Materials and Methods

Disabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein

MATERIALS AND METHODSBacterial growth and phage propagation. Pseudomonas aeruginosa was grown in LB liquid medium at 37°C with shaking or on LB agar plates at 37°C overnight, unless otherwise indicated. When necessary, media were supplemented with 50 μg/ml gentamicin to maintain the pHERD30T plasmid (30). Plasmids were introduced into P. aeruginosa SMC4386 by electroporation. Induction of the plasmid promoter for overexpression of anti-CRISPR genes was achieved by supplementing medium with 4 mM arabinose.Phages were propagated by mixing with P. aeruginosa SMC4386 ΔCRISPR-cas, a susceptible host. The phage-host mixture was added to LB containing 0.7% agar and 10 mM MgSO4 and poured onto thick LB agar (1.5%) plates containing 10 mM MgSO4. After overnight incubation at 30°C, plates with near-confluent lysis were soaked with SM buffer for 3 h for phage extraction, followed by centrifugation at 14,000 rpm to remove cell and agar debris. Phages were stored in SM buffer over chloroform at 4°C.Phage plaque assays. To measure type I-E CRISPR-Cas activity in P. aeruginosa SMC4386, phage plaque assays were conducted as described previously (4). P. aeruginosa SMC4386 cells containing pHERD30T plasmid expressing AcrE1 were mixed with 0.7% LB agar and overlaid onto LB agar (1.5%) plates containing 10 mM MgSO4, 50 μg/ml gentamicin, and (where indicated) 4 mM arabinose to induce anti-CRISPR gene expression. Tenfold serial dilutions of a CRISPR-targeted phage (JBD8) and a CRISPR-insensitive phage (JBD93a) were spotted on the surface, and the plates were incubated overnight at 30°C.Site-directed mutagenesis. Pairs of complementary oligonucleotides containing the codon to be mutated plus five codons on either side were synthesized by Eurofins Genomics. Pfu DNA polymerase was used to PCR amplify DNA from template plasmid pHERD30T containing the acrE1 gene. After template digestion with DpnI (New England BioLabs NEB), the DNA was concentrated and purified by ethanol precipitation and used to transform E. coli DH5α. Mutations were confirmed by DNA sequencing, and mutant plasmids were introduced into P. aeruginosa SMC4386 for phage plaque assays.AcrE1 expression and protein purification. The AcrE1 open reading frame was cloned by ligation into pET21d(+) with either an N-terminal (NHis) or C-terminal (CHis) noncleavable 6×His tag. E. coli BL21(DE3) cells carrying either plasmid maintained in 100 µg/ml ampicillin were grown to an optical density at 600 nm (OD600) of 0.5 to 0.6, and IPTG (isopropyl-β-d-thiogalactopyranoside) was added to a final concentration of 0.8 mM. After 4 h of shaking incubation at 37°C, cells were pelleted and resuspended in binding buffer (20 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM β-mercaptoethanol) with 5 mM imidazole added. Cells were lysed by sonication, and lysates were cleared by centrifugation for 20 min at 17,000 rpm. Nickel-nitrilotriacetic acid (Ni-NTA) beads (Qiagen) were incubated with the clarified lysate for 30 min at 4°C. Column purification at room temperature was performed with washes in wash buffer (binding buffer plus 30 mM imidazole), and the protein was eluted in elution buffer (binding buffer plus 300 mM imidazole). Binding buffer was used for overnight dialysis at room temperature. The protein was further purified by size exclusion chromatography (SEC) using either a Superdex 200 16/60 column (for large-scale purification) or a Superdex 75 10/30 column (for analytical SEC prior to CD spectroscopy) in binding buffer. The same purification methods and buffers were used to purify AcrE1Δ91–100 NHis. Selenomethionine (Se-Met)-labeled CHis AcrE1 was expressed using the methionine-auxotrophic E. coli BL21(DE3) B834 strain cultured in M9 minimal medium containing 0.2% glucose and trace metals supplemented with Se-Met and was purified using the same protocol as described above.Crystallization of AcrE1. Purified AcrE1 CHis or NHis (native) was initially screened with a 1:1 (protein/precipitant) ratio against the MCSG commercial suite