Inclusion of this indicator made it easier to

see the small recombinant colonies. Plates were seeded with 5 μl H. pylori liquid culture (forming a circle with 3 mm diameter) standardised to an OD600 nm of 1.0 and were incubated at 37°C for up to 7 days under the conditions described above. The motility halos were HDAC inhibitor recorded using a digital camera and the area of each halo was measured using a GS-800 Calibrated Densitometer (Biorad). Motility analysis was also carried out by direct observation under phase-contrast microscopy using a Nikon Eclipse E600 after cells were grown in co-culture conditions as used by Wand et al. [24]. Briefly, co-cultures of H. pylori-human gastric adenocarcinoma (AGS) cells were prepared NVP-LDE225 purchase (described below). After 24 h, 10 μl culture was placed onto a microscope slide and covered with a coverslip and freely-motile H. pylori cells were analysed under the microscope. Plate motility bioassay using chemically defined media (CDM) The liquid chemically defined media were prepared as previously described [15, 28]. 60 ml of sterile chemically defined media were added to 40 ml of molten 1% Oxoid No. 1 agar base to make 0.4% semi-solid chemically defined agar. Cysteine supplemented

plates (CSP) were made by adding cysteine to the https://www.selleckchem.com/Proteasome.html molten agar, shortly before it set. The final concentration of cysteine was 1.0 mM, which was non-limiting for H. pylori growth. The centre of each plate was seeded with one-day incubated H. pylori cells and was incubated for 5 Non-specific serine/threonine protein kinase days under the conditions described above. The motility halos were recorded using a digital camera and the area of each halo was measured using a GS-800 Calibrated Densitometer (Biorad). Motility assay with AI-2 complementation AI-2 was synthesised enzymatically as described previously using purified proteins LuxS

E. coli and Pfs E. coli [8]. For complementation of the ΔluxS Hp motility phenotype, soft motility agar plates (0.4% w/v) were made as previously described. Bioluminescence activity of the AI-2 product was quantified using the V. harveyi bioassay and compared to CFS from H. pylori wild-type broth culture standardised to an OD600 nm of 1.0 at the time point in the growth curve that maximal AI-2 activity was measured. 1/400 diluted in vitro synthesised AI-2 product shows the same level of bioluminescence as seen in the H. pylori wild-type CFS in the V. harveyi bioassay. Therefore, in the complementation experiment AI-2 was added to motility plates to a final concentration of 0.25% (v/v). 24 h H. pylori cultures were seeded individually onto the centre of each motility plate and incubated for 5 days. The area of outward migration was recorded with a digital camera and measured using a GS-800 Calibrated Densitometer (Biorad). Tissue culture and bacterial co-culture All chemicals were obtained from Gibco, UK.