DNA fragments were purified from agarose gel using a QIAquick gel extraction kit (QIAquick, UK) according to the manufacturer’s instruction. H. pylori genomic DNA was isolated as described previously [26]. DNA sequencing was conducted using

standard fluorescent dye terminator chemistries, and analysis performed using the Applied Biosystems 3730 DNA Analyzer system (Geneservice, Cambridge, UK, Applied Biosystems Inc, Foster City, CA.). Results were analysed using the Bioedit software suite [27]. Construction of the complemented ΔluxS + strain H. pylori J99 wild-type was transformed with the plasmid pGEMTluxSXN396 containing a km-sacB construct GS-1101 mw encoding kanamycin RG7112 in vitro resistance (Kmr) and (5%) sucrose sensitivity (Sucs) [17]. Disruption of the chromosomal luxS gene was accomplished by natural transformation, allelic exchange, and screening for kanamycin-resistance as previously described [15], resulting in the J99 ΔluxS mutant strain. The presence of the km-sacB cassette was verified by amplifying fragments of H. pylori chromosomal DNA using primers luxS-F/luxS-R (forward, 5′>GTG GCT TTA GCG GGA

TGT TTT<3'; reverse, 5'>GCGA ACA AAT CCC CGC TG<3') and DNA sequencing. The J99 ΔluxS was then transformed with plasmid pGEMTluxS (encoding wild-type luxS), and transformants in which km-sacB had been replaced with the introduced original luxS locus were selected for sucrose resistance on medium containing 5% sucrose and screened selleck chemicals llc for kanamycin sensitivity. The presence of the original luxS gene was verified by amplifying fragments on H. pylori chromosomal DNA using primers luxS-F/luxS-R and DNA sequencing.

Bacterial growth curves and V. harveyi bioluminescence assay Bacterial broth cultures were started from a blood agar plate culture, diluted to an OD600 nm of 0.05 in fresh BB medium, and grown at 37°C in a VAIN-cabinet with shaking. OD600 nm measurements were taken at the 6 h, 24 h, 48 h and 72 h time points, and at the same time cell suspensions were harvested and filtered through a 0.2 μm pore size filter. The AI-2 activity in cell free supernatants (CFS) was tested as previously described using the V. harveyi reporter strain BB170 [9, 22]. Briefly, an overnight V. harveyi culture was diluted 1:2500 Aspartate in fresh AB medium [23]. CFS samples were diluted 1:10 in the AB medium containing BB170 into the 96 well bioluminescence plates to give a final volume of 200 μl and were incubated at 30°C. The bioluminescence and optical density were determined at 30 min intervals for at least 8 h using a luminometer (Anthos Labtech LUCY 1.0). AI-2 activity alterations in bioluminescence were expressed as induction (n-fold) over the negative control. Motility assay Plate motility assay of H. pylori was performed in Brucella broth medium (BD Biosciences), supplemented with 7% (v/v) fetal bovine serum (Gibco), 0.35%-0.45% (w/v) agar (No.