Cross-genus rebooting of custom-made, synthetic bacteriophage genomes in L-form bacteria

Edited by Sankar Adhya, National Institutes of Health, National Cancer Institute, Bethesda, MD, and approved December 8, 2017 (received for review August 18, 2017)
January 3, 2018
115 (3) 567-572


The unique host specificity and antimicrobial activity of bacterial viruses have inspired many diagnostic and antibacterial applications in industry, agriculture, and medicine. Because of the rise in antibiotic-resistant infections, phage therapy is a reemerging field of interest. Many restrictions that are associated with the use of native, isolated phages can be overcome by genetic engineering. Thus, efficient genome-editing tools are needed to unleash the full potential of phage therapy and biotechnology. In vitro assembly of synthetic virus genomes and the rapid isolation of corresponding phages is an important step in this direction. By using L-form bacteria as rebooting compartments of synthetic genomes, we report a simple, highly efficient, and broadly applicable technology that enables engineering of diverse phage families.


Engineered bacteriophages provide powerful tools for biotechnology, diagnostics, pathogen control, and therapy. However, current techniques for phage editing are experimentally challenging and limited to few phages and host organisms. Viruses that target Gram-positive bacteria are particularly difficult to modify. Here, we present a platform technology that enables rapid, accurate, and selection-free construction of synthetic, tailor-made phages that infect Gram-positive bacteria. To this end, custom-designed, synthetic phage genomes were assembled in vitro from smaller DNA fragments. We show that replicating, cell wall-deficient Listeria monocytogenes L-form bacteria can reboot synthetic phage genomes upon transfection, i.e., produce virus particles from naked, synthetic DNA. Surprisingly, Listeria L-form cells not only support rebooting of native and synthetic Listeria phage genomes but also enable cross-genus reactivation of Bacillus and Staphylococcus phages from their DNA, thereby broadening the approach to phages that infect other important Gram-positive pathogens. We then used this platform to generate virulent phages by targeted modification of temperate phage genomes and demonstrated their superior killing efficacy. These synthetic, virulent phages were further armed by incorporation of enzybiotics into their genomes as a genetic payload, which allowed targeting of phage-resistant bystander cells. In conclusion, this straightforward and robust synthetic biology approach redefines the possibilities for the development of improved and completely new phage applications, including phage therapy.

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Proceedings of the National Academy of Sciences
Vol. 115 | No. 3
January 16, 2018
PubMed: 29298913


Submission history

Published online: January 3, 2018
Published in issue: January 16, 2018


  1. bacteriophage engineering
  2. synthetic biology
  3. L-form bacteria
  4. Listeria monocytogenes
  5. biotechnology


This article is a PNAS Direct Submission.



Institute of Food, Nutrition, and Health, Eidgenoessische Technische Hochschule Zurich, 8092 Zurich, Switzerland
Patrick Studer
Institute of Food, Nutrition, and Health, Eidgenoessische Technische Hochschule Zurich, 8092 Zurich, Switzerland
Christina Muessner
Institute of Food, Nutrition, and Health, Eidgenoessische Technische Hochschule Zurich, 8092 Zurich, Switzerland
Jochen Klumpp
Institute of Food, Nutrition, and Health, Eidgenoessische Technische Hochschule Zurich, 8092 Zurich, Switzerland
Martin J. Loessner
Institute of Food, Nutrition, and Health, Eidgenoessische Technische Hochschule Zurich, 8092 Zurich, Switzerland


To whom correspondence should be addressed. Email: [email protected].
Author contributions: S.K., P.S., and M.J.L. designed research; S.K., P.S., and C.M. performed research; S.K., P.S., C.M., J.K., and M.J.L. analyzed data; and S.K. and M.J.L. wrote the paper.

Competing Interests

Conflict of interest statement: The technology described in this manuscript is a pending patent.

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    Cross-genus rebooting of custom-made, synthetic bacteriophage genomes in L-form bacteria
    Proceedings of the National Academy of Sciences
    • Vol. 115
    • No. 3
    • pp. 433-E557







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