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Completing the BASEL phage collection to unlock hidden diversity for systematic exploration of phage–host interactions [1]

['Dorentina Humolli', 'Institute Of Food', 'Nutrition', 'Health', 'Ifnh', 'Eth Zürich', 'Zürich', 'Damien Piel', 'Enea Maffei', 'Biozentrum']

Date: 2025-04 

Research on bacteriophages, the viruses infecting bacteria, has fueled the development of modern molecular biology and inspired their therapeutic application to combat bacterial multidrug resistance. However, most work has so far focused on a few model phages which impedes direct applications of these findings in clinics and suggests that a vast potential of powerful molecular biology has remained untapped. We have therefore recently composed the BASEL collection of Escherichia coli phages (BActeriophage SElection for your Laboratory), which made a relevant diversity of phages infecting the E. coli K-12 laboratory strain accessible to the community. These phages are widely used, but their assorted diversity has remained limited by the E. coli K-12 host. We have therefore now genetically overcome the two major limitations of E. coli K-12, its lack of O-antigen glycans and the presence of resident bacterial immunity. Restoring O-antigen expression resulted in the isolation of diverse additional viral groups like Kagunavirus, Nonanavirus, Gordonclarkvirinae, and Gamaleyavirus, while eliminating all known antiviral defenses of E. coli K-12 additionally enabled us to isolate phages of Wifcevirus genus. Even though some of these viral groups appear to be common in nature, no phages from any of them had previously been isolated using E. coli laboratory strains, and they had thus remained largely understudied. Overall, 37 new phage isolates have been added to complete the BASEL collection. These phages were deeply characterized genomically and phenotypically with regard to host receptors, sensitivity to antiviral defense systems, and host range. Our results highlighted dominant roles of the O-antigen barrier for viral host recognition and of restriction-modification systems in bacterial immunity. We anticipate that the completed BASEL collection will propel research on phage–host interactions and their molecular mechanisms, deepening our understanding of viral ecology and fostering innovations in biotechnology and antimicrobial therapy.

Data Availability: All data generated or analyzed during this study are included in this published article. The raw data of every replicate experiment underlying the plots shown in Figs 5D , 6G , 7D , 8G , 9E , 10E , 11A , 11B , S1A and S1B, all calculations underlying the summary data in these figures, and additional analyses are compiled in S1 Data . Tree files of all phylogenies shown in this work and the underlying sequence alignments in NEXUS format are available in S3 Data file. The code used to run GAPS for the results presented in this work is available on Github ( https://github.com/hiller-lab/gaps ) and on Zenodo ( https://www.doi.org/10.5281/zenodo.14277981 ). The annotated genome sequences of all 37 newly isolated phages as well as Lederbergvirus isolates Dewey, Huey, and Louie ( Figs 10D and S6B) are available in NCBI BioProject PRJNA1207239 with GenBank accession numbers listed in S2 Table.

In the current study, we have addressed both limitations of our previous work by expanding the BASEL collection with 37 new phages that largely depend on (restored) O-antigen expression and of which some are inhibited by resident antiviral defenses in the K-12 genome in the form of O-antigen glycosylation. Notably, while almost none of these phages belong to groups of which other representatives have previously been found to infect E. coli K-12, all these phages do infect E. coli K-12 with a few easily implemented modifications and can thus be used for systematic fundamental research as an expansion of the original BASEL collection. Similar to our previous study, we have performed a systematic phenotypic and genomic characterization of these phages with a view to host receptors, bacterial immunity, and host range, and we make them accessible to the scientific community. These results extend some of our previous observations and complement them with new discoveries on previously understudied phage groups such as the Gordonclarkvirinae podoviruses that are abundant in nature, highly resistant to tested phage-defense systems, and have a comparably broad host range. The dependence of many newly included phages on O-antigen as host receptor highlights the dominant role of viral adsorption for E. coli phage host range and provides a valuable tool for studying diverse mechanisms of glycan recognition using the well-studied model strain E. coli K-12. Taken together, our findings provide novel insights into the ecology and evolution of bacteriophages as well as into their arms race with each other and bacterial immunity. The now completed BASEL collection comprises 106 phages that have been isolated from diverse sources and belong to more than 30 genera ( S1 and S2 Tables ). We anticipate that our work will serve as a reference point for future studies in fundamental phage biology and strengthen the BASEL collection as an effective and widely used tool in the scientific community.

