Overview

Currently, bacterial diseases cause annual crop yield losses of 20%-30% worldwide. Traditional pesticide-based control methods are associated with issues such as environmental pollution and the development of bacterial drug resistance. In recent years, bacteriophages have emerged as a research hotspot in the field of biological pesticides, owing to their highly specific ability to lyse target bacteria. However, the direct development of natural bacteriophages into biological agents is confronted with challenges, including lengthy screening cycles, unstable efficacy, and difficulties in standardized production.

To overcome the limitations of natural bacteriophages, we propose an innovative solution—large-scale production of phage-like particles in a controllable chassis organism, which retain the key structures for precise phage recognition and bacterial invasion but lack the ability to replicate autonomously in host bacteria or lyse bacteria. This approach combines the targeting advantages of phages with the controllability and safety of bioengineered products, offering the potential for precise, efficient, and controllable elimination of target pathogens.

We selected Escherichia coli MG1655—a strain derived from the genetically well-characterized E. coli K-12—as the chassis organism. E. coli MG1655 closely approximates the natural state of bacteria and is amenable to genetic modification, which enhances the reproducibility and universality of experimental results. Concurrently, we selected the T7 bacteriophage genome for modification. The T7 phage genome is a linear double-stranded DNA molecule, has been fully sequenced, and can be stably maintained within the chassis strain, ensuring the efficient and controllable production of phage-like particles.

System design and construction

Natural bacteriophages release large numbers of progeny phages while efficiently lysing host bacteria, posing risks such as uncontrollable titer and horizontal gene transfer. To achieve a safer and more controllable precision antibacterial strategy, we plan to produce phage-like particles that encapsulate toxic plasmid DNA.

Replacing the T7 genome with DNA concatemer constructed from toxic plasmid DNA to enable controlled toxin delivery

To address the uncontrollable self-replication of natural phages, we replace the natural genome within the head coat of wild-type T7 phages with a ~40 kb DNA fragment generated via rolling circle replication of toxic plasmid DNA. The resulting phage-like particles retain the ability to infect target bacteria and deliver toxin genes, leading to bacterial death. However, since this DNA fragment lacks genes for phage coat synthesis, it cannot produce progeny particles, enabling controlled bactericidal activity.
Our constructed plasmid incorporates several key elements that collectively achieve the above design.

1. The T7-packaging sequence. This sequence enables the rolling circle replication of the toxic plasmid and its packaging into the phage head capsid.

2. The P_TetA. The P_TetA is repressed when TetR (tetracycline repressor protein) binds to the operator site within the promoter.

3. The MazF toxin gene from E. coli. MazF encodes an ACA-specific endoribonuclease that cleaves ACA sequences in mRNA, thereby blocking bacterial protein synthesis. The endogenous MazF in E. coli MG1655 has been extensively studied, with its function and regulatory mechanisms well characterized. Therefore, MazF was selected for constructing the toxic plasmid.

Figure 1: The model of toxic plasmid

The Dual Safety Strategy Ensuring the Survival of the Chassis Organism

To prevent the host bacteria used for producing phage-like particles from being killed by excess toxins expressed by MazF, we drew inspiration from the E. coli MG1655 natural toxin/antitoxin system MazE-MazF. This Type II toxin/antitoxin system comprises a stable toxin protein and an unstable antitoxin protein.

We used a plasmid to integrated an antitoxin gene MazE, into the host strain’s genome. MazE acts as an antagonist to MazF, neutralizing its toxicity by inserting its C-terminal helical segment into the RNA-binding channel of MazF, thereby preventing MazF from binding to RNA. The J23119 promoter is a constitutive promoter. Under the normal culture conditions, the continuously expressed antitoxin neutralizes the toxin’s toxicity, ensuring the normal growth of the chassis organism. We also introduced the TetR gene, whose expressed TetR repressor binds to the P_TetA, inhibiting the expression of the downstream MazF gene. This provides a dual safeguard in the chassis organism, preventing the overexpression of MazF.