and JCSG+ commercial screen using sitting drop vapor diffusion at 10 mg/ml. AcrE1 CHis crystals were observed in 0.1 M sodium cacodylate buffer, pH 6.0, 0.1 M NaBr, and 25% polyethylene glycol (PEG) 3350. The crystals were further optimized with a 1:1-ratio sitting drop at 20°C under a precipitant condition composed of 0.07 M sodium cacodylate, pH 6.1, 0.5 M NaBr, 27% PEG 3350, and 15% glycerol, yielding single crystals in space group C2. Native NHis AcrE1 crystals were observed in 0.2 M ammonium citrate dibasic, 20% (wt/vol) PEG 3350. The crystals were further optimized with a 1:1-ratio sitting drop at 20°C under a precipitant condition composed of 0.2 M ammonium citrate dibasic, pH 7.0, 25% (wt/vol) PEG 3350, and 15% glycerol, yielding single crystals in space group C2.Data collection and structure determination. CHis and NHis AcrE1 crystallographic data were collected on crystals frozen at 105 K on 08B1-1 and 08ID-1 beamlines at Canadian Light Source (CLS), respectively. Diffraction data from a total of 360 images were collected at wavelengths of 0.9199 and 0.9795 using 1° oscillations. Data were processed with the XDS package to a resolution of 2.5 and 2.0 Å for CHis and NHis AcrE1, respectively. A complete model for CHis was solved by Br-SAD with additional anomalous signal from Se atoms using Phenix AutoSol. The final structure was obtained after multiple rounds of refinements and building cycles using Phenix Refine and Coot software packages, yielding a final Rwork/Rfree of 0.18/0.22. The structure of NHis AcrE1 was obtained by molecular replacement using CHis as a starting model using Phaser. The final model was generated after several rounds of model building and refinement using Coot and the Phenix Refine program using TLS (translation, liberation, screw), yielding a final Rwork/Rfree of 0.18/0.23 for native NHis AcrE1.CD spectroscopy. Circular dichroism (CD) was conducted using an 0.1-cm quartz cuvette at 25°C with a measurement range of 260 to 200 nm. The scanning speed was 50 nm/min with a bandwidth of 1 nm, response time of 2 s, and data pitch of 1.0 nm. Protein concentrations for both wild-type and mutant His-tagged AcrE1 were 16 µM in buffer containing 20 mM Tris-HCl, pH 7.5, 200 mM NaCl, and 5 mM β-mercaptoethanol. Triplicate measurements were recorded and averaged for each CD run. Three biological replicates were collected.In vivo formaldehyde cross-linking and affinity purification. One-hundred-milliliter cultures of P. aeruginosa strain SMC4386 containing plasmids encoding 6×His-tagged anti-CRISPR AcrE1 or empty vector were grown at 37°C in LB medium. At an OD600 of 0.5, 4 mM arabinose was used to induce plasmid expression, except for the uninduced samples in Fig. 3b, to which no arabinose was added. After a 2-h incubation, 200 μl of 37% formaldehyde solution was added, and cells were incubated with shaking for an additional 30 min at 37°C. Glycine was added to a final concentration of 100 mM to quench the cross-linking reaction, and cells were pelleted. One milliliter of Novagen BugBuster reagent was used to resuspend the cell pellet, followed by 1 h of shaking incubation at 25°C and centrifugation at 14,000 rpm for 5 min. We added 50 μl Ni-NTA beads (Qiagen) to each clarified lysate and purified with the same buffer system as described for AcrE1 purification above. One hundred microliters of elution buffer was added to the washed beads and incubated at 100°C for 15 min to elute proteins and reverse heat-labile formaldehyde cross-links (26). Proteins were analyzed by SDS-PAGE followed by silver staining and by SDS-PAGE followed by anti-His-tag Western blotting, as well as by trypsin digest followed by linear trap quadrupole (LTQ) (Thermo Scientific) liquid chromatography-tandem mass spectrometry (LC-MS/MS) (SPARC Biocentre, SickKids, Toronto, Canada).Transcriptional repression assay. Pairs of complementary oligonucleotides were designed to encode a CRISPR repeat-spacer-repeat unit in which the repeat sequence is the P. aeruginosa SMC4386 consensus repeat (5′-GTGTTCCCCACATGCGTGGGGATGAACCG-3′) and the spacer sequence matches