Production of AI-2 using crude cell-extracts Cell pellets were harvested from exponentially growingC. jejunicultures by centrifugation (3000 g for 20 min) and resuspended in an appropriate volume of 10 mM sodium phosphate JPH203 buffer (pH

7.7) containing freshly added lysozyme (100 μg/ml; Sigma-Aldrich UK) and ‘Bugbuster Benzonase’ nuclease (1 μl ml-1; Novagen UK). After 30 min incubation at 37°C, debris was pelleted by centrifugation (10000 g for 15 min) and the crude cell extracts transferred to a new microfuge tube. To VRT752271 assess LuxS activity, cell-extracts were added in a 1:1 ratio to 4 mM SAH in sodium phosphate buffer, or to 2 mM SRH that was enzymatically produced from SAH as previously described [26]. In each case the resulting mixture was incubated for 2 hours at 37°C, mixed with Selleckchem YH25448 an equal volume of chloroform, centrifuged, and the aqueous extract analysed for AI-2 activity usingV. harveyiBB170 strain as a bioluminescent reporter [13]. As positive and negative controls for LuxS activity, cell extracts ofE. coliMG1655 and DH5α, respectively, were used, as well asC. jejuniextracts incubated with buffer lacking the substrate. Addition of exogenous AI-2 toC. jejunicultures Cultures ofC. jejuniNCTC 11168 and LuxS01 were grown as described above. After 2.5 h,in vitro-produced AI-2 was added to test cultures and the AI-2 negative mix was added to the control cultures as

described above. This gave the cells time to reach exponential growth phase, and ensured AI-2 levels were maintained throughout the same growth period as is observed for the WT grown in MHB. Light assay samples were taken from controls and AI-2 samples immediately following addition of AI-2, then again at 8 h, before the cells were harvested and the RNA extracted for microarray expression analysis. Microarray Data Microarray data is available on the Gene Expression Omnibus (GEO) database,http://​www.​ncbi.​nlm.​nih.​gov/​sites/​entrez?​db=​gds. The accession number is GSE18455. Results C. jejuniproduces AI-2 in MHB but not

MEM-α In line with observations made in otherC. jejunistrains (NCTC 11168, 81116, and 81-176; [37,44,48], we found that in MHB, AI-2 production and motility byC. jejunistrain NCTC 11168 was abolished in an isogenicluxSmutant strain (LuxS01). We set out to understand the nature of Tyrosine-protein kinase BLK the phenotypes reported forC. jejuni luxSmutants, which have been attributed to AI-2 mediated quorum sensing [44,48], or more recently at least in part to the role of LuxS in central metabolism [37]. To do this, we monitored the extracellular AI-2 profile during growth ofC. jejuniNCTC 11168 and the isogenicluxSmutant strain (LuxS01) in a defined medium (MEM-α). As in the rich MHB media, disruption ofluxShad no effect on growth in MEM-α (Data not shown). Interestingly, however, the growth medium had a marked effect on AI-2 production.