Cross-genus rebooting of custom-made, syntheticbacteriophage genomes in L-form bacteriaSamuel Kilchera,1, Patrick Studera, Christina Muessnera, Jochen Klumppa, and Martin J. Loessnera

aInstitute of Food, Nutrition, and Health, Eidgenoessische Technische Hochschule Zurich, 8092 Zurich, Switzerland

Edited by Sankar Adhya, National Institutes of Health, National Cancer Institute, Bethesda, MD, and approved December 8, 2017 (received for review August18, 2017)

Engineered bacteriophages provide powerful tools for biotech-nology, diagnostics, pathogen control, and therapy. However,current techniques for phage editing are experimentally challeng-ing and limited to few phages and host organisms. Viruses thattarget 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 thatinfect Gram-positive bacteria. To this end, custom-designed, syn-thetic phage genomes were assembled in vitro from smaller DNAfragments. We show that replicating, cell wall-deficient Listeriamonocytogenes L-form bacteria can reboot synthetic phage ge-nomes upon transfection, i.e., produce virus particles from na-ked, synthetic DNA. Surprisingly, Listeria L-form cells not onlysupport rebooting of native and synthetic Listeria phage genomesbut also enable cross-genus reactivation of Bacillus and Staphylo-coccus phages from their DNA, thereby broadening the approachto phages that infect other important Gram-positive pathogens.We then used this platform to generate virulent phages by tar-geted modification of temperate phage genomes and demon-strated their superior killing efficacy. These synthetic, virulentphages were further armed by incorporation of enzybiotics intotheir genomes as a genetic payload, which allowed targeting ofphage-resistant bystander cells. In conclusion, this straightforwardand robust synthetic biology approach redefines the possibilitiesfor the development of improved and completely new phage ap-plications, including phage therapy.

bacteriophage engineering | synthetic biology | L-form bacteria |Listeria monocytogenes | biotechnology

Bacteriophages are viruses that exclusively infect bacteria andconstitute their natural enemies. Based on their extraordi-

nary host specificity and bacteriolytic potential, phages areconsidered for various medical and technological applicationsand are used as diagnostic tools for rapid and sensitive detectionof viable bacterial cells (1). Virulent/“strictly lytic” phages areespecially useful for biocontrol, targeting potential pathogens inagriculture and food production (2). In addition, the antibioticresistance crisis prompted reevaluation of phages as alternativeantimicrobials, and phage therapy approaches are beginning toshow promising results (3–5). Despite their high genus- andspecies-specificity, self-replicating nature, and low productioncost, wide-spread antibacterial and medical application of phagesis hindered by several challenges (6): Due to restricted host rangesof individual phages, mixtures of phages (cocktails) are often re-quired to cover all relevant strains of a pathogen, and the regulatoryframework for cocktail approval is unclear (7, 8). In addition,temperate phages can integrate into the host genome withoutinducing cell lysis, may contribute to the spread of antibioticresistance by transduction (9), or may increase bacterial viru-lence through lysogenic conversion, effectively excluding theiruse as biocontrol agents (10). Also, target cells may possess severalphage-resistance mechanisms, including receptor diversification,biofilm formation, restriction-modification systems, and CRISPRinterference (11). Such limitations can potentially be overcomeby tailored modification of phage genomes [reviewed by Pires

et al. (12)], which would also allow the introduction of additionalgenetic traits for diagnostics, biocontrol, and other applica-tions (12, 13). For example, phages have been engineered assequence-specific antimicrobials that selectively remove antibiotic-resistant bacteria from mixed populations (14, 15).However, targeted genome engineering of virulent phages is,

