We found that all strains tested produced FlaB at approximately the same level (Figure. 4). The reflective density of the FlaB bands of the wild-type, ΔluxS Hp mutant and the complemented ΔluxS Hp + mutant were (means ± SD) 0.210 ± 2.0E-03 RD, n = 4; 0.204 ± 5.8E-04 RD, n = 4; and 0.207 ± 5.8E-04 RD, n = 4, respectively. We expressed all other results

(FlaA and FlgE) relative to FlaB in each strain. Mutagenesis of LuxSHp reduced the expression of FlaA relative to FlaB (from mean 1.60 in the wild-type to 1.23 in the ΔluxS Hp mutant, p < 0.01), and complementation increased the ratio back to wild-type levels (mean 1.70 in the ΔluxS Hp + mutant, p < 0.01 compared with the ΔluxS Hp mutant). Next, we examined FlgE expression, and a similar trend was found (wild-type FlgE:FlaB ratio mean 0.74; ΔluxS Hp mutant 0.51; complemented ΔluxS Hp + mutant 0.77; p < 0.01 for differences between ΔluxS Hp mutant and wild-type phosphatase inhibitor and complemented stains). These data show that FlaA and FlgE synthesis was reduced relative to FlaB

in the ΔluxS Hp mutant and these changes were restored by genetic complementation. AI-2 regulates the transcription of flagellar genes Previous reports have provided evidence that luxS Hp-dependent QS may occur to modulate motility via transcriptional regulation of flaA or flhA [20]. We utilised quantitative APO866 RT-PCR (qRT-PCR) to screen for alterations in transcription of these and other genes involved in flagellar assembly to extend our understanding of the regulatory mechanisms that might be involved. Selleck Regorafenib To exclude an effect of learn more cysteine biosynthesis, exogenous addition of cysteine was also undertaken. The concentration of cysteine was non-limiting to H. pylori growth. 16 S rRNA transcription

was used for normalization and ureA served as a non-flagella linked gene control (Figure. 5D). Figure 5 luxS Hp /DPD modulates H. pylori flagellar gene transcription. Transcript levels of (A) flhA, motA, motB; (B) flaB, flgE, flaA; (C) fliI were determined by qRT-PCR normalised to the levels of the 16 S rRNA gene. (D) Relative expression of ureA was utilised as a non-flagella gene control. The Y axis shows the relative transcriptional level of each gene in each strain normalised to the level of the same gene in the strain control (which is J99 wild-type in every case). Values are mean activities of triplicate RNA samples of each strain. Transcript levels were measured in wild-type and ΔluxS Hp cultures grown with or without DPD (150 μM) and in ΔluxS Hp + cultures grown without DPD. (E) AI-2 activity (using the previously-described V. harveyi BB170 bioluminescence assay [4]) in DPD solution (at concentrations of 50 μM, 150 μM or 500 μM) and in cell free culture supernatant (24 h) of H. pylori wild-type, ΔluxS Hp and ΔluxS Hp + strains grown in the Brucella broth (starting OD600 nm of 0.05).