Visual observation of H2S production was performed using lead-acetate paper (Macherey-Nagel) that turned black following the incubation for up to 3 h at 37°C. Intracellular concentrations of amino acids and other ninhydrin-reactive compounds were estimated using high-pressure

liquid chromatography (HPLC). Briefly, cells were suspended GDC-0449 molecular weight in a sulfosalicylic acid buffer (3% final concentration) and disrupted using a FastPrep apparatus (Bio101). Supernatant samples were analyzed by cation-exchange chromatography, followed by ninhydrin postcolumn derivatization as previously described [8]. Intracellular metabolite concentrations were estimated assuming a cell volume of 4 μl per mg of proteins or a C. perfringens intracellular volume of 3 μm3 [31]. Metabolite concentration was estimated PCI-32765 cell line with the ratio between total quantity of a metabolite and the total cellular volume. The mean value is calculated from three independent experiments. A statistical CH5183284 mw Wilcoxon test was realized giving a p-value < 0.05. RNA isolation, Northern blot analysis and quantitative RT-PCR We extracted total RNA from strains 13, TS133 or TS186 grown in minimal medium with 0.5 mM cystine or 1 mM homocysteine as sole sulfur source. Cells were harvested at an OD600 nm of 0.6 (homocysteine) or 0.8 (cystine) by centrifugation for 2

min at 4°C. The cells were first broken by shaking in a Fastprep apparatus (Bio101) for 2 × 30 sec in the presence of one gram of 0.1-mm diameter glass beads (Sigma), then treated with Trizol

reagent, chloroform/isoamylalcohol and precipitated with isopropanol. The pellet was resuspended in 100 μL of TE buffer (Tris 10 mM, EDTA 0.1 mM). For Northern blot analysis, 10 μg of total RNA was separated in a 1.5% denaturing agarose gel containing 2% formaldehyde, and transferred to Hybond-N+ membrane (Amersham) in 20 × SSC buffer (3 M NaCl, 0.3 M sodium citrate pH 7). Prehybridization was carried out for 2 h at 68°C in 10 ml of prehybridization buffer ULTRAHyb (Ambion). Hybridization was performed overnight at 68°C in the same buffer in the presence of a single strand RNA [α-32P]-labeled probe. The probes were synthesized from a buy 5-Fluoracil PCR product containing a T7 phage promoter sequence on one of its extremities. One probe is located in the 5′ untranslated region of the cysP2 gene (-326 to -181 relative to the cysP2 translational start point) and the second probe hybridizes with the coding region of cysP2 (+71 to +299 relative to the cysP2 translational start point). 1 μg of each PCR product was used as a matrix for in vitro transcription reaction with phage T7 RNA polymerase, 0.5 mM each ATP, GTP, CTP, and 50 μCi of [α-32P]UTP using Maxiscript kit (Ambion). The probe was then treated with TURBO DNAse I and purified on “”Nucaway spin column”" (Ambion). After hybridization, membranes were washed twice for 5 min in 50 ml 2× SSC 0.1%SDS buffer and twice for 15 min in 50 ml 0.1 × SSC 0.1% SDS buffer.