Fig. 3
creg motif of PhrS is essential for crRNA regulation of CRISPR-Cas adaptive immunity. a Diagram of the sRNA PhrS and mutations. creg motif is marked with bold. Eleven nucleotides of creg were deleted in the plasmid pJT-phrSΔcreg. pJT-phrScmut plasmid contained site mutations in creg motif. pJT-phrScreg represent regions with expression of creg motif. b LacZ activity as determined in the PA14 ΔphrS background strain harboring the PhrS expression plasmid pJT-PhrS, plasmid pJT-PhrSΔcreg, plasmid pJT-phrScmut, or plasmid pJT-phrScreg with toluate (2 mM final concentration). c Northern blot of CRISPR2 crRNA production in PA14 ΔphrS background strain containing the PhrS expression plasmid pJT-phrS, plasmid pJT-phrSΔcreg, plasmid pJT-phrScmut, or plasmid pJT-phrScreg. d Transformation efficiency of CR2-sp1 vector in PA14 ΔphrS background strain within the plasmid pJT-phrS, pJT-phrSΔcreg, pJT-phrScmut, or pJT-phrScreg. e Phage plaque of CRISPR-sensitive phage JBD18 on PA14 ΔphrS background harboring the plasmid pJT-phrS, pJT-phrSΔcreg, pJT-phrScmut, or pJT-phrScreg. f IntaRNA (Freiburg RNA tools) prediction of PhrS interactions with CRISPR2 leader target (upper). A diagram of leader binding site with phrS knockout or mutant with red parts mark (lower). g GFP fluorescence in PA14 strains or ΔphrS transformed with pleaderCRISPR2-GFP, pleaderCRISPR2-Δ-GFP, pleaderCRISPR2-mut1-GFP, or pleaderCRISPR2-mut2-GFP plasmid, respectively. GFP mode is on the left and the visible light of light mode is on the right). h Western blot analysis of GFP in the PA14 WT or ΔphrS transformed with pleaderCRISPR2-GFP, pleaderCRISPR2-Δ-GFP, pleaderCRISPR2-mut1-GFP, or pleaderCRISPR2-mut2-GFP plasmid, 5 μg total proteins were used to western blotting analysis. Results are shown with mean ± SEM from three independent experiments. **P < 0.01, *P < 0.05, one-way ANOVA plus Tukey test
