A Jumbo Phage Forms a Nucleus-like Compartment to Evade Bacterial Defense Systems

T battle for domination between bacteria and bacteriophages has resulted in innovative defense mechanisms and transformative molecules, including CRISPR/Cas9 and restriction enzymes. Bacteria deploy CRISPR proteins to combat phage infection, and phages have evolved to counter these using anti-CRISPR proteins. In their recent paper, Mendoza and colleagues note that φKZ, a “jumbo phage” with >200 kb DNA, does not contain any anti-CRISPR genes, which is surprising considering their large genome. This prompted the question of how φKZ evades the bacterial defense systems. The authors demonstrated that φKZ is resistant to a variety of CRISPR-Cas endonucleases and restriction endonucleases. They found that although φKZ contains cut sites for these enzymes, these cut sites cannot be accessed by enzymes when inside the host Pseudomonas aeruginosa because the φKZ DNA is segregated by a nucleus-like compartment (Figure 1). This nucleus-like compartment is an assembled protein barrier around the genome that protects the phage DNA, a finding in line with other reports that the phage DNA is surrounded by proteins in jumbo phages. The authors found that φKZ is resistant to multiple subtypes of CRISPR-Cas enzymes (Cas3, Cas9, and Cas12) and restriction endonucleases (types I and II, HsdRMS and EcoRI, respectively). In the presence of CRISPR enzymes and single-guide RNAs targeting the φKZ DNA, φKZ could still infect P. aeruginosa, while the control phage JBD30 could not. Similarly, in the presence of restriction endonucleases HsdRMS and EcoRI, φKZ was still infectious while JBD30 was not. Localization studies on CRISPR enzymes revealed that these endonucleases do not localize to the phage DNA, though phage protein ORF152 and host protein topoisomerase I do. Questions about what other phage or host proteins reside in or near this core and what factors determine the selectivity of access to this nucleus-like compartment remain. To confirm that the phage DNA is not protected from enzymatic cleavage outside the protective core, the authors performed in vitro assays with extracted phage DNA. The extracted DNA was susceptible to cleavage by Cas9 and several restriction enzymes, including EcoRI. To “go undercover” to cut the DNA in cellulo, they rationally fused the phage protein ORF152, which resides within the core, with bacterial EcoRI to generate a fluorescently tagged EcoRI-ORF152. As visualized by fluorescence imaging, the fusion protein entered the core of φKZ-infected cells, whereas EcoRI alone was occluded. ORF152 fused to wild type EcoRI effectively reduced the phage infectivity, while phage treated with a fusion containing the catalytically dead mutant EcoRI (E111G) remained