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. Author manuscript; available in PMC: 2020 Sep 23.
Published in final edited form as: Nat Microbiol. 2020 Mar 23;5(5):679–687. doi: 10.1038/s41564-020-0691-3

Figure 3: AmrZ is a surface-activated repressor of CRISPR-Cas immunity.

Figure 3:

a. Efficiency of immunity (EOI) against phages DMS3acrIE3 (non-targeted) and DMS3macrIF4 (CRISPR-targeted). Plaque forming units (PFUs) were quantified on ΔamrZ or the complemented strain, then represented as a ratio of the number of PFUs measured on WT PA14. ΔamrZ + EV shows increased EOI against DMS3macrIF4 relative to WT (P = 7.3 × 10−4). EOI measurements are represented as the mean of 3 biological replicates +/− SD. b. Growth curves of PA14 WT and ΔamrZ infected with 106 PFU of virulent DMS3macrIF4 alongside uninfected controls. c. EOI against virulent DMS3macrIF4 in liquid culture of WT and ΔamrZ strains (CRISPR active) or WT csy3::lacZ and ΔamrZ csy3::lacZ (CRISPR inactive). PFUs were quantified after 24 h from ΔamrZ or amrZ csy3::lacZ, then represented as a ratio of PFUs from WT or WT csy3::lacZ, respectively. OD600 and EOI measurements are represented as the mean of 3 biological replicates +/− SD. ΔamrZ and ΔamrZ csy3::lacZ show no significant difference of EOI relative to WT and WT csy3::lacZ, respectively (ΔamrZ, P = 0.6 ΔamrZ csy3::lacZ, P = 0.08). d, e. Timecourse of the fluorescence levels of Csy1-sfCherry reporter strains during surface-association (d) or planktonic growth (e). ΔamrZ has increased Csy1-sfCherry levels during surface association relative to WT (10 h, P = , 8.9 × 10−4, 15 h, P = 1.5 × 10−3, 20 h, P = 2.0 × 10−2, 25 h, P = 2.2 × 10−4 , 30 h , P = 7.0 × 10−4) f. Normalized fluorescence measurements of WT Cas3-sfCherry (red) or Csy1-sfCherry (yellow) overexpressing the indicated transcription factor after 10 h growth in liquid culture. AmrZ and AlgU overexpression reduced Cas3-sfCherry (AmrZ, P = 1.5 × 10−3, AlgU, P = 7.8 × 10−3) and Csy1-sfCherry (AmrZ, P = 7.5 × 10−6, AlgU, P = 9.9 × 10−5) levels relative to WT. Fluorescence measurements are represented as the mean of 3 biological replicates +/− SD. Two-tailed unpaired Student’s T-test was used to calculate P values, ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001.