For the culture, a cysteine production medium [composition (per liter): a quantity of 12 g of ammonium chloride, 1.5 g of potassium dihydrogenphosphate, 1 g of magnesium sulfate heptahydrate, 0.1 mg of thiamine hydrochloride, 1.7 mg of ferrous sulfate heptahydrate, 0.15 mg of sodium molybdate dihydrate, 0.7 mg of cobalt chloride
hexahydrate, 1.6 mg of manganese chloride tetrahydrate, 0.3 mg of zinc sulfate heptahydrate, 0.25 mg of copper sulfate pentahydrate, 0.6 g of tryptone, 0.3 g of yeast extract, 0.6 g of sodium chloride, 20 g of calcium carbonate, 135 mg of l-histidine monohydrochloride MK-2206 nmr monohydrate, 4 g of sodium thiosulfate, 2 mg of pyridoxine hydrochloride, 40 g of glucose, 12.5 mg of tetracycline] (Nonaka, 2010) was used. For the cultivation with thiosulfate or sulfite as a sulfur source, 8 g L−1 of sodium thiosulfate or 2.6 g L−1 of sodium sulfite was added, respectively.
For the cultivation with sulfate selleck as a sulfur source, 15 g L−1 of ammonium sulfate was added instead of ammonium chloride. The BW26424/pACYC-DES and the BW25113/pACYC-DES strains were each applied and spread onto LB agar medium containing tetracycline, and precultured overnight at 34 °C. The streak cells corresponding to about 7 cm on the plate were scraped with an inoculating loop of 10 μL size (NUNC Blue Loop), and inoculated into 2 mL of the cysteine production medium. The culture was grown at 32 °C with shaking for 42 h, and the amount of cysteine accumulated in the medium was quantified. The quantification of cysteine was performed by the method described by Gaitonde (1967). The experiment was performed in hexaplicate for both the strains, and averages and standard
deviations of the accumulated cysteine amounts were calculated. Escherichia coli cells grown in 10 mL of LB medium Farnesyltransferase were harvested by centrifugation and resuspended in 0.2 mL 8 M urea/lysis buffer (8 M urea, 50 mM Tris-HCl, pH 8.0 at 4 °C, and 100 mM NaCl), and sonicated. Cell extracts (10 μg) were subjected to 10% SDS-PAGE and blotted on to polyvinylidene difluoride (PVDF) membranes using iBlot semi-dry transfer apparatus (Invitrogen). Membranes were first immuno-detected with anti-β-galactosidase (Progema) and horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Nacalai tesque) antibodies and then developed with a chemiluminescence kit (Nacalai tesque). The image was analyzed with a LAS-4000 IR multi color (Fuji Film). The transcriptome analysis of E. coli response to metal-shock indicated that the genes for cysteine biosynthesis including cysK are regulated by several metals (Yamamoto & Ishihama, 2005a, b; Hobman et al., 2007). Since a set of the metal stimulon genes are regulated by some of two-component system (TCS) (Yamamoto & Ishihama, 2006; Yamamoto et al., 2008), we tested possible influence of TCS knock-out on cysK expression. For this purpose, we used the collection of TCS deficient E. coli mutants (Oshima et al.