BW25113 GhoS for RNase activity

To provide more evidence of the specificity of the RNase activity of GhoS, we performed a whole-transcriptome study for the production of GhoS vs. an empty plasmid so that we could investigate all of the cell’s transcripts for cleavage with GhoS, in vivo (i.e., BW25113/pCA24N-ghoS vs. BW25113/pCA24N with 1 mM IPTG induction of ghoS for 90 min). Under these conditions, only 20 genes were found to be repressed by more than 4-fold; there were no induced genes. These GhoS-repressed genes were all involved in the biosynthesis/transport of purines and pyrimidines; among them, pyrI was most highly repressed (-20 fold). These results suggest that GhoS selectively cleaves only a few cellular targets. Overnight cultures of E. coli strains BW25113-pCA24N-ghoS and BW25113-pCA24N (emtpy plasmid) were inoculated into fresh LB medium to a turbidity of 0.05 at 600 nm and grown at 37°C. IPTG (1 mM) induction was performed when turbidity reached 0.2 and continued for 90 min. Total RNAs were isolated from the cultures and converted to cDNA for the application on E. coli genome 2.0 array.