Article Figures & Data
Figures
- FIG. 1.
H. pylori produces ppGpp upon nutrient downshift. Three wild-type strains of H. pylori, J99, 26695, and G27, were grown to early log phase in rich medium (brucella broth plus 10% fetal bovine serum), shifted to either new rich medium or minimal medium lacking carbon sources and phosphate (MOPS-MGS without mannitol or phosphate: 50 mM MOPS [pH 7.4], 1 mM MgSO4, 0.25 mM CaCl2, 19 mM glutamic acid, and 0.004 mM biotin), and immediately labeled with 32Pi. Nucleotides were resolved by TLC.
- FIG. 2.
Nutrient downshift of H. pylori causes a protein synthesis-dependent decrease in sRNA (A) and a relative increase in spoT RNA versus 16S rRNA (B). (A) Strain G27 was grown to early log phase in rich medium and then transferred either to new rich medium, to minimal medium lacking carbon sources and phosphate (MOPS-MGS without mannitol or phosphate), or to minimal medium plus 25 μg/ml chloramphenicol and incubated microaerobically at 37°C for 3 h. Samples were harvested at the indicated times and assayed for total RNA (∼90% of which is sRNA) and viability (CFU counts). RNA was quantified by spectrophotometric analysis. Average amounts (femtograms) of RNA per CFU are shown. Samples from rich medium are solid black; samples from minimal medium are gray; samples from minimal medium plus chloramphenicol are hatched gray lines. CFU counts in minimal medium-shifted samples remained constant throughout the experimental time course (data not shown). (B) G27 was grown as described above and shifted to either rich or minimal medium for the indicated times. RNA was harvested, reverse transcribed, and subjected to quantitative PCR analyses using the TaqMan system from ABI. Primers used were 16S-fwd (5′-CAGCCATGTTGCGGTGAAT-3′), 16S-rev (5′-TGTGACGGGCGGTGAGTA-3′), 16S probe (5′-6FAM-CGTTCCCGGGTCTT-3′), spoT-fwd (5′-ACCTCGTTTCATTTGGATGGAT-3′), spoT-rev (5′-TGGATGCGCAAATGGTTTT-3′), and spoT probe (5′-6FAM-AGCTTAAAACTTCTAAGGCT-3′).
- FIG. 3.
H. pylori produces (p)ppGpp upon pH downshift. H. pylori strain G27 was grown to early log phase in rich medium at neutral pH and then diluted 1:1 either in rich medium at neutral pH (final pH 7.0) or in rich medium at pH 3.0 (final pH 4.5) and labeled with 32Pi. Nucleotides were resolved by TLC. (p)ppGpp corresponding to guanosine penta- and tetraphosphate in the pH 4.5 sample is indicated by the arrow. This spot was quantified by densitometer analysis and found to be ∼4.5-fold higher than in the pH 7.0 control.
