A diluted in vitro synthesised AI-2 sample was utilised as a qualitative positive control [8]. Error bars indicate standard deviation. The flagellar genes tested included several from different regulatory hierarchy positions in flagellar synthesis [33]: class 1 genes flhA (encodes flagellar regulator component), motA and motB (encode flagellar motor proteins); class 2 genes flaB (encodes hook-proximal minor flagellin) and flgE (enodes flagellar hook protein); and class 3 gene flaA (encodes major flagellin). fliI (encodes membrane-associated export ATPase of the flagellar basal body) was also examined (Figure. 5). For class 1 genes tested, flhA showed a consistent

pattern of 1.75 fold reduced transcription (p < 0.001), and both motA and motB showed a consistent pattern of 2 fold (p < 0.001) reduced transcription in the ΔluxS Hp mutant compared to the wild-type (Figure. 5A). For class 2 genes tested, flgE was 1.5 Sotrastaurin fold (p < 0.001) down-regulated in the ΔluxS Hp mutant; while flaB did not exhibit any significant change. flaA was the only class 3 gene tested, which was 3.5 fold (p < 0.001) down-regulated in the ΔluxS Hp mutant compared to the wild-type

(Figure. 5B). Additionally, the transcript of fliI was also significantly (1.5 fold, p < 0.001) decreased in the mutant (Figure. 5C). The reduced transcription of flhA, motA, motB, flgE, flaA and fliI was restored genetically by the complementation Napabucasin concentration of the mutant with the wild-type luxS Hp gene. Also, 150 μM DPD was sufficient to restore the transcription of these genes in the ΔluxS Hp mutant to levels similar to the wild-type (Figure. 5E). Although Figure 5E shows that 50 μM and 150 μM DPD induced why almost the same level of bioluminescence as the wild-type, we chose to use 150 μM DPD in the complementation experiment because this concentration was shown to be more reproducible (it has the smaller error bar). In wild-type cells, addition of DPD markedly increased transcription

of motA, motB, flaA and flaB, whilst flhA, flgE and fliI only showed a marginal increase. Exogenous addition of GW-572016 solubility dmso cysteine to the ΔluxS Hp mutant did not significantly increase transcription of any of the genes studied; suggesting that addition of cysteine was not able to restore the transcription of flagellar genes (data not shown). Consistent with the analysis of protein levels, these RT-PCR data indicate that luxS Hp disruption has a greater effect upon transcription of flaA than of flaB. Taken together, these data suggest that the effect of LuxS in cysteine metabolism does not regulate expression of flagellar genes, and that the effects on flagellar gene transcription are likely through AI-2 production. Discussion The function of luxS Hp is controversial due to putative roles both in signalling and metabolism. Disruption of cysteine biosynthesis by independent mutations that had no influence on AI-2 production did not alter motility. In contrast, the motility defect of a ΔluxS Hp mutant of H.