at best, a difficult and labor-intensive process (12). Currently, themost broadly applicable approach is based on modification ofphage genomes during infection by homologous recombination.Because recombination rates are relatively low (10−4 to 10−10),screening for recombinant phages is very time consuming andoften requires the coincorporation of selectable marker genesinto virus genomes (12). To accelerate the isolation of modifiedphages, recombinants can alternatively be enriched by negativeselection using CRISPR-Cas systems. To this end, sequence-specific RNA-guided nucleases are designed to cleave the WTallele while leaving recombinant genomes intact. So far, thisapproach was used to modify virulent phages of Escherichia coli,Lactococcus, and Streptococcus (16–19). CRISPR-Cas allowsmarker-free phage engineering but is limited to host strains forwhich such a system is available. For negative selection to work,recombination rates need to exceed the frequency of naturallyoccurring CRISPR escape mutants (typically 10−6 to 10−5) (16,18). In addition, editing templates and CRISPR-RNA vectorsneed to be constructed and transformed into the host bacteriumfor each planned modification. Thus, CRISPR-based phage en-gineering remains relatively time-consuming and is currentlylimited to a few bacterial hosts and phages. Multiple modifica-tions can only be introduced sequentially, hampering the con-struction of more complex engineered viruses. Efficient, faster,

Significance

The unique host specificity and antimicrobial activity of bac-terial viruses have inspired many diagnostic and antibacterialapplications in industry, agriculture, and medicine. Because ofthe rise in antibiotic-resistant infections, phage therapy is areemerging field of interest. Many restrictions that are associ-ated with the use of native, isolated phages can be overcomeby genetic engineering. Thus, efficient genome-editing toolsare needed to unleash the full potential of phage therapy andbiotechnology. In vitro assembly of synthetic virus genomesand the rapid isolation of corresponding phages is an impor-tant step in this direction. By using L-form bacteria as reboot-ing compartments of synthetic genomes, we report a simple,highly efficient, and broadly applicable technology that en-ables engineering of diverse phage families.

Author contributions: S.K., P.S., and M.J.L. designed research; S.K., P.S., and C.M. per-formed research; S.K., P.S., C.M., J.K., and M.J.L. analyzed data; and S.K. and M.J.L. wrotethe paper.

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

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence should be addressed. Email: samuel.kilcher@hest.ethz.ch.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1714658115/-/DCSupplemental.

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and more broadly applicable methods are required to fully de-velop the potential of phage engineering. One intriguing option isthe reactivation of synthetic bacteriophage DNA, a process alsoknown as “genome rebooting.” Some phage genomes can berebooted either using cell-free systems (in vitro transcription-translation) (20, 21) or in E. coli cells transfected with full-length phage genomic DNA (gDNA) (22, 23). Based on the lat-ter approach, Ando et al. (22) have presented an elegant platformtechnology to genetically modify phages, which is based on as-sembly and capture of synthetic genomes into yeast artificialchromosomes (YAC) and subsequent rebooting of YAC-phageDNA in E. coli recipient cells. While in vitro- and yeast-basedapproaches allow rapid phage genome engineering, their usewith phages of Gram-positive bacteria has not been demonstrated.So far, phages that target Gram-positive bacteria could not be

modified by any synthetic approach, because virus genomescould not be transferred back into the thick-walled Gram-positive recipient cells for rebooting. Here, we overcome thislimitation by employing L-form bacteria as genome recipientsand rebooting compartments. We present a robust and broadlyapplicable platform technology for rapid phage engineering anddemonstrate that fully tailor-made phage genomes can bedesigned on the drawing board, synthesized, and rebooted within

as few as 6 days. As proof of concept, we created synthetic, virulentphages with specifically tailored antimicrobial properties.

ResultsRebooting Genomic Bacteriophage DNA in Listeria monocytogenesL-Form Cells. L-form bacteria are pleomorphic, wall-deficientcells that undergo division and feature metabolic activity (24).L-forms have the ability to take up large molecules of DNA (25,26) and should be able to replicate, transcribe, and translatenewly introduced genetic information. Based on these assump-tions, we evaluated L-form cells of Listeria monocytogenes asrecipients and rebooting compartments for native, purifiedphage gDNA. Listeria L-form cells can be generated by pro-longed subcultivation in the presence of cell wall-active antibi-otics in an osmoprotective environment (24, 27). We used aL. monocytogenes EGDe strain variant designated “Rev2,” whichcan rapidly be induced to grow as an L-form (designated“Rev2L”) (27). To provide a proof of concept for the rebootingapproach, we used gDNA of the virulent L. monocytogenes phageP35. Its 35,822-bp linear gDNA (28) is too large for electro-poration into walled Listeria cells, which only accept supercoiledplasmids of up to 10 kb with low efficiency (29). We devised aworkflow for PEG-mediated transfection of Rev2L bacteria with