The value of these BASEL phages lies in their “assorted diversity,” providing access to a broad spectrum of biological diversity both within and between phage groups when exploring any aspect of phage–host interactions [ 6 , 10 ]. For example, the BASEL collection has been used to study phage host recognition [ 17 ], viral dependence on host codons [ 18 ], porin functionality [ 19 ], phage replication rates [ 20 ], and bacterial immunity [ 21 – 25 ]. A key prerequisite for broad applicability of the BASEL phages is that they have deliberately been isolated using the E. coli K-12 laboratory strain as host, the most important model organism in fundamental microbiology research [ 9 , 26 ]. However, phage isolation with E. coli K-12 as host has also conflicted with the goal of assorted phage diversity by introducing inherent biases that prevented us from sampling a complete overview of E. coli phage diversity [ 10 ]. Firstly, the domesticated E. coli K-12 laboratory strain has lost the O-antigen glycan chains on the lipopolysaccharide (LPS) core that form a dense barrier on wild E. coli and shield cell surface receptors from viral recognition [ 27 – 30 ]. While the absence of this barrier made it easier to isolate multiple phages from different viral groups, it naturally excluded the isolation of phages that depend on the O-antigen as host receptor [ 31 ]. Furthermore, a second level of sampling bias may have been caused by known (and possibly unknown) antiviral defenses encoded by E. coli K-12, although we had deleted some of them already in the host strain used to isolate and study the original BASEL collection [ 10 ].

Bacteriophages, the viruses infecting bacteria, have attracted great interest due to their abundance and diversity in all ecosystems, their key roles as inspiration for biotechnology, and their therapeutic potential in the fight against resilient bacterial infections [ 1 – 4 ]. Most cultured phages are so-called tailed phages or Caudoviricetes that characteristically use a tail structure at the virion to inject their genome into host cells. Morphologically, tailed phages can be classified as myoviruses (contractile tail), siphoviruses (long, flexible, noncontractile tail), and podoviruses (short stubby tail). Following the “phage treaty” declared by Max Delbrück in 1944 [ 5 ], a few tailed phages infecting Escherichia coli have become key model systems for studying the molecular biology of phage–host interactions [ 6 ]. We therefore know comparably much about the molecular mechanisms underlying infections of E. coli with these seven “T phages” T1–T7 [ 7 , 8 ] and a few additional ones infecting E. coli or other major model organisms like Bacillus subtilis. While this work has resulted in many major breakthroughs since the “golden era” of molecular biology [ 3 , 9 ], the limited number and diversity of these model phages is a severe limitation for systematic research that could inform the effective use of natural phage diversity for therapeutic or biotechnological applications. We and others have therefore proposed broader sets of model phages infecting commonly studied host organisms such as E. coli [ 10 – 12 ], Klebsiella [ 13 ], or Pseudomonas aeruginosa and other pseudomonads [ 14 – 16 ]. Our previous work had presented 68 well-characterized new E. coli phages as the BASEL (BActeriophage SElection for your Laboratory) collection which we have made accessible to the academic community.

