MATERIALS AND METHODSPlasmid and strain constructionAll strains (Supplementary Table S1) are based on KD263 (23) or KD349 (an F-derivative of KD263) that were engineered with a Red recombinase-based procedure (24). Both strains contain a g8 spacer targeting a protospacer in the M13 genome. The same protospacer is present in plasmids with cloned fragments of lytic phage genomes. The cas3 gene expression is driven by the lac promoter in both strains. The remaining cas genes are transcribed from the araB8 promoter.The pG8mut, a pT7Blue-based plasmid carrying a 209-bp M13 fragment with the g8 protospacer (genome positions 1311–1519) with an escape mutation C1T at the first position of the protospacer is described in (25,26) and was used to clone fragments of phage DNA. Phage DNA was purified from lysed cultures by a protocol described in (27).To construct pG8mut-based plasmids containing λ, T5 or T7 fragments, the plasmid was treated with NdeI and ligated with phage DNA digested with MseI. Plasmids containing phage DNA inserts of desired size (2–4 kbp) were selected for further use. To construct pG8mut-based plasmids containing fragments of T4 or R1–37 DNA 2–4 kb regions of phage genomes were amplified using appropriate primers and individually cloned in pG8mut. The sequences of primers are listed in Supplementary Table S2.Construction of KD263 or KD349 derivatives harboring phage targeting CRISPR spacers using plasmids containing cloned phage DNA fragments was performed as described in (25) and as summarized in Supplementary Figure S1. We use the following nomenclature to refer to strains carrying spacers targeting phage protospacers: ‘phage name—temporal class of the gene carrying targeted protospacer, protospacer #—protospacer orientation’. For example, ‘T7-M3-F’ refers, depending on the context, to a strain or a protospacer from phage T7 middle (M) gene in the forward orientation, i.e. the sequence of the spacer in crRNA or the protospacer in phage genome matches the genomic sequence deposited in the Genbank. ‘R’ indicates reverse orientation when crRNA spacer is complementary to the GenBank sequence. To distinguish spacers in different strains and targeted protospacers, they are numbered (in the example above we refer to strain or protospacer number 3 of the four distinct strains targeting middle T7 genes that are present in our collection, see Supplementary Table S1). All phage targeting strains are listed in Supplementary Table S1.CRISPR interference assaysTo assay for plasmid interference, cells were grown in LB medium at 37°C in the presence or in the absence of 1 mM arabinose and 1 mM IPTG until the culture OD600 reached 0.6. Electrocompetent cells were prepared using a standard protocol (28) and transformed with 5 ng of plasmids containing protospacers. After 1-h 37°C outgrowth in 500 μl LB without antibiotic, 100 μl aliquots of serial dilutions of transformation mixtures were plated onto LB agar plates containing 100 μg/ml ampicillin. Plates were incubated at 37°C overnight. For each plasmid, transformation was repeated three times. Efficiency of transformation was determined as a ratio of transformants formed by induced and uninduced cells.To determine interference with phage infection, aliquots of induced and uninduced cultures grown as described above were combined with serial dilutions of phage lysates, combined with soft LB agar and overlaid on LB plates with or without 1 mM arabinose and 1 mM IPTG. Each experiment was repeated at least three times. After drying, plates were incubated at 37°C overnight. The efficiency of plaquing was determined as a ratio of phage plaques formed by induced and uninduced cells.Monitoring phage infectionThe growth of induced and uninduced cultures with or without the phage was continuously monitored in EnSpire Multimode Plate Reader (PerkinElmer). At least three growth curves were determined for each strain at every condition. Induced (1 mM arabinose and 1 mM IPTG) and uninduced cells were grown in LB at 37°C to OD600 ≈ 0.4 and infected with phages at various multiplicities. Cultures growth at 37°C was continued for 2–3 h with intensive aeration.To determine phage burst size, infections were carried out essentially as described in (29). Cultures grown to OD600 0.6 were infected at MOI of 0.1. After 3–7 min at 37°C to allow phage adsorption, cells were separated from unbound phage by centrifugation, resuspended in fresh medium with or without inducers and incubation at 37°C was continued. At