L-form+ Φ genomic DNA

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mix L-forms andpropagation strain

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Fig. 1. Rebooting genomic bacteriophage DNA in L. monocytogenes L-form cells. (A) The workflow for rebooting of phage genomes in Listeria L-form strain Rev2L isshown (see SI Materials andMethods and Fig. S1A for more details). (B) Rebooting was initially assessed using purified gDNA of Listeria phage P35. L-form transfectionreactions were prepared as indicated, incubated at 32 °C, and tested for plaque formation on the indicator strain at 24 h posttransfection. Soft-agar overlays of therebooting reactions are shown. DNaseI indicates a 30-min predigestion of P35 gDNAwith DNaseI. (C) Efficiency of transfection and rebooting was determined using adilution series of P35 gDNA. (D) Listeria phages P70, A511, and B035 were rebooted in Rev2L using 500–1,000 ng gDNA and were detected using L. ivanovii WSLC3009 as an indicator. (E) Rebooting kinetics in Rev2L were assessed for phages P35, P70, and A511 over a period of 96 h. Data are mean ± SD (n = 3). Φ, phage.

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full-length phage DNA. To this end, purified gDNA was mixedwith cells from a growing penicillin G (PenG)-induced Rev2Lculture, and gDNA uptake was mediated by addition of PEG-8000. Following dilution in osmotically stabilized L-form me-dium (modified DM3), the transfection reaction was incubatedto allow rebooting, i.e., DNA uptake, phage gene expression, andvirus assembly. Subsequently, we assayed the transfection re-action for the presence of reactivated phages using a suitablephage propagation strain as an indicator. A workflow is shown inFig. 1A. We found that P35 could be rebooted in a gDNA-,L-form-, and PEG-dependent process (Fig. 1B). To maximizephage yield, each step of the rebooting protocol was optimized(Fig. S1 A–F). We found a linear correlation between input

DNA quantity and phage production (Fig. 1C), with a lowerdetection limit of 2.6 pg of P35 gDNA. This corresponds toabout 66,000 genomes, assuming that the DNA used consistedsolely of intact, infectious gDNA (the quality of phage DNA wasassessed by pulsed-field gel electrophoresis) (Fig. S1G). BesidesP35, the L-form transfection protocol allowed rebooting of sevenother Listeria phages (Fig. 1D and Fig. S2) (30). These includedboth temperate (B025, B035, B056, PSA) and virulent (P70,P100, A511) phages, some with large genomes (>130 kb; P100,A511), and morphologically diverse phages featuring eithercontractile (Myoviruses; P100, A511) or noncontractile (Siphoviruses;all other) tails. The phage genomes used featured either single-stranded overlapping “cohesive” ends (hereafter, “cos” phages;B025, PSA) or terminally redundant DNA molecules, with orwithout circular permutation (A511, P100, P70, P35). This sug-gests that rebooting is largely independent of virus morphology,genome structure, DNA packaging mode, replication strategy,and genome size. Moreover, we were able to produce Listeriaphages that would normally not infect the walled Rev2 strain(P70, B025, B035, B056, and PSA) (Fig. S1H). These findingsindicate that virus attachment and/or genome translocation arethe major barriers restricting the infectious range of these phagesamong the different serovars of Listeria. The diverse features ofthe reactivated Listeria phages (summarized in Table S1) in-dicate that Rev2L represents a suitable rebooting compartmentfor all Listeria phage genomes. The rebooting kinetics of phagesP35, P70, and A511 (Fig. 1E) revealed that virus productionpeaked at 24 h posttransfection. The large volume of the L-formcells and their attenuated metabolic activity (24, 27) may explainwhy rebooting is a slow process compared with infection ofwalled cells. Because L-forms are devoid of a cell wall, progenyphages are released by osmotic disruption when transfectedRev2L cells are diluted with nonstabilized medium. Disruption

