SDS–PAGE was used to separate 3.5 µg of total proteins and SpxSPA was revealed by western-blot using anti-M2 antibodies. Total protein concentration was assayed with Bradford’s method using BSA as control. Cells were harvested before (t0) and 5 or 15 min after treatment and then disrupted by sonication. Log-phase cultures of strain carrying spx-spa translational fusion were treated with 0.5 mM diamide or not. (B) Effect of diamide on SpxSPA protein levels. Serial 10-fold dilutions were then plated on LB supplemented with 0.05 mM diamide (right) and no diamide (left). Cells were grown aerobically in LB medium until stationary phase. subtilis 168 wt strain, Δspx mutant and strain carrying spx-spa translational fusion (SpxSPA). subtilis spx-spa and Δspx strains and SpxSPA protein levels under diamide stress. Genes and its complex interplay with other stress responses. Our study globally characterized the Spx regulatory network, revealing its role in the basal expression of some In vitro transcription confirmed activation by oxidized Spx of seven newly identified promoters, of which one was also activated by Strongly correlated with observed activation. Spx-controlled promoters exhibited an extended −35 box in which nucleotide composition at the −43/−44 positions In wild-type and Δspx strains exposed or not to diamide revealed that 144 transcription units comprising 275 genes were potentially under direct Correlation of Spx–RNAP-binding sites with gene differential expression In parallel with the number and occupancy of DNA sites. Were located near Sigma(A)-dependent promoters, and upon diamide treatment, the fraction of the Spx–RNAP complex increased Here we identified 283 discrete chromosomal sites potentiallyīound by the Spx–RNA polymerase (Spx–RNAP) complex using chromatin immunoprecipitation of Spx. Defects in Spx were previously shown to lead to differential expression of numerous genesīut direct and indirect regulatory effects could not be distinguished. Dimension 5 Limitless Dimension EP 1999 eMi (18.87 MB)įile name: Dimension 5 Limitless Dimension EP 1999 eMiĭimension 5 tribes of the moon and limitless dimension vinyl 1998 goa (23.83 MB)įile name: dimension 5 tribes of the moon and limitless dimension vinyl 1998 goaĭimension 5 tribes of the moon and limitless dimension vinyl 1998 goa (23.The transcriptional regulator Spx plays a key role in maintaining the redox homeostasis of Bacillus subtilis cells exposed to disulfide stress.
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