Methods

A chimeric nuclease substitutes CRISPR-Cas: A phage weaponizes laterally acquired specificity to destroy subviral parasites
chimeric nuclease CRISPR/CASMolecular Biology

Strains and Culture conditions

V. cholerae strains used in this study are derived from E7946. Bacteria were routinely grown on LB agar plates and in LB broth with aeration at 37°C. Antibiotics were supplemented as appropriate at the following concentrations: 75 μg/ml kanamycin, 100 μg/ml spectinomycin, 1.25 or 2.5 μg/ml chloramphenicol (V. cholerae for broth or plate conditions, respectively), 25 μg/ml chloramphenicol (E. coli), 100 μg/ml streptomycin. A detailed list of all strains used throughout this study can be found in the Key Resource table.

Phage titers were determined using a soft agar overlay method wherein ICP1 was allowed to adsorb to V. cholerae for 10 min at room temperature before the mixture was added to molten LB soft agar (0.5%) and poured onto 100 mm × 15 mm LB agar plates. Plaques were counted after overnight incubation at 37°C. Prior to phage infection for purposes of quantification or qPCR or spot assay analysis, V. cholerae was grown on plates overnight and then inoculated into 2mL LB liquid cultures. Liquid cultures were grown to an OD greater than 1, then back diluted in fresh media to OD600 = 0.05, and then grown to OD600 = 0.3, at which point they were infected.

Generation of Mutant Strains and Constructs

V. cholerae mutants were generated through natural transformation as described previously (Dalia et al., 2014). For gene knockouts, splicing by overlap extension (SOE) PCR was used to generate deletion constructs with a spectinomycin resistance cassette flanked by frt recombination sites. Following selection of spectinomycin resistant mutants, a plasmid bearing an IPTG inducible Flp recombinase was mated into transformants and Flp expression was induced to generate in-frame deletions. The plasmid was cured by growing mutants under inducing conditions with 300μg/ml streptomycin. For unmarked replication origin swapped constructs, mutants were generated through natural transformation by cotransformation (Dalia et al., 2014). For plasmid expression constructs, a derivative of the pMMB67EH vector with a theophylline inducible riboswitch was used as previously described (McKitterick and Seed, 2018). All constructs were confirmed with DNA sequencing over the region of interest and primer sequences are available upon request.

Phage infection spot assays

V. cholerae was added to molten 0.5% LB top agar and poured over LB plates. Following solidification of the top agar, 3μL of serially 10-fold diluted phage were spotted onto the plate. Once phage spots dried, plates were incubated for at 37°C for 2 hours and then overnight at room temperature before visualization.

Real time quantitative PCR

qPCR experiments were performed as previously described (Barth et al., 2020b; O’Hara et al., 2017). Briefly, liquid cultures were infected with ICP1 at a multiplicity of infection (MOI) of 2.5 at OD600 = 0.3. Samples were taken at 0 and 20 minutes (min) post-infection and boiled before serving as templates for IQ SYBR (Biorad) qPCR reactions. For assays involving induction of repA, 2 ml cultures were grown with 1.25 μg/ml chloramphenicol for plasmid maintenance and induced for 20 min prior to infection using a final concentration of 1.5 mM theophylline and 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) starting at OD600 = 0.17. All conditions were tested in biological triplicate, and each reported data point is the mean of two technical replicates. A single primer set (Key Resources table) which amplifies a conserved region in all PLEs was used to detect PLE replication by qPCR.

Protein purification

E. coli BL21 cells containing a His6-SUMO fusion to WT or E185A Gp88 were grown to OD600 = 0.5 at 37°C and induced with IPTG to a final concentration of 0.5 mM. The culture was grown for two hours and harvested by centrifugation at 4000xg for 20 min. The pellet was resuspended in lysis buffer (50 mM Tris–HCl pH 8, 200 mM NaCl, 1 mM BME, 0.5% Triton-X 50 mM imidazole, 1 Pierce™ Protease Inhibitor Mini Tablet (Thermo Scientific) and sonicated. Cell debris was removed by centrifugation (29 097xg for 40 min). The lysate was applied to a HisTrap HP column (Cytiva). The column was washed with wash buffer (50 mM Tris–HCl pH 8, 200 mM NaCl, 1 mM BME, 50 mM imidazole) and a high salt wash (50 mM Tris–HCl pH 8, 2 M NaCl, 1 mM BME, 50 mM imidazole) was used to remove residual DNA. The protein was eluted using an elution buffer (50 mM Tris–HCl pH 8, 200 mM NaCl, 1 mM BME, 300 mM imidazole), and then the eluate was applied to a HiTrap Heparin HP column (Cytivia) for further purification. Following elution from the HiTrap Heparin colum, the protein was dialyzed using a 10k Slide-A-Lyzer Dialysis cassette (ThermoFischer) in 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1 mM Dithiothreitol (DTT). Concomitant with dialysis, the His6-SUMO tag was cleaved using SUMO protease. The SUMO tag was removed using Dynabeads (Invitrogen).

Sequence analysis

All genomes were visualized and compared in CLC Main Workbench 7. Multiple sequence alignments were performed using the Multiple Sequence Alignment (MUSCLE) tool with default settings (Edgar, 2004). Conservation of PLE sequence was compared and visualized using Mauve (Darling et al., 2004).

Nuclease assays

Nuclease assays were performed with 100ng of DNA probes and up to 500nM of purified Gp88 in 20μL reactions with 50mM Tris, 10mM MgCl2, 50mM NaCl, 1mM DTT reaction buffer. Reactions proceeded at 30°C for 30 minutes, and were visualized on 0.8% agarose gels ran at 80V for 30 minutes, and stained with GelRed (Biotium). For smaller probes (Figure 6), 25ng of probe was included in reactions, and the product was visualized on 2% agarose gels ran at 120V for 20 minutes and stained with GelGreen (Biotium). Primers used for probe amplification can be found in the Key Resource table..

Article TitleA chimeric nuclease substitutes CRISPR-Cas: A phage weaponizes laterally acquired specificity to destroy subviral parasites

Abstract

Mobile genetic elements, elements that can move horizontally between genomes, have profound effects on their hosts fitness. The PLE is a mobile element that integrates into the chromosome of Vibrio cholerae and parasitizes the bacteriophage ICP1 to move between cells. This parasitism by PLE is such that it abolishes the production of ICP1 progeny and provides a defensive boon to the host cell population. In response to the severe parasitism imposed by PLE, ICP1 has acquired an adaptive CRISPR-Cas system that targets the PLE genome during infection. However, ICP1 isolates that naturally lack CRISPR-Cas are still able to overcome certain PLE variants, and the mechanism of this immunity against PLE has thus far remained unknown. Here we show that ICP1 isolates that lack CRISPR-Cas encode an endonuclease in the same locus, and that the endonuclease provides ICP1 with immunity to a subset of PLEs. Further analysis shows that this endonuclease is of chimeric origin, incorporating a DNA binding domain that is highly similar to some PLE replication origin binding proteins. This similarity allows the endonuclease to bind and cleave PLE origins of replication. The endonuclease appears to exert considerable selective pressure on PLEs and may drive PLE replication module swapping and origin restructuring as mechanisms of escape. This work demonstrates that new genome defense systems can arise through domain shuffling and provides a greater understanding of the evolutionary forces driving genome modularity and temporal succession in mobile elements.


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