testosteroni S44 was cultured in LB broth with 1 mM Se(IV) at 26°C with shaking at 180 rpm, harvested at both log phase and stationary phase. Samples that were grown without Se(IV) were find more used as controls. Cultured samples were fixed using 2% v/v glutaraldehyde in 0.05 M sodium phosphate buffer (pH 7.2) for 24 h and were then rinsed three times in 0.15 M sodium cacodylate buffer (pH 7.2) for 2 h. The specimens were dehydrated in graded series of ethanol (70%, 96% and 100%) transferred to propylene oxide and embedded in Epon according to standard procedures. Sections, approximately 80 nm thick, were cut with a Reichert-Jung Ultracut E microtome and collected

on copper grids with Formvar supporting membranes. The sections were stained or unstained with uranyl acetate and lead citrate and then TEM-STEM-EDX (TITAN 120 kV) and EDS Mapping (QUANTA 200 F) were performed, respectively. Tungstate test on Se(IV) and Se(VI) reduction C. testosteroni S44 cells were incubated in CDM (chemically defined medium) [50], LB and TSB plates supplemented with 0.2 mM sodium

selenite, 5.0 mM sodium selenate, respectively, and with or without 10 mM tungstate (Na2O4W.2H2O) at 26°C under aerobic condition for two days. The inhibiting effect of tungstate was shown by appearance or absence of the specific red color of SeNPs in comparison with control in absence of tungstate. Cellular fractionations and determination of Se(IV)-reducing activity Log-phase (12 hr) and stationary phase Birinapant molecular weight (20 hr) cells ADP ribosylation factor of C. testosteroni S44 were obtained by growth at 26°C, shaking at 180 rpm in 20 ml LB broth. The modified method was based on protocol of method No. 5 for subcellular fractionation [51]. All further parts of the procedure were carried out at 0 to 4°C unless differently noted. The cells in 20 ml LB cultures were harvested by

centrifugation for 20 min at 4,500 × g, and then the supernatant was removed. After being harvested, the cells were suspended in 2.0 ml 1 × PBS buffer (pH 7.0), GSK2118436 centrifuged three times for 10 min at 4,500 × g. The cells were then suspended in 1.0 ml 1 × PBS buffer (pH 7.0) containing 5% glycerol (v/v, final concentration). The suspension was treated with 1.0 mg ml−1 (final content) lysozyme for 5 min at room temperature and afterwards centrifuged for 20 min at 20,000 × g. The supernatant was periplasmic protein. In order to separate the membranes from the cytoplasm, the pellet was suspended in 1.0 ml 1 × PBS buffer containing 5% glycerol (v/v) and 125 units per ml (final concentration) DNase I. The suspension was treated with ultrasound for 20 min (20 amplitude microns, 5 s /5 s, Sanyo Soniprep). The broken-cell suspension was centrifuged for 6 min at 6000 × g to remove unbroken cells. The supernatant was centrifuged for 60 min at 20,000 × g. The supernatant contained the cytoplasmic fraction and the pellet contained the crude membranes (outer membrane and cytoplasmic membrane).