B. cereusHER 1399

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S. aureusATCC 19685

INDICATOR:gDNA / L-form: - / Rev2L ΦK / - ΦK / Rev2L

INDICATOR:A

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Fig. 2. Listeria L-forms reboot phage genomes from other Gram-positivebacteria. To test for cross-genus rebooting of Bacillus (A) and Staphylococcus(B) phage genomes in Listeria L-forms, Rev2L cells were transfected with500–1,000 ng gDNA, and rebooted phages were detected on suitable in-dicator strains. Reactions lacking either L-forms or phage gDNA served ascontrols. Soft-agar overlays of the rebooting reactions are shown.

f1 f2 f3 f4 f5 f6ΦP35 fragments

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Fig. 3. A synthetic platform for rebooting of in vitro-assembled bacteriophage genomes in Listeria L-forms. (A) The workflow for assembly and rebooting ofsynthetic genomes is depicted. Genome fragments can be generated by either PCR or gene synthesis. Red arrows indicate PCR primers. (B–E) To createsynthetic phages with nonmodified sequences, phage DNA was extracted from Listeria phage P35 and Bacillus phage TP21-L. (B and D) Overlapping PCRfragments covering the full P35 and TP21-L genomes were amplified by PCR and assembled in vitro to generate circular DNA. (C and E) Rev2L cells weretransfected with assembled DNA and rebooting reactions plated on suitable indicator strains (labeled on the right) to probe for infectious phage formation.Incomplete assemblies served as controls. circ, circular; f, genome fragment; m, molecular weight standard. (Scale bar in C: 35 mm.)

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releases any viral particles produced from heterologous DNA,including phages that cannot bind or infect Rev2, and/or whosecell wall lytic enzymes (endolysins) are unable to degrade Listeriapeptidoglycan.

Listeria L-Forms Reboot Phage Genomes from Other Gram-PositiveBacteria. By transfecting L-form bacteria, we were able to by-pass the first and last steps of the phage infection cycle butnot virus genome replication, gene expression, and virion mor-phogenesis. This system allowed us to explore whether ListeriaL-forms could possibly reactivate genomes of phages specific forother Gram-positive pathogens (but which cannot infect Lis-teria). We show that the 37.46-kb Siphovirus TP21-L (31) ofBacillus cereus (Fig. 2A) as well as the large, 153.96-kb MyovirusBastille (32) of Bacillus thuringiensis (Fig. S3A) can be rebootedin Rev2L and subsequently infect their original Bacillus hostcells. In addition, Listeria L-forms also supported rebooting ofStaphylococcus aureus phage 2638A (33), a 41.32-kb Siphovirus(Fig. S3B), and phage K (34), a large 127.40-kb Myovirus (Fig.2B). We de novo sequenced two reactivated L. monocytogenesphage P35 clones as well as two S. aureus phage 2638A clonesand compared their sequence to the transfected DNA. Only onenoncoding point mutation in one 2638A clone was found,

suggesting that facilitating mutations are not required forrebooting. Our data show that Rev2L represents a highly ver-satile system for uptake and cross-genus rebooting of phagesfrom linear gDNA.

Efficient Rebooting of Synthetic, in Vitro-Assembled Phage Genomes.To enable phage engineering, we tested rebooting of fully syn-thetic phage genomes (workflow in Fig. 3A). For this, we am-plified overlapping segments of phage DNA and reassembledthem using the Gibson method (35). Because most phages re-quire circular genome replication intermediates (36), overlapsbetween fragments were designed to allow end-joining (circularclosure). First, we used six fragments of about 6 kb to assemblesynthetic genomes of Listeria phage P35 (Fig. 3B). An in-complete assembly lacking fragment three (−f3) served as con-trol. We found that Rev2L efficiently reboots phage P35 fromin vitro-assembled DNA (Fig. 3C). Surprisingly, synthetic DNAyielded a higher rebooting efficiency than native phage gDNA(detection limit, 1.1 pg DNA) (Fig. S4). This may be explained bythe fact that synthetic gDNA is already circularized, whereasnative genomes need to be closed inside the L-form cell aftertransfection. Alternatively, the fraction of infectious genomes innative phage DNA preparations may have been low. In additionto P35, we assembled and rebooted synthetic genomes of tem-perate Listeria phages PSA (37) and B025 (28) (Fig. S5). Todemonstrate reactivation of synthetic phages from other genera,assembly and rebooting of B. cereus phage TP21-L was alsoestablished (Fig. 3 D and E).Rebooting synthetic phage genomes in L-form bacteria offers

