MATERIALS AND METHODSBacterial and phage growth. All bacterial and phage growth was performed as previously described (9). Phage lysogens in P. aeruginosa strain SMC4386 were created by streaking cells from the center of a phage plaque to single colonies. The presence of a prophage was confirmed by the development of resistance to superinfection by the phage in question and the production of that phage from the lysogenic strain.Transformation efficiency assay. Oligonucleotides encoding 32-nucleotide CRISPR spacers with five nucleotides upstream and downstream (encompassing the PAM sequence), flanked by NcoI and HindIII sites were synthesized and cloned into pHERD30T (22). Known concentrations of each of the protospacer-containing plasmids were used to transform P. aeruginosa via electroporation. One milliliter of overnight culture was collected by centrifugation, washed twice with 300 mM sucrose, and resuspended in 100 µl of sucrose. Aliquots of cells (100 µl) were transformed with 15 to 50 ng of plasmid DNA, diluted in 1 ml Luria broth, and incubated for 40 min at 37°C to allow for recovery. Serial dilutions of each transformation reaction were plated on LB agar containing gentamicin (50 µg/ml) and incubated overnight at 37°C. The following day, the colonies were counted and normalized according to the dilution factor and amount of plasmid DNA introduced during electroporation. The data were expressed as a percentage of colonies transformed with the empty-vector pHERD30T in the same experiment.P. aeruginosa plaque assays. One hundred fifty microliters of overnight culture grown in LB was added to 3 ml LB agar containing 10 mM MgSO4, 1.5% agar, and top plated on LB agar plates containing 50 µg/ml gentamicin, 10 mM MgSO4, and 0.7% agar. Tenfold serial dilutions of phage lysates were spotted on the surface. The plates were incubated overnight at 30°C, and the numbers of plaques were counted.E. coli M13 plaque assays. E. coli strains BW40114 and BW40119 contain an isopropyl-β-d-thiogalactopyranoside (IPTG)- and arabinose-inducible type I-E CRISPR-Cas system, and strain BW40119 contains a crRNA spacer that targets phage M13 (14). These cells were transformed with pHERD30T plasmids expressing each anti-CRISPR gene. Overnight cultures were diluted 1:100 into LB with 15 µg/ml gentamicin, grown to an optical density at 600 nm (OD600) of 0.6, and then induced with both 1 mM arabinose and 1 mM IPTG. After 3-h induction, cells were pelleted by centrifugation, resuspended in 3 ml soft agar, and then poured onto thick LB agar plates containing 1 mM arabinose and 1 mM IPTG. Serial dilutions of M13 phage were spotted on the surface. After incubation overnight at 30°C, plaques visible at the lowest dilution were counted, and the efficiency of plating (EOP) was calculated for each anti-CRISPR construct and for the pHERD30T empty vector in both strains.Confirmation of anti-CRISPR expression in E. coli. E. coli strain BW40119 (14) transformed with various anti-CRISPR constructs in pHERD30T were subcultured 1:100 into LB containing the appropriate antibiotic, grown for 4 h, and then either induced with 3 mM arabinose or inhibited with 0.2% glucose. After 2 h of induction, 1 ml of culture was collected by centrifugation, and cells were resuspended in 100 µl SDS running buffer and analyzed by SDS-PAGE on a 15% Tris-Tricine gel, followed by Coomassie staining.
Article TitleA New Group of Phage Anti-CRISPR Genes Inhibits the Type I-E CRISPR-Cas System ofPseudomonas aeruginosa
CRISPR-Cas systems are one of the most widespread phage resistance mechanisms in prokaryotes. Our lab recently identified the first examples of phage-borne anti-CRISPR genes that encode protein inhibitors of the type I-F CRISPR-Cas system ofPseudomonas aeruginosa. A key question arising from this work was whether there are other types of anti-CRISPR genes. In the current work, we address this question by demonstrating that some of the same phages carrying type I-F anti-CRISPR genes also possess genes that mediate inhibition of the type I-E CRISPR-Cas system ofP. aeruginosa. We have discovered four distinct families of these type I-E anti-CRISPR genes. These genes do not inhibit the type I-F CRISPR-Cas system ofP. aeruginosaor the type I-E system ofEscherichia coli. Type I-E and I-F anti-CRISPR genes are located at the same position in the genomes of a large group of relatedP. aeruginosaphages, yet they are found in a variety of combinations and arrangements. We have also identified functional anti-CRISPR genes within nonprophagePseudomonasgenomic regions that are likely mobile genetic elements. This work emphasizes the potential importance of anti-CRISPR genes in phage evolution and lateral gene transfer and supports the hypothesis that more undiscovered families of anti-CRISPR genes exist. Finally, we provide the first demonstration that the type I-E CRISPR-Cas system ofP. aeruginosais naturally active without genetic manipulation, which contrasts withE. coliand other previously characterized I-E systems.