Student Theses and Dissertations

Date of Award

2023

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Marraffini Laboratory

Abstract

Bacteriophages, or simply phages, are viruses that infect bacteria. They are the most abundant biological entity on our planet and outnumber bacteria 10:1 in the ocean. In response to this threat, bacteria have evolved a diverse battery of immune systems that prevent infection, which in turn has resulted in the development of numerous counter-defense mechanisms by phages. This evolutionary arms race drives molecular innovations and presents exciting avenues for the discovery of new molecular biology and new biotechnology tools, such as restriction enzymes andCRISPR-Cas9. My thesis investigates how mechanisms of DNA repair, specifically recombination and base excision, have been co-opted by phages and bacteria to execute non-canonical immune and counter-immune functions in prokaryotic host-virus genetic conflicts. CRISPR-Cas adaptive immune systems, found in nearly half of all bacteria, use sequence-specific guide RNAs to cleave the genetic material of infecting phages. Bacteria and some phages encode recombination systems that could repair the cleaved viral DNA. At the outset of my PhD thesis, it was unknown whether phages could counteract CRISPR-Cas cleavage of phage DNA by repairing CRISPR-induced DNA breaks. Bacteriophage λ, which infects Escherichia coli, encodes the Red system (gam-exo-bet) to promote recombination between related phages. Here, using molecular genetics and sequencing, I show that λ Red mediates evasion of CRISPR-Cas targeting in E. coli. Gam inhibits the host E. coli RecBCD recombination system, allowing recombination and repair of the cleaved DNA by the phage Exo-Beta exonuclease-recombinase. Repair by Exo-Beta promotes mutations, deletions, and genomic rearrangements within the target sequence in phage DNA to prevent recognition by CRISPR. I find that λ Red recombination is strikingly more efficient than the host’s RecBCD-RecArecombination pathway in the production of large numbers of phages that escape CRISPR targeting. These findings establish recombination-mediated DNA repair as a novel viral “anti-CRISPR” strategy that, rather than binding CRISPR-Cas nucleases and impeding their activity, provides a solution to evade the CRISPR-Cas immune response after it has been set off. While recombinases canonically function in DNA repair, my findings reveal an additional role for Red-like recombination systems in countering bacterial immunity, through the protection of phages against sequence-specific nucleases. Based on these findings, I speculate that the counter-immune advantage imparted by Red-like systems may facilitate their spread across bacteriophage genomes. For the second half of my thesis, I set out to discover novel defense systems in bacteria, specifically those that target viral DNA. To achieve this, I pioneered a new screening methodology to discover anti-phage defense systems from unculturable microorganisms using diverse bacterial metagenomic DNA libraries. These metagenomic libraries contain millions of DNA sequences from different microorganisms that are absent in available genetic databases. While bioinformatic mining has led to the discovery of many new bacterial immune systems, the genetic screening of DNA libraries has the advantage of:(i) examining unsequenced DNA, including the “dark matter” of microbial genomes, and (ii) discovering novel defense systems that cannot be predicted via computational analyses. By subjecting the metagenomic libraries (cloned in E. coli) to phage infection and isolating resistant colonies, I discovered a novel bacterial DNA glycosylase that I named Brig1(bacteriophage replication inhibition DNAglycosylase1). Brig1provides immunity against phages that carry “hypermodified” DNA nucleobases, specifically alpha-glucosyl-hydroxymethylcytosine nucleobases. Brig1 excises these nucleobases from the genome of T-even phages, such as coliphage T4, to generate abasic sites that inhibit DNA replication, which constitutes a novel anti-phage defense mechanism. Many phages have evolved to introduce DNA modifications to avoid recognition and cleavage by bacterial nucleases, including CRISPR-Cas and restriction endonucleases. Brig1 supplies the next step on the bacterial side of the arms race, reestablishing the restriction of phages with modified genomes. Structural predictions suggest that Brig1ispart of a novel family of bacterial anti-phage DNA glycosylases that evolved from uracil DNA glycosylases involved in base excision repair, a pathway that removes mis-incorporated uracil bases from DNA. Interestingly, Brig1homologs are present in multiple phage defense loci across distinct clades of bacteria, and these will be the focus of future studies. Future work will also employ the same metagenomic screening approach, but infecting with phages harboring different DNA modifications, to drive the discovery of new DNA glycosylases, restriction enzymes and DNA repair modules that target modified DNA. Overall, my thesis establishes DNA repair proteins as emerging players in prokaryotic host-virus warfare and will spur future work that explores the co-option of these proteins in varying immune and counter-immune contexts in both bacteria and their viruses.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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