Figure 2

The σ³² level is increased during the expression of BnTR1. (a) Distribution of σ³² regulons among the common DEGs (right panel). The expression level of rpoH measured by microarray is denoted as an orange dot. (b) Validation of microarray data by qRT-PCR and two-tailed Student’s t-test was used for the comparison (*p-value < 0.05 and **p-value < 0.001). Six genes were selected, including three heat shock genes (dnaK, groEL, and ipbA), one down-regulated DEG (prfH), and two genes without significance (hyfF and rpoH). (c) Whole-cell extracts from pET and pET-BnTR1 cells cultured at 37 °C with 0.1 mM IPTG were analysed using Western blotting (probed with anti-DnaK, anti-σ³², and anti-His for BnTR1). (d) Wild-type E. coli W3110 and C600 cells harbouring the pBAD24 empty vector or pBAD-BnTR1 were grown at 30 °C with 0.1% L-arabinose. Whole-cell extracts taken at the indicated time were loaded and probed with anti-σ³² and anti-His for BnTR1. (e) Whole-cell extracts were from E. coli C600 cells induced with different concentration of L-arabinose for 30 min at 30 °C. Immunoblots were shown and probed with antibodies indicated. (f) Western blotting (probed with anti-σ³² and anti-His for BnTR1) of whole cell, supernatant, and pellet proteins that were extracted as described in (d). OmpC was detected in parallel by anti-OmpC and used as a loading control in (c,d and e). The data are presented as means ± s.d. of three independent experiments (b). Full-length blots are presented in Supplementary Figure 6.