Article Figures & Data
Figures
- FIG. 1.
AI-2 production in H. pylori is luxS dependent. AI-2 production in the wild-type strain G27 (A), the ΔluxS mutant (B), and the complemented luxS* strain (C) was measured as the ability of CFS harvested at the indicated times after inoculation of liquid H. pylori cultures to induce bioluminescence in the V. harveyi reporter strain BB170 (relative light units [RLU]; bars). The cell densities of the cultures across the growth curves were determined by viable plate counts (CFU/ml; diamonds). Error bars indicate standard deviations of triplicate or duplicate samples for the bioluminescence and cell density measurements, respectively.
- FIG. 2.
The ΔluxS mutant has a motility defect that is rescued by wild-type CFS. Wild-type, ΔluxS, and luxS* bacteria were seeded onto soft agar plates composed of normal medium or supplemented with CFS from wild-type or ΔluxS cultures. After 6 days of incubation, the halos of migration were visualized (A) and quantified (B). Error bars indicate the standard deviation of halo sizes of triplicate cultures in one experiment.
- FIG. 3.
AI-2 modulates flagellar morphogenesis. Representative flagella, as visualized by TEM, are shown for each of the genotypes indicated grown in normal medium (A to C, E, F, I, J, M, and N) or for 4 h in the presence of 0.1 mM DPD (G, K, and O). In all cells with flagella, the base is marked by an arrow, and the tip is marked by an arrowhead. Scale bars, 1 μm. The flagellar morphologies were scored for at least 100 cells for each treatment group (D, H, L, and P).
- FIG. 4.
AI-2 modulates flagellar gene transcription. Transcript levels of flgS, fliA, flgM, and flaA (A) and of flhA (B) were determined by qRT-PCR normalized to the levels of ureA in each of the genotypes indicated and are represented as the log2 difference relative to the levels in the wild-type strain. Levels of the same transcripts were determined in the indicated genotypes and normalized to the levels of the 16S rRNA gene in each sample (C). AI-2 activity in CFS from wild-type early-stationary-phase H. pylori was measured relative to different concentrations of DPD (D). flhA transcript levels were measured as in panel B in wild-type and ΔluxS cultures untreated or treated for 4 h with 0.1 mM DPD (E) and in ΔluxS cultures treated with a range of DPD concentrations (F). Error bars indicate the standard deviations for three triplicate samples.
- FIG. 5.
Model of AI-2 modulation of the H. pylori flagellar regulon. The role of AI-2 in transcriptional regulation of components of the flagellar gene hierarchy of H. pylori is shown, based on Neihus et al. (36). AI-2 signals upstream of FlhA, which, in turn, regulates branching pathways of gene transcription regulated by FlgS, FlgM, and FliA/σ28.
Tables
- TABLE 1.
Strains used in this study
H. pylori strain Description Reference or source Wild type G27 14 luxS strain luxS::kan sacB This study ΔluxS strain Deletion of HP0105 This study luxS* strain rdx::luxS and deletion of luxS This study fliA strain fliA::kan This study fliA ΔluxS strain fliA::kan and deletion of luxS This study flgS strain flgS::kan This study flgS ΔluxS strain flgS::kan and deletion of luxS This study flgM strain flgM::kan This study flgM ΔluxS strain flgM::kan and deletion of luxS This study flhA strain flhA::kan This study flhA ΔluxS strain flhA::kan and deletion of luxS This study - TABLE 2.
Primers used for cloning and RT-PCR
Primer Sequence (5′-3′) LuxSSpeR AAAATCATTCCCCAAACCAATGATCAGGG LuxSEcoF CCGGAATTCAGAGCAAGCGTTCGCTAAAA TopoXbaF GCTCTAGACTGAGATCATCCGCAACCAT TopoBlgR GAAGATCTTTGGCATGTCCATGTGATCT FBglIIpKan+ GAAGATCTTCCCGGGCGAACCATTTGAGGTGA PFA3 GCTCTAGACCCGGGTATAAGCCCATTTTCATGC FLS2Bam CGGGATCCGAGGCGGTTGCTTTTGTAGA LuxS3AB CGACATAACTGGCATGGTTGGCGAACGCTTGCTCTAAA RLS2Xho2 TTTATCTTCTCGAGCGCCCATGAATGTCTGAAGT RdxA1F TAAACGAGCGCCATTCTTG RdxA1LR GATTAAAAGCGCTTCAGCGTGAGGCGGTTGCTTTTGTAGA RdxA2LF CTTCAGACATTCATGGGCGATTCAACCACAGCATGCAAA RdxA2R CGATCAAGCATGCGATTTTA FliaAF TGCTTCGTTTCAACCATGAG FliaAK AAGCAACAACACCACCATCACAGGAAACAGCTATGACA FliaBK CTGGCCGTCGTTTTACTCGCGCATTTCTCAAATC FliaBR ACTCACACGATTAGGGCGTTT HP0244AF CGATGAGATTTATGCGTCCA HP0244AK CCTTATTTGAAGCGTTCCCACAGGAAACAGCTATGAC HP0244BK ACTGGCCGTCGTTTTACGAGAGCGAACAGGGTCAA HP0244BR GCTAACCCTAGGCCGTTACC FlgMAF GTTGTGGTTGCTCATGTTCG FlgMAK AATGCCGTTTCTTCTCTTGCAGGCAACAGCTATGA FlgMBK ACTGGCCGTCGTTTTACTCTCACAAAATGGCAAAGGA FlgMBR TCCTAACTTCATGATTGA FlhAIntF CCTAGTGCGGTGAGCGATATTATC FlhAIntR GCTCACGCTAAATTCCCCAA UreAIntF AAAGACTGCGGCTGAATTG UreAIntR CCATCACATCATCCGGTTT FlhABgl2 GAAGATCTACCTCACAAGAACCCGTATCGT FlhAXho2 ATTCCGCTCGAGCAATGGAAACTCTCAGCCACTTC M13Nhe1 CTAGCTAGCGTAAAACGACGGCCAGT M13Acs1 TTGGCGCGCCCAGGAAACAGTTATGAC 1320f CCATGAAGTCGGAATCGCTAG 1431r ACTCCCATGGTGTGACGG