various times, aliquots were taken and phage titer was determined.DNA extractionInduced or uninduced cultures were grown to OD600 of 0.6 and infected at MOI of 2. Cells were collected shortly before lysis and DNA was isolated by phenol-chloroform method from cell pellet as described (27). The DNA was next digested with restriction endonucleases recognizing multiple sites in phage DNA and analyzed by agarose electrophoresis.CRISPR adaptation assayInduced cells cultures (OD600 = 0.6) were infected at the MOI of 0.001–1.0 with wild-type or escape phages and grown for 16 h at 37°C. Culture aliquots were withdrawn and cells were subjected to PCR with primers annealing to the CRISPR array leader sequence and to M13 g8 spacer. Amplification reaction products were analyzed as described in (23).High throughput sequencing and data analysisPCR-products corresponding to expanded CRISPR array were subjected to high-throughput sequencing with MiSeq Illumina system. The resulting data were analyzed using ShortRead (30) and BioStrings (31) Bioconductor packages. Sequences located between two CRISPR repeats were considered as spacers. They were mapped on phage genomes with no mismatches allowed. R scripts were used for statistical analysis and Circos (32) was used for graphical representation of the data.To sequence T7 phage genome in infected cells, total DNA was extracted from infected cells (27) several minutes before expected lysis time and treated with restriction endonucleases BamHI, EcoRV, HindIII, PstI (do not recognize T7 DNA) to destroy host DNA. Samples were loaded on an 0.8% agarose gel, resolved by electrophoresis and a high-molecular weight band of phage genomic DNA was extracted from gel with GeneJET Extraction Kit (ThermoFisher Scientific) and sequenced with MiSeq Illumina system. Reads were trimmed and mapped on the phage genome with no mismatches allowed.MicroscopyAll microscopy experiments were performed using Zeiss AxioImager.Z1 upright microscope equipped with a custom made incubation system to maintain cells at 37°C. Semrock mCherry-40LP filter set was used for propidium iodide fluorescence detection. Images were collected using Cascade II 1024 back-illuminated EM-CCD camera (Photometrics). Autofocusing, multichannel and multifield time-lapse acquisition were implemented using MicroManager (33) with custom scripts. Image analyses were performed using Fiji (ImageJ) (34,35).Microscope chambers were prepared using previously described procedure (36) to provide time-lapse imaging. 1.5% agarose (Helicon) diluted in LB (Amresco) was used as a medium to monitor bacterial growth during microscopy. One microscope chamber allowed simultaneous cultivation and monitoring of cells with and without cas gene induction by using two or more separate agarose pads with and without inducers. Cells were mixed with phage and propidium iodide (Molecular Probes), which was added to the final concentration of 20 μM and placed in microscope chamber (0.5 μl on each pad). When cells were absorbed onto agarose pads the chamber was sealed and installed in the microscope. Typically 4–10 fields of view were visualized in a single experiment. Each field was imaged every 5–10 min during 1–4 h in transmitted light channel and propidium iodide fluorescence channel.
Article TitleThe action ofEscherichia coliCRISPR–Cas system on lytic bacteriophages with different lifestyles and development strategies
CRISPR–Cas systems provide prokaryotes with adaptive defense against bacteriophage infections. Given an enormous variety of strategies used by phages to overcome their hosts, one can expect that the efficiency of protective action of CRISPR–Cas systems against different viruses should vary. Here, we created a collection ofEscherichia colistrains with type I-E CRISPR–Cas system targeting various positions in the genomes of bacteriophages λ, T5, T7, T4 and R1-37 and investigated the ability of these strains to resist the infection and acquire additional CRISPR spacers from the infecting phage. We find that the efficiency of CRISPR–Cas targeting by the host is determined by phage life style, the positions of the targeted protospacer within the genome, and the state of phage DNA. The results also suggest that during infection by lytic phages that are susceptible to CRISPR interference, CRISPR–Cas does not act as a true immunity system that saves the infected cell but rather enforces an abortive infection pathway leading to infected cell death with no phage progeny release.