Bacteria and growth conditions
Bacteria (Table S4) were cultured in lysogeny broth36 at 37°C. pCA24N-based plasmids37 were retained in overnight cultures via chloramphenicol (30 μg/mL), and kanamycin (50 μg/mL) was used for deletion mutants, where applicable.
To attempt to delete the CRISPR-Cas spacer region, the one-step inactivation method for single gene deletions38 was utilized in which primers that included the sequences flanking the spacer region (Table S5) were used with plasmid pKD4 to allow for insertion of the kanamycin resistance cassette (1.5 kb) and FRT sites.
Exponentially-growing cells (turbidity of 0.8 at 600 nm) were converted nearly completely to persister cells18,20 by adding rifampicin (100 μg/mL) for 30 min to stop transcription, centrifuging, and adding LB with ampicillin (100 μg/mL) for 3 h to lyse non-persister cells. To remove ampicillin, cells were washed twice with 0.85% NaCl then re-suspended in 0.85% NaCl. Persister concentrations were enumerated via a drop assay39.
Single-cell persister resuscitation
Persister cells (5 μL) were added to 1.5% agarose gel pads containing M9 glucose (0.4 wt%) medium40, and single-cell resuscitation was visualized at 37°C via a light microscope (Zeiss Axio Scope.A1, bl_ph channel at 1000 ms exposure). For each condition, at least two independent cultures were used with 150 to 300 individual cells used per culture.
Membrane integrity assay
To determine membrane integrity, the persister cells were analyzed with the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Inc., Eugene, OR, catalog number L7012). The fluorescence signal was analyzed via a Zeiss Axioscope.A1 using excitation at 485 nm and emission at 530 nm for green fluorescence and using excitation at 485 nm and emission at 630 nm for red fluorescence.
To quantify transcription from the cryptic prophage lytic genes, RNA was isolated from persister cells that were resuscitated by adding M9 glucose (0.4%) medium for 10 min then washed with 0.85% NaCl and from exponential cells grown to a turbidity of 0.8; samples were cooled rapidly using ethanol/dry ice in the presence of RNA Later. RNA was isolated using the High Pure RNA Isolation Kit (Roche). The following qRT-PCR thermocycling protocol was used with the iTaq™ universal SYBR® Green One-Step kit (Bio-Rad): 95 °C for 5 min; 40 cycles of 95 °C for 15 s, 60 °C for 1 min for two replicate reactions for each sample/primer pair. The annealing temperature was 60°C for all primers (Table S5).
To quantify prophage excision and the levels of DNA flanking the CRISPR-Cas cleavage sites, total DNA (100 ng) was isolated from exponentially-growing and persister resuscitating cells using an UltraClean Microbial DNA Isolation Kit (Mo Bio Laboratories). Excised cryptic prophage was quantified using primers for each prophage excisionase (Table S5) that only yield a PCR product upon prophage excision, and the relative amount of each target gene was determined using reference gene purM. The level of cryptic prophage flanking the CRISPR-Cas cleave site was quantified using primers that flank each site (Table S5). The qPCR reaction performed using CFX96 Real Time System. The reaction and analysis was conducted using the StepOne Real-Time PCR System (Bio-Rad).
Article TitleCRISPR-Cas Controls Cryptic Prophages
The bacterial archetypal adaptive immune system, CRISPR-Cas, is thought to be non-functional in the best-studied bacterium, Escherichia coli K-12. Instead, we demonstrate here that the E. coli CRISPR-Cas system is active and inhibits its nine defective (i.e., cryptic) prophages. Specifically, deactivation of CRISPR-Cas via deletion of cas2, which encodes one of the two conserved CRISPR-Cas proteins, reduces growth by 40%, increases cell death by 700%, and prevents persister cell resuscitation; hence, CRISPR-Cas serves to inhibit the remaining deleterious effects of these cryptic prophages. Consistently, seven of the 13 E. coli spacers contain matches to the cryptic prophages, and, after excision, CRISPR-Cas cleaves cryptic prophage CP4-57 and DLP-12 DNA. Moreover, we determine that the key genes in these cryptic prophages that CRISPR-Cas represses by cleaving the excised DNA include lysis protein YdfD of Qin and lysis protein RzoD of DLP-12. Therefore, we report the novel results that (i) CRISPR-Cas is active in E. coli and (ii) CRISPR-Cas is used to tame cryptic prophages; i.e., unlike with active lysogens, CRISPR-Cas and cryptic prophages may stably exist.