8% agarose gel, extracted with phenol and ether, and then precipitated with ethanol. The DNA fragments were used for the following assays. Assays were performed in 15-μL reaction mixtures in the absence or presence of 2 μM T. thermophilus SdrP by basically the same process as that described previously (Shinkai et al., 2007). The template DNA was preincubated with
or without SdrP at 55 °C for 5 min. Thermus thermophilus RNA polymerase-σA holoenzyme purified as described previously (Vassylyeva et al., 2002) was added, and then the mixture was further incubated EPZ015666 mouse for 5 min. Transcription was initiated by the addition of 1.5 μCi [α-32P]CTP and unlabeled ribonucleotide triphosphates. After further incubation for 10 min, the reaction was stopped, and the sample was analyzed on a 10% polyacrylamide gel containing 8M urea, followed by autoradiography. Primer extension analysis with RNA transcribed in vitro was performed by basically
the same method as that described Selleck Ruxolitinib previously (Shinkai et al., 2007). The nucleotide sequence of the template DNA was determined by the dideoxy-mediated chain termination method (Sanger et al., 1977). Samples were analyzed on an 8% polyacrylamide gel containing 8M urea, followed by autoradiography. A blast search was performed at http://blast.ncbi.nlm.nih.gov/Blast.cgi. In the previous study, we observed that the growth of an sdrP gene-deficient (ΔsdrP) strain was more significantly affected by diamide treatment, which forms non-native disulfide bonds (Leichert et al., 2003; Nakunst et al., 2007), in comparison with that of the wild type (Agari et al., 2008). In order to determine whether oxidative stress induces expression of the sdrP gene, we treated the wild-type T. thermophilus HB8 strain in the logarithmic growth phase with diamide or H2O2. RT-PCR analysis showed that expression of the sdrP gene increased with the addition of a final concentration of 2 mM diamide
or 10 mM H2O2 (Fig. 1), which was supported by DNA microarray aminophylline analysis results that showed that expression of the gene increased 27-fold (q-value=0.00) and 11-fold (q-value=0.00) in response to diamide and H2O2 treatment, respectively (Table 1). Next, we examined whether other environmental or chemical stresses, such as heavy metal ion (ZnSO4 and CuSO4), antibiotic (tetracycline), high-salt (NaCl), and organic solvent (ethanol) stresses, induce expression of the sdrP gene. RT-PCR (Fig. 1) and DNA microarray (Table 1) analyses indicated that expression of the sdrP gene was induced by all of these stresses. In the ΔcsoR strain, in which excess Cu(I) ions may accumulate due to a significant decrease in the expression of the probable copper efflux P-type ATPase gene copA (Sakamoto et al., 2010), the effect of excess CuSO4 on expression of the sdrP gene was more significant than that in the wild-type strain (Fig. 1 and Table 1). We found that expression of sdrP drastically changed depending on the environmental conditions.