Landscape of New Nuclease-Containing Antiphage Systems in Escherichia coli and the Counterdefense Roles of Bacteriophage T4 Genome Modifications

Abstract

Many phages, such as T4, protect their genomes against the nucleases of bacterial restriction-modification (R-M) and CRISPR-Cas systems through covalent modification of their genomes. Recent studies have revealed many novel nuclease-containing antiphage systems, raising the question of the role of phage genome modifications in countering these systems. Here, by focusing on phage T4 and its host Escherichia coli, we depicted the landscape of the new nuclease-containing systems in E. coli and demonstrated the roles of T4 genome modifications in countering these systems. Our analysis identified at least 17 nuclease-containing defense systems in E. coli, with type III Druantia being the most abundant system, followed by Zorya, Septu, Gabija, AVAST type 4, and qatABCD. Of these, 8 nuclease-containing systems were found to be active against phage T4 infection. During T4 replication in E. coli, 5-hydroxymethyl dCTP is incorporated into the newly synthesized DNA instead of dCTP. The 5-hydroxymethylcytosines (hmCs) are further modified by glycosylation to form glucosyl-5-hydroxymethylcytosine (ghmC). Our data showed that the ghmC modification of the T4 genome abolished the defense activities of Gabija, Shedu, Restriction-like, type III Druantia, and qatABCD systems. The anti-phage T4 activities of the last two systems can also be counteracted by hmC modification. Interestingly, the Restriction-like system specifically restricts phage T4 containing an hmC-modified genome. The ghmC modification cannot abolish the anti-phage T4 activities of Septu, SspBCDE, and mzaABCDE, although it reduces their efficiency. Our study reveals the multidimensional defense strategies of E. coli nuclease-containing systems and the complex roles of T4 genomic modification in countering these defense systems. IMPORTANCE Cleavage of foreign DNA is a well-known mechanism used by bacteria to protect themselves from phage infections. Two well-known bacterial defense systems, R-M and CRISPR-Cas, both contain nucleases that cleave the phage genomes through specific mechanisms. However, phages have evolved different strategies to modify their genomes to prevent cleavage. Recent studies have revealed many novel nuclease-containing antiphage systems from various bacteria and archaea. However, no studies have systematically investigated the nuclease-containing antiphage systems of a specific bacterial species. In addition, the role of phage genome modifications in countering these systems remains unknown. Here, by focusing on phage T4 and its host Escherichia coli, we depicted the landscape of the new nuclease-containing systems in E. coli using all 2,289 genomes available in NCBI. Our studies reveal the multidimensional defense strategies of E. coli nuclease-containing systems and the complex roles of genomic modification of phage T4 in countering these defense systems.

Keywords: E. coli defense systems; bacteriophage; genome modifications; phage T4.

FIG 1

Identification of antiphage systems with nuclease domains in E. coli. (A and B) Identification of E. coli Gabija, Septu, Shedu, mzaABCDE, and type III Druantia systems. The domain organization (A) of each system is shown, and their defense activities (B) were determined using E. coli MG1655 against 111 different phages as described in Materials and Methods. (C) The abundance of all 17 experimentally confirmed defense systems containing nuclease domains in the 2,289 E. coli genomes.

FIG 2

Genome modifications of phage T4 cannot abolish the antiphage activities of Septu, SspBCDE, and mzaABCDE. (A) Genetic compositions and defense activities of E. coli Septu, SspBCDE, and mzaABCDE systems. The nuclease domains of each system are highlighted in red, and the putative active sites or the conserved amino acids in the nuclease domain are indicated with red stars. The representative results of plaque assays show the defense activities of each system against T4 (ghmC), T4 (hmC), and T4 (C). The number of phages (10¹ to 10⁶ PFU) used for plaque assays is indicated on top of each panel. (B to D) The antiphage activities of wild-type and mutated Septu (B), SspBCDE (C), and mzaABCDE (D) systems against phages T4 (ghmC), T4 (hmC), and T4 (C) are presented as fold reduction in efficiency of plating (EOP) (see Materials and Methods for the details). Data are represented as mean ± SD from three independent assays. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (analysis of variance).

FIG 3

Glucosylation of hmC in phage T4 genome abolishes Gabija and Shedu defense activities. (A and B) Genetic compositions and defense activities of E. coli Gabija (A) and Shedu (B) against T4 (ghmC), T4 (hmC), and T4 (C). The nuclease domains are highlighted in red, and the putative active sites in the nuclease domain are indicated with red stars. The representative results of plaque assays are shown. (C and D) The antiphage activities of wild-type (WT) and mutated Gabija (C) and Shedu (D) against phages T4 (hmC) and T4 (C) are presented as fold reduction in EOP compared to the empty vector control. Data are represented as mean ± SD. **, P < 0.01; ****, P < 0.0001 (Student’s t test).

FIG 4

The Restriction-like defense system specifically targets phage T4 (hmC). (A) The cartoon shows the genetic composition and protein domain of the Restriction-like system. The main functional domains are indicated in different colors. (B) The representative results of plaque assays show the defense activity of the Restriction-like system against T4 (ghmC), T4 (hmC), and T4 (C). The number of phages (10¹ to 10⁶ PFU) used for plaque assays is indicated on top of each panel. (C) Phage plaque assays on E. coli DH10B cells expressing wild-type or mutated Restriction-like systems. E. coli cells containing empty vectors were used as controls. The mutation sites Q109A, G132A, K135A, D230A, H233A, and T269A in the nuclease domain are indicated.

FIG 5

Druantia and qatABCD systems confer protection only against phage T4 (C). (A and B) The genetic composition and defense activities of E. coli Druantia (A) and qatABCD (B) against T4 (ghmC), T4 (hmC), and T4 (C). The genetic composition of each system, the nuclease domains (highlighted in red), and the putative nuclease active sites (red stars) are shown on top of each panel. The representative results of plaque assays are shown at the bottom. (C and D) The defense activities of wild-type and mutated Druantia (C) and qatABCD (D) against phage T4 (C) are presented as fold reduction in EOP compared to the empty vector control. Data are represented as mean ± SD from three independent assays. **, P < 0.01; ****, P < 0.0001 (Student’s test or analysis of variance).

FIG 6

E. coli Zorya, hhe, ppl, AVAST type 4, Retron Ec78, and Retron Ec67 systems lack defense activities against phage T4. (A) The genetic composition and defense activities of each system against phages T4 (ghmC), T4 (hmC), and T4 (C). The genetic compositions, the main functional domains, and the putative nuclease active sites (red stars) are shown on the left side. The antiphage activities were determined by plaque assays, and representative results are shown. (B to F) Antiphage activities of wild type and mutants of each system against phage T7 or phage #76. Data are represented as mean ± SD from three independent assays. *, P < 0.05; **, P < 0.01; ***, P < 0.001 (Student’s test or analysis of variance).