For in vitro assay of Cr(VI) reductase activity, NADH was used as the electron donor. When equal amounts of protein were used in the reactions, the cytoplasmic fraction showed slightly higher activity than the crude extract. After 1 h of reaction at 65 °C, the cytoplasmic fraction was found to be 3-fold more active than the membrane fraction (data not shown). When extracts were prepared from cells

grown at 37 °C, the cytoplasmic fraction showed higher Cr(VI) reduction activity at 65 °C than that at 37 °C (Fig. 1c). However, such activity in the cytoplasmic fraction prepared from cells grown at 65 °C and assayed at the same temperature was even higher (Fig. 1c). Silmitasertib datasheet The results indicated that Cr(VI) reduction activity by TSB-6 cells was greater at 65 °C than that at 37 °C not just because of an increase in the reduction efficiency of the putative reductase(s)

but possibly also because of production of such factor(s) in greater amounts in cells growing at the higher temperature. To determine whether heat exerted oxidative stress on TSB-6 and, consequently, affected its growth and Cr(VI) reduction activity, cells grown in LB at 37 °C check details were transferred to 65 °C. With time of incubation, the control cells at 37 °C produced gradually decreasing amount of ROS (Fig. 2a). However, ROS produced by the cells transferred to 65 °C at 2, 4, 6, and 24 h was found to be, respectively 24, 78, 75, and 38% greater than control cell (Fig. 2a). The cell density started decreasing immediately after the transfer and continued to decrease for about 4 h. OD600 nm values of the both 37 °C and 65 °C cultures at different time points could be well correlated with viable counts (data not shown). Thereafter, the cells resumed growth, but at a slower rate, and the final OD600 nm of the culture at 65 °C tended to be lower than that at 37 °C (Fig. 2b). Cr(VI) reduction activity of the cells at 65 °C remained unchanged till 4 h post-transfer, but was 35% and 57% higher than that of the cells at 37 °C at 6 and 24 h, respectively (Fig. 2c).

Proteins in whole cell extracts from TSB-6 cultures ifenprodil grown at 37 and 65 °C were separated by two-dimensional gel electrophoresis. A relative change of ≥ 2 in abundance of proteins was considered to be significant. Comparison of the spots using this criterion showed that 18 proteins were upregulated in 65 °C, whereas 12 were downregulated (Fig. 3). MALDI-TOF analysis identified 14 of the upregulated and 11 of the downregulated spots and found that the upregulated set included proteins involved in cellular metabolism of sugar, nucleotide, amino acids, lipids and vitamins, oxidoreductase activity, and protein folding (Table 1). The downregulated proteins are also involved in cellular metabolism, DNA binding, and environmental signal processing (Table 1). Mesophilic bacteria can adapt themselves to survive in thermophilic environments (Dowben & Weidenmüller, 1968; Droffner et al., 1995).