Covalent Modifications of the Bacteriophage Genome Confer a Degree of Resistance to Bacterial CRISPR Systems

Abstract

The interplay between defense and counter-defense systems of bacteria and bacteriophages has been driving the evolution of both the organisms leading to their great genetic diversity on the planet. Restriction-modification systems are well-studied defense mechanisms of bacteria, where phages have evolved covalent modifications as a counter-defense mechanism to protect their genomes against restriction. Here, we present evidence that these genome modifications might also have been selected to counter, broadly, the CRISPR-Cas systems, an adaptive bacterial defense mechanism. We found that the phage T4 genome modified by cytosine hydroxymethylation and glucosylation (ghmC) exhibits various degrees of resistance to the type V CRISPR-Cas12a system, producing orders of magnitude more progeny than the T4(C) mutant that contains unmodified cytosines. Furthermore, the progeny accumulated CRISPR-escape mutations allowing rapid evolution of mutant phages under CRISPR pressure. A synergistic effect on phage restriction was observed when two CRISPR-Cas12a complexes were targeted to independent sites on the phage genome, another potential counter mechanism by bacteria to more effectively defend themselves against modified phages. These studies suggest that the defense-counter defense mechanisms by bacteria and phages while affording protection against one another also provide evolutionary benefits for both. IMPORTANCE Restriction-modification (R-M) and CRISPR-Cas systems are two well-known defense mechanisms of bacteria. Both recognize and cleave phage DNA at specific sites while protecting their own genomes. It is well-accepted that T4 and other phages have evolved counter-defense mechanisms to protect their genomes from R-M cleavage by covalent modifications such as the hydroxymethylation and glucosylation of cytosine. However, it is unclear whether such genome modifications also provide broad protection against the CRIPSR-Cas systems. Our results suggest that genome modifications indeed afford resistance against CRISPR systems. However, the resistance is not complete, and also variable, allowing rapid evolution of mutant phages that escape CRISPR pressure. Bacteria in turn could target more than one site on the phage genome to more effectively restrict the infection of ghmC-modified phage. Such defense-counter defense strategies seem to confer survival advantages to both the organisms, one of the possible reasons for their great diversity on the planet.

Allosteric communication in DNA polymerase clamp loaders relies on a critical hydrogen-bonded junction

“…We could not delete these genes using CRISPR-cas9 mediated genome engineering ( Tao et al, 2017 ). Our inability to edit the T4 phage genome with CRISP-cas9 is consistent with the observation that the wild-type T4 genome, with extensive hydroxymethylation and glucosylation, is recalcitrant to cas9 cleavage ( Bryson et al, 2015 ; Liu et al, 2020 ; Tao et al, 2017 ; Vlot et al, 2018 ). We used CRISPR-cas12a since it was reported to efficiently cleave the genome of T4 phage in vitro ( Vlot et al, 2018 ).…”

A Universal Bacteriophage T4 Nanoparticle Platform to Design Multiplex SARS-CoV-2 Vaccine Candidates by CRISPR Engineering

“…First, a series of recombinant phages containing SARS-CoV-2 gene insertions were created in days, by CRISPR genome engineering using a combination of type II Cas9 and type V Cpf1 nucleases. This combination provided built-in choices for spacers as well as for efficient cleavage of T4 genome that is extensively modified by cytosine hydroxymethylation and glycosylation, to attain near 100% success 21, 22 .…”

“…The expression vector pET28b (Novagen, MA) was used for donor plasmid construction and protein expression plasmid construction. The spacer plasmids LbCpf1 and SpCas9 were constructed as described previously 21, 22 . The DNA fragments containing NP and RBD were codon-optimized for E.coli expression and synthesized by GeneArt (Thermo Fisher).…”

“…CRISPR-LbCpf1/SpCas9 plasmid was constructed based on the streptomycin-resistant plasmid DS-SPCas (Addgene no. 48645) as described previously 21, 22 . The spacer containing fragments were prepared by annealing and extension of two amplified DNA fragments containing 26 bp complementary nucleotides (overlap extension PCR).…”

“…The above constitutes an ideal architecture to develop a universal vaccine design template. Further, our four decades of genetic, biochemical, and structural studies on phage T4 including the recently developed CRISPR ( c lustered r egularly i nterspaced s hort p alindromic r epeats) phage engineering 21, 22 provide an extraordinary resource. The atomic structures of all the capsid proteins including Soc, Hoc, as well as the entire capsid have been determined 15, 18, 19, 23, 24 .…”