a 32-nucleotide sequence in the phzM promoter element flanked by a consensus protospacer adjacent motif (PAM), 5′-NTT-3′. The sense-strand crRNA sequences (with repeats in lowercase and spacer in uppercase) used in this study are crRNA 1 (5′-gtgttccccacatgcgtggggatgaaccgAATAAAATTACAACTTGGCTACAACCTCCGGCgtgttccccacatgcgtggggatgaaccg-3′) and crRNA 2 (5′-gtgttccccacatgcgtggggatgaaccgCTGATGCTTCCTGCAATGCCGGAGGTTGTAGCgtgttccccacatgcgtggggatgaaccg-3′). Once the oligonucleotides were annealed by heating to 95°C and slow cooling to 10°C, sticky ends were formed that could ligate into pHERD30T predigested with EcoRI and HindIII restriction endonucleases. Ligated plasmids were sequence confirmed and then used to transform SMC4386 cells by electroporation. A dramatic reduction in transformation efficiency of the crRNA-containing plasmids compared to the empty vector indicated that the crRNAs were expressed and processed and successfully targeted the CRISPR-Cas system to the host genome. The indicated mutant or lysogenic strains of SMC4386 were transformed with the crRNA plasmid or empty vector. To ensure that the observed phenotypes were not due to the prophage insertion site (which, for these phages, is random), all results were confirmed using a second, independently isolated lysogen (data not shown). Colonies were used to inoculate King’s A liquid medium (20 g/liter peptone, 10 g/liter potassium sulfate, 1.64 g/liter anhydrous magnesium chloride, and 1% glycerol) supplemented with 50 µg/ml gentamicin to maintain pHERD30T and grown at 37°C. After 10 h of growth, plasmid expression of crRNAs was induced by the addition of 4 mM arabinose, and induction was allowed to proceed for 16 h at 37°C.Construction of SMC4386 cas3 H89A/D90A mutant. The cas3 gene was cloned by ligation into pHERD30T (30), and site-directed mutagenesis was performed to introduce the double mutation of H89A and D90A, which comprise the active site residues of the HD nuclease domain of Cas3. The resultant plasmid was used to transform P. aeruginosa SMC4386, and cells were grown in LB under 50-μg/ml gentamicin selection for 48 h and then passaged in antibiotic-free LB medium for 72 h to cause plasmid loss. A type I-E CRISPR locus containing spacers targeting highly conserved regions of several essential genes in P. aeruginosa, including RNA polymerase and DNA polymerase, was ligated into pHERD20T, a plasmid identical to pHERD30T but with a carbenicillin (instead of gentamicin) resistance cassette. This plasmid was used to select for cells in which recombination of the mutated cas3 gene had occurred and CRISPR-Cas activity was lost due to the lack of nuclease function. The plasmid should not be able to transform cells with an active CRISPR-Cas system, as they would target and cleave their own genome at several key locations that are, in combination, unescapable by mutation. Sequencing was used to confirm that the correct mutation in cas3 had caused the loss of CRISPR-Cas activity.

Article TitleDisabling a Type I-E CRISPR-Cas Nuclease with a Bacteriophage-Encoded Anti-CRISPR Protein

Abstract

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas adaptive immune systems are prevalent defense mechanisms in bacteria and archaea. They provide sequence-specific detection and neutralization of foreign nucleic acids such as bacteriophages and plasmids. One mechanism by which phages and other mobile genetic elements are able to overcome the CRISPR-Cas system is through the expression of anti-CRISPR proteins. Over 20 different families of anti-CRISPR proteins have been described, each of which inhibits a particular type of CRISPR-Cas system. In this work, we determined the structure of type I-E anti-CRISPR protein AcrE1 by X-ray crystallography. We show that AcrE1 binds to the CRISPR-associated helicase/nuclease Cas3 and that the C-terminal region of the anti-CRISPR protein is important for its inhibitory activity. We further show that AcrE1 can convert the endogenous type I-E CRISPR system into a programmable transcriptional repressor.


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