These data suggested that either AI-2 is not released from the cell in MEM-α, or that part of the AMC is not active under these conditions. To distinguish between the two possibilities, cell extracts ofC. jejuniNCTC 11168 were prepared from cells harvested after 5 h growth and

analysed for LuxS activity (see Methods for details). As positive and negative controls, cell extracts selleck chemical fromE. colistrain MG1655 and strain DH5α containing aluxSframe shift mutation were used. Whole cell lysates were prepared and SRH added. Conversion to homocysteine and DPD were assessed using Ellmans reagent and theV. harveyibioassay respectively. In agreement with previous studies [26,49] crude extracts ofE. coliMG1655 contained detectable levels of homocysteine and DPD indicating LuxS activity (data not shown). However, neither compound was detectable in cell extracts ofE. coliDH5αluxSmutant (negative control) orC. jejuniNCTC 11168. Neither growth in MHB nor MEM-α to the point when extracellular AI-2 levels are high in MHB (5 h) yieldedC. jejuniNCTC 11168 extracts capable of converting SRH to homocysteine and DPD (i.e. exhibiting LuxS activity), suggesting either lack of DPD production (with detection limit for AI-2 of approx 6 μM) or rapid turnover. Mutation ofluxSalters gene expression in a medium-dependent fashion Microarrays were employed to compare the transcriptomes ofC. jejuniwild type

andluxSmutant grown in either MHB or MEM-α. This analysis, which was performed with cells harvested in late exponential growth (8 h after inoculation), revealed a number of differentially expressed PFT�� purchase genes

[see Additional Files 1 and 2). Interestingly, most of the observed Carbohydrate differences were media-dependent and associated with metabolic functions (i.e. catabolism, Selleck VX-689 anabolism, transport, and energy production). There were also considerably more differentially expressed genes when the mutant and wild type strains were grown in MHB rather than in MEM-α (131 and 60 genes with a greater than twofold change respectively). 20 genes (comprising 14 probable transcription units) were differentially expressed in both media (thus comprising a third of the changes seen in MEM-α), suggesting that they were linked to loss ofluxSfunction. These included genes with (putative) roles in amino acid and lactate uptake (Cj0982c andlctP, respectively), electron transport and respiration (Cj0037, Cj0073, Cj0074, Cj0075,sdhC) and oxidoreductase reactions (Cj1199, Cj0415). Some of the identified genes are known to play a role in anabolic pathways such as amino acid (e.g.trpA,trpB,glnA) and fatty acid (fabI) biosynthesis or central metabolism such as the tricarboxylic acid cycle (e.g.sdhC). Interestingly, gene Cj0982c has recently been shown to be involved in cysteine uptake. The upregulation of this gene in theluxSmutant is in agreement with the hypothesis thatluxSmutants have an increased requirement for sulphur-containing amino acids [50]. In MEM-α, Cj0982 transcript levels were increased 7.