a broadly applicable platform technology for phage engineeringthat enables full control of sequence composition and organi-zation. By modification, removal, addition, or reorganization ofindividual fragments in the assembly, any genome configurationcan be realized in a single step. Since the input DNA is defined,all emerging phages have the desired sequence, which eliminateslengthy screening procedures. Because Rev2L cells also rebootphage genomes across otherwise conserved genus boundaries,the technology is applicable to other relevant Gram-positivepathogens, such as bacilli and staphylococci. Overall, engineeringis fast, requiring only 6 d from in silico phage design to isolationof the synthetic virus (Fig. S6).

Modifying Temperate Phage Genomes to Create Synthetic, VirulentPhages. Next, we applied the synthetic platform to create tailor-made phages. Any antibacterial phage application, includingphage therapy, requires strictly lytic viruses. To date, virulentcandidate phages that feature the desired host range, killing ef-ficiency, and physical properties must be identified from naturalisolates. Here, we applied the rebooting platform to rapidlycreate virulent phages using temperate phages as genetic back-bones, a process we call “virulent conversion.” To this end, we denovo assembled the genome of temperate Listeria phage B025(28) but removed the gene cluster encoding the lysogeny controlfunctions that mediate genome integration and prophage main-tenance. In B025, these genes are located on a 2.7-kb geneticmodule (28), the lysogeny control region (LCR). We designedsynthetic genomes to lack either just the repressor of the lyticcycle (B025 Δrep) or the entire LCR, including the integrase(B025 ΔLCR). The assembly strategy is shown in Fig. 4A, andfragments for the assembly of mutant phage genomes are shownin Fig. 4B. Engineered genomes were then rebooted, plaquephenotypes visualized (Fig. 4C), and phage genomes validated byPCR (LCR-PCR) (Fig. S7) and sequencing of the LCR. We usedthe soft-agar overlay method to visualize multiplicity of infection(MOI)-dependent host-cell killing by the engineered B025phages (Fig. 4D). As expected, synthetic B025 WT phages in-tegrated readily, and lysogenization effectively prevented sub-sequent killing of the host, especially at higher MOIs. As a result,a seemingly unperturbed bacterial lawn, which consisted ofhomoimmune WSLC 3009::B025 lysogens (Fig. S8), was obtainedafter overnight incubation (B025 WT 105/106 pfu) (Fig. 4D). In

A

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Fig. 4. Engineering of a strictly lytic virus by converting the life-style oftemperate phage B025. Genome assembly and rebooting were used toproduce mutants of the temperate Listeria phage B025 lacking either therepressor of the lytic genes (Δrep) or the complete lysogeny control region(ΔLCR). (A) The phage engineering strategy is shown. Arrows indicate pri-mers for fragment amplification. Genome fragments f3 and f4 weredesigned to exclude the repressor or the complete LCR, yielding (B) five PCRfragments for genome assembly. Synthetic genomes were rebooted in Rev2Land tested for plaque formation on L. ivanovii WSLC 3009. (C) Plaque phe-notypes of synthetic WT and mutant phages are shown. (D) To visualize MOI-dependent host cell infection and killing, 200 μL of a WSLC 3009 culture wasinfected with increasing MOIs of B025 WT, Δrep, and ΔLCR phages using thesoft-agar overlay technique and plates were incubated for 24 h, unless in-dicated otherwise. int, integrase; rep, repressor of the lytic cycle.

