The array was then washed successively with Gene Expression Wash Buffer 1 and 2 (Agilent). We realized arrays scanning with a GenePix 4200L dual-channel (635 nm and 532 nm) laser

scanner (GenePix). The complete experimental data set was deposited in the GEO database with accession numbers GSM480613 to GSM480620. All slides were analyzed using R and limma software (Linear Model for Microarray Data) from CB-839 research buy Bioconductor project http://​www.​bioconductor.​org. For each slide, we corrected background with the ‘normexp’ method [34], resulting in strictly positive values and reducing variability in the log ratios for genes with low levels of hybridization signal. Then, we normalized each slide with the ‘loess’ method [35]. In order to identify genes differentially expressed, we used the bayesian adjusted t-statistics and we performed a multiple testing correction of Benjamini & Hochberg [36] based on the false discovery rate. A gene was considered as differentially expressed when the p-value is < 0.05. Stress response analysis Disk diffusion

assays were performed as follows: 20 ml calibrated agar plates were poured on a horizontal plane. C. perfringens strain 13 was grown in minimal medium containing 0.5 mM cystine or 1 mM homocysteine until it reached an OD600 nm of 0.5. The cells were then spread onto solid minimal medium containing the same sulfur source. After absorption, a sterile 6 mm disk was placed on the agar and 10 μl of 1 M H2O2, 1 M diamide or CAL 101 0.2 M paraquat was added to the disk. The plates were incubated 48 h at 37°C and the diameters of growth inhibition were measured. These experiments were repeated 5-fold and a Wilcoxon test was realized giving a p-value < 0.05. Results and Discussion Reconstruction of sulfur metabolism in C. perfringens We performed a systematic search in the C. perfringens genomes for genes known to

be involved in assimilation pathways of sulfur-containing compounds. This tentative reconstruction is shown in Fig. 1. We also tested the ability of C. perfringens strain 13 to grow in a sulfur-free minimal medium in the presence of various sulfur sources in order to support the metabolic reconstruction performed Urocanase and to obtain new insights about the physiology of this bacterium. We first tested the growth in the presence of the sulfur-containing amino-acids, methionine or cystine, the dimer of cysteine. This strain can grow in the presence of 0.5 mM cystine as sole sulfur source (Fig. 2) indicating a conversion of cysteine to methionine. Surprisingly, the genes required for methionine biosynthesis via transsulfuration or thiolation in other bacteria (metA, metI, metC, patB, metY, metH, metE, metF) [6, 9] are absent in the genome of C. perfringens strain 13 [21]. This suggests the existence of an atypical methionine biosynthetic pathway in C. perfringens, which remains to be characterized. Figure 2 Growth of C. perfringens strain 13 in the presence of various sulfur sources.