5 0.036 ± 0.018 5 × 105 0/4 0 5 × 104 0/4 0 ↵ a Mice were infected with various doses of H. pylori SS1 or SS1 ΔmotB as noted above. After three days the mice were sacrificed and their stomach contents were plated to determine the number of CFU per gram of stomach.
b Values are means ± standard deviations of CFU × 106 per gram of stomach of infected mice.