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contrast, the virulent B025 derivatives B025 Δrep and B025 ΔLCRdisplayed strongly enhanced killing efficacy and lysed almost allhost cells at an identical MOI (105/106 pfu) (Fig. 4D). Only a fewsurvivors were able to regrow upon infection with the mutants after48 h (Fig. 4D, 3-fold magnified areas). These survivors were notgenetically resistant to B025 but regained sensitivity within onepassage. We found that they featured an empty integration site (Fig.S8), confirming the lack of lysogenization by LCR-mutant phages.A similar approach was applied to B. cereus phage TP21-L, fromwhich we also removed either the repressor or the complete LCR(Fig. S9). Taken together, these results show that virulent conver-sion represents a fast and broadly applicable method to increase thearsenal of phages with strong lytic activity, an important prerequisitefor applications in biocontrol and especially for phage therapy.

Genetic Arming Enables Control of Phage-Resistant Cells. The oc-currence and selection of phage-resistant cells within a bacterialtarget population can hamper the potential application of bac-teriophages. As proof of concept, we addressed this limitationusing phage PSA. This temperate virus specifically infects L.monocytogenes serovar 4b cells (38). Using the same strategy asfor B025, we first removed the LCR of PSA to yield a virulentderivative that can no longer integrate into the tRNA attB site(PSA ΔLCR, virulent conversion) (37, 39). To target phage-re-sistant cells, we introduced an additional effector gene intosynthetic PSA WT and PSA ΔLCR phage genomes as geneticpayload, a strategy we refer to as “arming.” The effector issynthesized during the lytic cycle, coreleased upon host cell lysis,and subsequently acts on neighboring cells. Here, PSA WT andPSA ΔLCR phages were armed with an additional, heterologousendolysin gene derived from Listeria phage A511 (ply511). Incontrast to PSA’s endogenous endolysin (PlyPSA), Ply511 effi-ciently degrades cells of all Listeria serovars, including serovar1/2 (40). The strategy for LCR deletion and incorporation of ply511

into the late gene cluster of PSA and PSA ΔLCR and the validationof recombinants are shown in Fig. S10. We found that expression ofply511 from modified PSA genomes results in the formation of verylarge plaques with a characteristic halo (Fig. 5A) caused by thediffusion and enzymatic activity of the A511 endolysin effector. Asfor phage B025, deletion of the PSA LCR induced a strong lyticphenotype in plate culture (Fig. 5B, 106 pfu). Compared with PSAΔLCR, the combination of LCR deletion with ply511 expression(PSA ΔLCR ply511) significantly reduced the occurrence of survi-vors (Fig. 5B, magnified areas). Next, we tested the ability of allPSA derivatives to kill target bacteria in liquid culture. A PSA-sensitive culture of L. monocytogenes serovar 4b cells was infected atan MOI of 0.1, and OD was monitored over time (Fig. 5C, Upper).Alternatively, to determine the control of Listeria cells that are re-sistant to phage PSA infection, the sensitive host strain was mixedwith a PSA-resistant strain (serovar 1/2) at a sensitive:resistant ratioof 10:1 (Fig. 5C, Lower). Because of rapid lysogenization and sub-sequent growth of homoimmune cells, both PSA WT and thearmed but nonvirulent PSA derivative (PSA ply511) were unable tocontrol bacterial growth in both assays. While virulent conversionwas sufficient to control phage-sensitive cells, the sensitive:resistanthost cell mixture could not be cleared by the virulent PSA derivative(Fig. 5C, PSA ΔLCR). However, by targeting via the released cellwall hydrolase payload, the combination of virulent conversionand enzymatic arming (Fig. 5C, PSA ΔLCR ply511) equipped thesynthetic phage with the ability to efficiently control native hostcells and PSA-resistant serovar 1/2 cells in mixed culture. Inconclusion, our results demonstrate that rational design of syn-thetic phages can provide antimicrobial agents with specificallytailored killing functions.