Figure 2: The model of dual safety

Split the T7 genome and construct an inducible expression system for controlled production of phage-like particles

When the toxic plasmid is introduced, the complete wild-type T7 phage genome remains in the chassis bacteria. The wild-type T7 phage genome also undergoes rolling-circle replication and is packaged into phage capsids, resulting in wild-type T7 phage progeny contaminating the final product. To produce pure phage-like particles, the generation of wild-type T7 phages must be prevented. To achieve this, we integrated the T7 phage genome — excluding the T7-packaging sequence and the gp17 gene — into the host genome. Simultaneously, to ensure the controlled production of phage-like particles, we designed an inducible expression pathway.

We amplified the T7 phage genomic fragments excluding the gp1 gene, the T7-packaging sequence, and the gp17 gene via PCR and cloned them into the pkD46-SacB vector. Using homologous recombination double-exchange technology, we integrated the phage genome into the E. coli MG1655 genome, enabling this integration to achieve stable expression of phage capsid proteins in the chassis organism.

Figure 3: The method of integration of T7 phage genomes

The gp1 gene encodes the phage RNA polymerase, which transcribes the DNA sequence downstream of the T7 promoter within the T7 phage genome. If gp1 is not expressed, the T7 phage coat protein cannot be synthesized, thereby preventing the initiation of phage-like particle production. We inserted a lactose operator upstream of the gp1 gene to construct an inducible expression pathway, enabling controlled production of phage-like particles by artificially adding IPTG to trigger the expression of gp1.

Figure 4: The model of gp1 recombination

To insert the truncated T7 genome into the chassis organism, we constructed the pKD46-SacB vector. The Amp and SacB resistance markers on this vector serve as two selection criteria for homologous recombination.

The pKD46-SacB plasmid carries the exo, beta, and gam genes, encoding the Exo, Beta, and Gam proteins respectively. These proteins are induced for expression upon exposure to a specific concentration of arabinose. The constructed genomic homologous fragment undergoes homologous recombination with the E. coli MG1655 genome, facilitated by these three proteins. Concurrently, the pKD46-SacB plasmid uses the pSC101 replication origin and is a temperature-sensitive plasmid that spontaneously loses its stability at 42℃. SacB encodes levansucrase, rendering bacteria sensitive to sucrose for growth. In the presence of the sucrose, the bacteria die.

Figure 5: The model of pKD46-SacB

We performed seamless cloning of the segmented T7 genomic DNA fragments with pKD46-SacB to construct four vectors for homologous recombination.

Figure 6: Four models of T7 phage genome recombination

After transforming the homologous recombination vector into E. coli MG1655, we induced the production of Exo, Beta, and Gam proteins using L-arabinose while cultured at 30°C. Under Amp-containing and 42°C culture conditions, we selected strains that underwent homologous single-exchange. Strains exhibiting normal growth were identified as having completed the homologous single-exchange.

The strains selected through single-homologous exchange were further screened in antibiotic-free LB medium containing sucrose. Surviving strains were identified as having completed double-homologous exchange, while unsuccessful strains were lethal due to D-sucrase activity expressed by SacB.

Through the above single and double-homologous exchange screening, DNA fragments are successfully inserted into the host genome. Constructing four plasmids and performing four rounds of single and double-exchange screening enables the insertion of the deleted T7 genome into the E. coli MG1655 genome, thereby achieving the integration of the truncated T7 genome in E. coli MG1655.

Following IPTG induction, the modified chassis bacteria initiate phage coat synthesis, with the coat exclusively packaging DNA fragments from toxic plasmid rolling circle replication, thereby producing pure phage-like particles.

Modular Reprogramming of Tail fiber to Expand Host Targeting Range

Nevertheless, the narrow host range of wild-type T7 phage necessitates precision targeting against diverse pathogens, so we designed the modular tail fiber replacement strategy by substituting the native tail fiber genes of T7 with those from phages specific to target pathogens. This modular design transforms our chassis bacterium into a highly flexible engineered platform. By swapping different tail fiber genes, distinct phage-like particles can be generated to recognize and kill diverse pathogenic bacteria, thereby upgrading a system originally specific toE. coli into a programmable, broad-spectrum precision antibacterial platform.