Results

Overview: Including phages inhibited by resident immunity of E. coli K-12 in the BASEL collection Bacteriophages encounter multiple layers of antiviral defenses outside and inside their host cells that must be overcome for successful viral replication [40]. Some of these defenses prevent phage adsorption or DNA ejection, while others directly attack the invading virus such as restriction-modification (RM) systems, which cleave foreign DNA based on self/ non-self recognition through certain DNA modifications. Beyond direct attack, a deeper layer of defense systems protects the bacterial population through abortive infection, i.e., the altruistic suicide of infected cells prior to successful phage replication [41]. Recent work on anti-phage defense systems has vastly expanded our knowledge about their abundance and diversity through the discovery of many new systems that target various aspects of phage biology and are countered by phages in diverse ways [40–45]. The phages of the original BASEL collection had been sampled with a derivative of the commonly used E. coli K-12 MG1655 laboratory strain in which we had knocked out all known RM systems, i.e., the EcoKI type I RM system and three type IV restriction systems [10]. This enabled broad sampling of E. coli phages, including several taxonomic groups of phages that displayed considerable sensitivity to EcoKI and would otherwise have been excluded [10]. However, the literature and commonly used prediction tools such as DefenseFinder and PADLOC indicate that E. coli K-12 encodes several additional bona fide defense systems that might limit the range of phages accessible with this genomic background [46,47] (see details at Bacterial strains in Materials and methods). We had not considered this to be a widely relevant factor when composing the original BASEL collection because phage defenses beyond RM systems were thought to be rather specific. However, our own results showing broad antiviral defense by the Fun/Z system and the work of others, e.g., on the Zorya system revealed that this is not necessarily the case [10,24]. Notably, all these bona fide defense systems are encoded on the cryptic prophages of E. coli K-12 that are genomic islands descended from once active prophages, i.e., the facultatively host-integrated form of so-called temperate phages [46,47]. This is in line with the common observation that anti-phage defenses are frequently encoded on mobile elements and genomic islands [48–50]. To overcome possible sampling biases caused by these bona fide phage-defense systems (and possibly others that are yet unknown), we thus performed phage isolation experiments with a variant of E. coli K-12 in which a previous study had removed all cryptic prophages, E. coli K-12 BW25113 Δ9CP (both with and without restored O-antigen) [51] (Fig 2C). Similar to previous work in Vibrio that had used an analogous approach [50], these experiments indeed resulted in the isolation of an additional diversity of phages both from groups already covered in the BASEL collection and from a genus that had never been isolated before using any E. coli laboratory strain (Wifcevirus myoviruses).

Properties of siphoviral genus Kagunavirus Small siphoviruses like the Drexlerviridae family (relatives of phage T1) or genus Dhillonvirus are frequently isolated using E. coli as a host and typically have genomes of 40−50kb size. The diverse representatives of these groups in the original BASEL collection mostly showed considerable sensitivity to RM systems and—as expected from previous work—targeted different porins as terminal host receptors [10,60,61]. Only one of these phages, JakobBernoulli (Bas07), is able to infect E. coli K-12 with restored O-antigen expression, suggesting that its lateral tail fibers can target O16-type O-glycans [10] (see also S1A Fig and below). Our targeted search for phages that depend on O16-type O-antigen resulted in the isolation of various small siphoviruses of genus Kagunavirus within the subfamily of Guernseyvirinae (Fig 5A–5D). These phages have small genomes of ca. 41−44kb, which makes them attractive models for synthetic biology applications, e.g., in the case of phage CM001 [62]. While all our isolates fully depend on restored O-antigen expression, only some of them (Bas70−73) infect a regular E. coli K-12 host while others (Bas74−76) could only be isolated using a K-12 host devoid of cryptic prophages (Fig 5D). Phenotypically, both groups of Kagunavirus isolates behave very similarly and display a high sensitivity to RM systems and a variable sensitivity to the Old antiphage defense system that is typical for small siphoviruses [10] (Fig 5D). The dependence on glycans as host receptor seems to be a general property of Guernseyvirinae phages, including, e.g., the prototypic Kagunavirus K1G targeting the K1 capsule of E. coli [15] or the Jerseyvirus SETP3 and several relatives targeting O-antigen of Salmonella enterica [63]. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Overview of Guernseyvirinae genus Kagunavirus phages. (A) Schematic model of host recognition by Kagunavirus phages (O-antigen chains in blue). In analogy to other siphoviruses infecting E. coli like lambda or T5, we anticipate that these phages also use a phage-derived conduit for DNA injection across the outer membrane after the tail fibers recognizing the terminal host receptor have bent off [110,141]. (B) Representative TEM micrograph of phage SonjaBuckley (Bas74). (C) Maximum-likelihood phylogeny of Kagunavirus phages and relatives based on a curated whole-genome alignment with bootstrap support of branches shown if > 70/100. Newly isolated phages of the BASEL collection are highlighted by red phage icons. The phylogeny was rooted between the genera Kagunavirus and Jerseyvirus. (D) The results of quantitative phenotyping experiments with Kagunavirus phages regarding sensitivity to altered surface glycans and bacterial immunity systems are presented as efficiency of plating (EOP). Small notes of “ΔRM” or “Δ9CP” indicate on which host strain the respective phage has been characterized (see Materials and methods). Data points and error bars represent the average and standard deviation of at least three independent experiments. Raw data and calculations are available in S1 Data. https://doi.org/10.1371/journal.pbio.3003063.g005

