, 2008 [37] To do this, we analysed the transcriptome ofC. jejuniNCTC 11168 and its isogenicluxSmutant grown in both defined and complex media. Furthermore, exogenousin vitro-produced AI-2 was added back to growing cultures of theluxSmutant to monitor the transcriptional response induced by this extracellular signal. Methods C. jejunistrains and growth

conditions The bacterial strains used in this study were kindly donated by Simon Park and Karen Elvers (University of Surrey).C. jejunistrain NCTC 11168 (wild type) and its isogenicluxSmutant, LuxS01 [35] were routinely grown at 42°C under microaerobic conditions (10% CO2, 85% N2, 5% O2; all vol/vol) on Skirrow agar plates, in Mueller Hinton selleck products broth (MHB; Oxoid, Basingstoke, UK), or in MEM-α medium (Invitrogen, UK) on an orbital shaker (380 rpm) inside a MACS-MG-1000 controlled atmosphere workstation (Don Whitley Scientific, UK). When required, kanamycin at a final concentration of 25 μg ml-1(Sigma-Aldrich, UK) was added to the medium. To test for AI-2 activity, CRT0066101 purchase 50 ml of MHB or MEM-α was inoculated withC. jejuniwild type orluxSmutant grown on Skirrow agar and incubated overnight (16-18 h). A 3% inoculum was then used to inoculate a fresh 50 ml broth and grown to late www.selleckchem.com/products/H-89-dihydrochloride.html logarithmic phase (approx. 8 h; determined

by viable counts and OD600). Samples were taken at intervals (typically 8 h) during the logarithmic growth phase to test for AI-2 activity using theV. harveyibioassay. For this assay 1 ml was removed from each culture and centrifuged at 12000g, 4°C for 10 min. The supernatant was then filter-sterilised with a 0.2 μm filter unit (Millipore) and stored at -80°C before analysis. Motility Assays Motility assays were performed as described by Elvers and Park [35] using MHB and MEM-α broth, respectively, both containing 0.4% (wt/vol) agar. Plates were incubated at 37°C and 42°C and motility halos were examined after 16 h, 24 h and 48 h. Parallel experiments were performed on cultures

grown in the presence or absence of exogenous AI-2. Analysis of culture supernatants for AI-2 activity Cell-free culture supernatants were prepared by centrifugation and 0.2 μm filtration. AI-2 activity in supernatants was analysed as Succinyl-CoA described by Bassleret al. 1997, using 20 μl AI-2 extract and 180 μl 1:5000 diluted overnight culturedV. harveyiBB170 in AB medium [13]. Changes in bioluminescence upon addition of AI-2 were determined at 30°C every 30 min using a combined, automated luminometer-spectrometer (Anthos Labtech Lucy1). AI-2 activity was defined as the fold increase in light production in comparison with medium or buffer controls. For a single experiment, theV. harveyibioassay was performed at least in duplicate for each sample. Experiments were repeated at least three times. RNA isolation and purification Cultures were grown in triplicate as described above and bacteria were harvested during late logarithmic phase of growth (approximately 8 h, OD 0.