Taking the bacterial spot disease in tomatoes as an example, the wild-type T7 phage is nearly incapable of targeting its pathogen, Pseudomonas syringae. To enhance the phage’s recognition specificity and infection efficiency against the target pathogen, we expressed the HOQ98_gp43 gene from the Pseudomonas syringae phage phiPsa 17 genome within the toxic plasmid. This gene encodes VO98_215, a key tail fiber protein for phage phiPsa 17 host recognition, thereby modifying the tail fiber protein of the phage-like particles. This enhancement significantly improves their binding affinity to the outer membrane receptors of target pathogens.

Based on these principles, we have developed a highly controllable, safe, and efficient precision antibacterial platform. This platform not only achieves targeted elimination of specific pathogenic bacteria but also transforms a natural bacteriophage into a SMART pesticide through genome fragmentation, induced expression, and modular tail reprogramming. This breakthrough offers a novel, sustainable solution for combating increasingly severe bacterial diseases.

Figure 7: The model of Tail Fibrils modification

References

[1]Aizenman, E., Engelberg-Kulka, H., & Glaser, G. (1996). An escherichia coli chromosomal “addiction module” regulated by guanosine [corrected] 3’,5’-bispyrophosphate: A model for programmed bacterial cell death. Proceedings of the National Academy of Sciences of the United States of America, 93(12), 6059–6063.

[2]Akiyama, K., Fujisawa, K., Kondo, H., Netsu, Y., Nishikawa, K., Takata, Y., Nakamura, Y., Kino, Y., Ayukawa, S., Yamamura, M., Hayashi, N., Tagawa, Y., & Nakashima, N. (2020). MazF activation causes ACA sequence-independent and selective alterations in RNA levels in escherichia coli. Archives of Microbiology, 202(1), 105–114.

[3]Chung, Y.-B., & Hinkle, D. C. (1990a). Bacteriophage T7 DNA packaging: I. Plasmids containing a T7 replication origin and the T7 concatemer junction are packaged into transducing particles during phage infection.Journal of Molecular Biology, 216(4), 911–926.

[4]Chung, Y.-B., & Hinkle, D. C. (1990b). Bacteriophage T7 DNA packaging: II. Analysis of the DNA sequences required for packaging using a plasmid transduction assay. Journal of Molecular Biology, 216(4), 927–938.

[5]Chung, Y.-B., Nardone, C., & Hinkle, D. C. (1990). Bacteriophage T7 DNA packaging: III. A “hairpin” end formed on T7 concatemers may be an intermediate in the processing reaction. Journal of Molecular Biology, 216(4), 939–948.

[6]Taslem Mourosi, J., Awe, A., Guo, W., Batra, H., Ganesh, H., Wu, X., & Zhu, J. (2022). Understanding bacteriophage tail fiber interaction with host surface receptor: The key “blueprint” for reprogramming phage host range. International Journal of Molecular Sciences, 23(20), 12146.

[7]Tsuchida, S.-I., Kokubo, H., Tasaka, M., & Fujisawa, H. (1996). DNA sequences responsible for specificity of DNA packaging and phage growth interference of bacteriophages T3 and T7. Virology, 217(1), 332–337.

[8]Yoichi, M., Abe, M., Miyanaga, K., Unno, H., & Tanji, Y. (2005). Alteration of tail fiber protein gp38 enables T2 phage to infect escherichia coli O157:H7. Journal of Biotechnology, 115(1), 101–107.

[9]Yosef, I., Goren, M. G., Globus, R., Molshanski-Mor, S., & Qimron, U. (2017). Extending the host range of bacteriophage particles for DNA transduction. Molecular Cell, 66(5), 721-728.e3.