Hyphomicrobium sulfonivorans S1T was grown in a batch culture on dimethylsulfone as described previously by Boden et al. (2011) and R. sulfidophilum was grown photoorganoautotrophically on DMS according to McDevitt et al. (2002). Sagittula stellata was grown in
steady-state chemostats at a range of dilution rates (D) between 0.01 and 0.15 h−1 on fructose (12 mM) and between 0.01 and 0.10 h−1 on succinate (2 mM) with or without the addition of DMS (1 mM). Kinetic parameters were determined as described previously (Boden et al., 2010). Five volume changes at each steady state occurred before the kinetic parameters were determined. Cells were harvested for enzyme assays by centrifugation at 13 000 g for 30 min at 4 °C. Natural Product Library screening selleck inhibitor Cells were washed and resuspended in 50 mM PIPES-HCl, pH 7.4, containing 50 mM magnesium sulfate. If not used immediately, cells were snap-frozen in liquid nitrogen and stored at −80 °C. Spectrophotometric enzyme assays were routinely conducted at 30 °C in an Ultrospec 3100pro UV/Visible Spectrophotometer
(Amersham). Each reaction was conducted in a sevenfold replicate against a blank. Cell-free extracts were prepared by three passages through a French pressure cell (120 MPa), with debris removed by centrifugation (13 000 g, 30 min, 4 °C). Protein was quantified using the method of Bradford (1976). DMS dehydrogenase Cetuximab research buy activity was assayed using a modification of the method of McDevitt et al. (2002). Two milliliters of 500 mM Tris-HCl, pH 8.0, 300 μL of 35 mM phenazine methosulfate and 300 μL of 100 μM 2,6-dichlorophenolindophenol (DCPIP) were placed in a 3 mL modified Thunberg cell (Baumberger, 1933) and degassed by bubbling with oxygen-free nitrogen for 10 min before adding 100 μL cell-free extract (containing 5–10-mg protein). Sixty microliters of 100 mM DMS solution in ethanol was placed in the bulb of the side-arm and the cell was assembled. The cell was evacuated on ice for 10 min before sealing and the reaction was initiated by pouring
the contents of the side-arm into the main chamber. The A600 nm was monitored and the rate of reduction of DCPIP was determined using the millimolar extinction coefficient for the oxidized form of 21.5 mM−1 cm−1. Cell-free extracts prepared from R. sulfidophilum SH1 grown photoorganoautotrophically with DMS as an energy source were used as a positive control (McDevitt et al., 2002). An alternative assay was performed using 300 μL of 3 mM potassium ferricyanide in place of DCPIP solution and reduction was monitored at 420 nm with a millimolar extinction coefficient of 1.0 mM−1 cm−1. DMSO reductase was assayed in the same way using a reaction mixture comprising 150 μL of 1.0 M Tris-HCl, pH 7.6, 20 μL of cell-free extract and 1.03 mL of MilliQ water in the main chamber of the cell. These were degassed in situ before adding 1.