Properties of other O16-dependent small siphoviruses and Dhillonvirus Besides phages of the genus Kagunavirus, we also obtained different other small siphoviruses that depend on restored O16-type O-antigen to infect E. coli K-12 and new isolates of the Dhillonvirus genus (Fig 6A–6G). Some of the new O16-dependent isolates belong to a cluster of siphoviruses formed primarily by the genus Skatevirus (Bas77–81, Fig 6C and 6D). These viruses have genomes of 46–48 kb, which is well within the typical range for small siphoviruses. Additionally, we present phage KarinMuster (Bas82), belonging to the rarely reported Nonanavirus genus comprising relatives of the more commonly known Salmonella phage 9NA (Fig 6E) [64]. The genome size of this phage is ca. 58.5 kb and thus around 10 kb larger than that of the other small siphoviruses presented here, reminiscent of the Queuovirinae characterized in our previous work [10]. This latter group encodes a 7-deazaguanosine modification pathway in their additional genome content which provides considerable resistance to RM systems [10,65]. However, there is no recognizable additional locus in KarinMuster and its relatives that could encode such supplemental biological functions [64], though their distant relationship to other known phages and limited knowledge about their biology makes it difficult to draw firm conclusions. Very similar to small siphoviruses characterized previously and the Kagunavirus phages shown above, all these phages are markedly sensitive to RM systems and to the Old defense system, which in the latter case results in quantifiable decrease of plaque counts only for some phages (Fig 6G). The additional genome size of KarinMuster therefore does not seem to make this phage more robust than other small siphoviruses when facing the tested examples of bacterial immunity. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Overview of other O16-dependent siphoviruses of genera Skatevirus and Nonanavirus as well as new isolates of siphoviral genus Dhillonvirus. (A) Schematic model of host recognition by O16-dependent small siphoviruses just as shown for Kagunavirus phages in Fig 5A. (B) Schematic model of host recognition by small siphoviruses of the Dhillonvirus genus that use first a lateral tail fiber to overcome the O-antigen barrier before binding a porin receptor with the central tail fiber (instead of only glycan-targeting tailspikes for the LPS-dependent small siphoviruses; see also our previous work [10]). (C) Representative TEM micrograph of Skatevirus ClaudiaMuster (Bas77). (D) Maximum-likelihood phylogeny of the Skatevirus genus and relatives based on a curated whole-genome alignment with bootstrap support of branches shown if > 70/100. The phylogeny was rooted between genus Roufvirus and the others. Note that most reference phages presented here as Skatevirus have no formal ICTV classification while others are scattered over several seemingly paraphyletic genera, prompting us to show all phages as Skatevirus [68]. (E) Maximum-likelihood phylogeny of the Nonanavirus genus and relatives based on a curated whole-genome alignment with bootstrap support of branches shown if >70/100. The phylogeny was rooted between phage 9NA and the other relatives based on their much closer relationship. Note that Sasha and Sergei are formally classified as Sashavirus while 9NA and AVIO78A are classified as Nonanavirus [68], which—according to this phylogeny—would be paraphyletic. (F) Maximum-likelihood phylogeny of the Dhillonvirus genus based on a curated whole-genome alignment with bootstrap support of branches shown if > 70/100. The phylogeny was rooted as previously [10] between the clade containing phage WFI and the others. In (D-F), newly isolated phages of the BASEL collection are highlighted by red or orange phage icons. The color code of phage names in (F) refers to porin host receptors with blue indicating FhuA, red LptD, green BtuB, and orange LamB [10] (see also S2B Fig). (G) The results of quantitative phenotyping experiments with Skatevirus, Nonanavirus, and new Dhillonvirus isolates regarding sensitivity to altered surface glycans and bacterial immunity systems are presented as efficiency of plating (EOP). Small notes of “ΔRM” or “Δ9CP” indicate on which host strain the respective phage has been characterized (see Materials and METHODS). Data points and error bars represent average and standard deviation of at least three independent experiments. Raw data and calculations are available in S1 Data. https://doi.org/10.1371/journal.pbio.3003063.g006 Finally, the phage isolation using a host without cryptic prophages identified additional members of the Dhillonvirus genus (Bas83−85), which—unlike their relatives of the original BASEL collection (Bas14−18)—fail to form plaques on regular E. coli K-12 (Fig 6B, 6F, and 6G). These new isolates do not cluster separately within the Dhillonvirus genus but are rather split widely across this group, suggesting that no vertically inherited feature but rather highly variable aspects of phage biology are responsible for this difference (Fig 6F). Phenotypically, the three additional phages selected for this work behave mostly similar to the original Dhillonvirus phages, e.g., by showing only moderate sensitivity to tested RM systems [10] (Fig 6G). Like the other siphoviruses characterized for the original BASEL collection, these Dhillonvirus phages display the typical arrangement of two separate types of tail fibers with distinct receptor-binding proteins [10,34] (see below and S2A Fig). Their central tail fiber has a bona fide target recognition module that is predicted to encode LptD specificity (S2B Fig) [10].