DiscussionSynthetic genome rebooting allows rapid, selection-free engi-neering and production of genetically modified phages. It should

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Fig. 5. Control of phage-resistant cells using a virulent and genetically armed PSA derivative. (A) Plaque phenotypes of PSA WT, PSA ΔLCR, PSA ply511, andPSA ΔLCR ply511 at 48 h postinfection. (B) To visualize host lysis in plate culture, 200 μL of a L. monocytogenesWSLC 1042 overnight culture was infected withincreasing MOIs of WT, ΔLCR, and ply511-armed phages using the soft-agar overlay technique. Magnifications (2.6-fold) show surviving colonies that grewupon infection with ΦPSA ΔLCR and ΦPSA ΔLCR ply511. (C, Upper) To monitor host killing by PSA-derived phages in liquid medium infections, WSLC 1042 cells(Listeria serovar 4b) were infected with the indicated phages at an MOI of 0.1, and turbidity was monitored for 15 h. (Lower) To assess indirect targeting ofphage-resistant cells by the released Ply511 effector, a mixture of PSA-sensitive (serovar 4) and PSA-insensitive (serovar 1/2, WSLC 1001) cells was exposed toPSA-derived phages in the same assay. Data are mean ± SD (n = 3). ply, phage endolysin.

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be noted that, unlike CRISPR-based editing and other recom-bination-based strategies, this approach offers complete andunrestricted freedom of genome design and editing, includingmajor rearrangements, hybrid phages, and even fully tailor-madegenomes. Multiple modifications can be incorporated at once, ina single assembly. The method is fully independent of trans-formation and recombination efficiencies of the host organismand circumvents cloning of genes potentially toxic to an in-termediate host cell. Moreover, Rev2L L-forms can be employednot only for rebooting of Listeria phage genomes but also forreactivation of viruses infecting Bacillus, Staphylococcus andlikely other Gram-positive hosts. Thus, the synthetic platform isbroadly applicable and does not require any additional special-ized strains for recombinant phage construction.The virulent conversion and arming approach demonstrates

the utility of rebooting for the construction of phages with cus-tomized antimicrobial properties. This technology will facilitatemodifications that have previously been very difficult or impos-sible, such as incorporation of reporter genes for pathogen detectionor host-range alterations by modification of receptor-binding pro-teins. It will also allow the incorporation and phage-mediated pro-duction of any desired genetic payload. This may be of particularinterest for the delivery of therapeutic genes to specific bacterialpopulations within complex environments, such as the gut micro-biome. Because rebooting of synthetic genomes is highly efficient(Fig. S4), this platform technology will also permit screening of DNA

fragment libraries in a directed evolution approach, for exampleusing error-prone PCR. Potentially, directed evolution of definedgenome fragments may allow the generation of phages with alteredhost range or increased stability and circulation half-life. Finally,many aspects of basic phage biology are still poorly understood,mostly due to the lack of efficient genetic tools for mutation, de-letion, and molecular tagging of phage genes. We are convinced thatthe approach presented here will contribute substantially to an en-hanced understanding of the biology of these versatile self-replicat-ing units and will pave the way for novel phage-based applicationsbeyond their use as biocontrol and detection agents.

Materials and MethodsL. monocytogenes strains WSLC 1042, WSLC 1001, and Mack, Listeria ivanoviiWSLC 3009, B. thuringiensis HER 1211, and B. cereus HER 1399 were grownat 30 °C in 0.5× Brain Heart Infusion (BHI, Biolife Italiana) medium. S. aureusstrains ATCC 19685 and 2638A were grown at 37 °C in 0.5× BHI medium. L.monocytogenes L-form strain Rev2L was grown at 32 °C in modifiedDM3 medium (5 g/L tryptone, 5 g/L yeast extract, 0.01% BSA, 500 mM suc-cinic acid, 5 g/L sucrose, 20 mM K2HPO4, 11 mM KH2PO4, 20 mM MgCl2, pH7.3) (41). Detailed methods for bacteriophage propagation and DNA ex-traction, L-form transfection and rebooting, in vitro assembly of syntheticgenomes, time-course turbidity assay, and de novo phage sequencing aredescribed in SI Materials and Methods. All primers used in this study arelisted in Table S2.

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