Properties of myoviruses in genus Wifcevirus Phages of the genus Wifcevirus within Caudoviricetes have been described comparably rarely with currently less than 20 sequenced isolates in NCBI GenBank that all infect E. coli or Shigella (Fig 9A–9E). They have genomes of ca. 66–72kb and were described as a genus when two new isolates, WFC and WFH, had been linked to several phages that had been sequenced earlier [58]. Interestingly, distant relatives of these phages in the Pbunavirus genus are among the P. aeruginosa phages most used for personalized therapy [84,85]. Based on clear homology, the cryogenic electron microscopy structure of virions of a Pbunavirus has greatly helped us to annotate the structural genes of Wifcevirus phages [86]. No Wifcevirus infecting E. coli laboratory models K-12 or B has ever been reported, limiting our knowledge about the biology of these phages and their interaction with the host. However, one previous study on two Wifcevirus isolates reported remarkably fast replication with only 3–5 min between adsorption and release of progeny [87]. This is much faster than even mutants of phage T7—another phage considered to be fast—which had been evolved for rapid replication but did not get faster than seven minutes in comparable assays [88]. This property of Wifcevirus phages could be of great interest for biotechnology or clinical applications, but the underlying molecular mechanisms have not been explored. PPT PowerPoint slide

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TIFF original image Download: Fig 9. Overview of Wifcevirus phages. (A) Schematic model of host recognition by Wifcevirus myoviruses targeting host O-antigen (blue) using tail fibers. Note that these phages carry two sets of tail fibers (see below) whose relationship is unclear, so only a simple schematic with one set is shown. (B) Maximum-likelihood phylogeny of the Wifcevirus genus and relatives based on a curated whole-genome alignment with bootstrap support of branches shown if > 70/100. The phylogeny was midpoint-rooted between the distantly related Wifcevirus and Pbunavirus genera. Phages of the BASEL collection are highlighted by little green phage icons. (C) Representative TEM micrograph of phage HermannAdler (Bas98) with a virion morphology very similar to other representatives that have been visualized previously [58]. (D) Wifcevirus isolate ManiWeber (Bas99) was streaked for single plaques on E. coli K-12 strains with and without cryptic prophages. Kuravirus SkaterBoy (Bas90; see Fig 7) is shown as a control without sensitivity to the presence of cryptic prophages. (E) The results of quantitative phenotyping experiments with Wifcevirus phages regarding sensitivity to altered surface glycans and bacterial immunity systems are presented as efficiency of plating (EOP). Small notes of “ΔRM” or “Δ9CP” indicate on which host strain the respective phage has been characterized (see Materials and methods). Data points and error bars represent average and standard deviation of at least three independent experiments. Raw data and calculations are available in S1 Data. https://doi.org/10.1371/journal.pbio.3003063.g009 Our four isolates (Bas98−101) belong to both major subgroups of Wifcevirus phages (Fig 9B) and all depend totally on restored O16-type O-antigen as host receptor (Fig 9E), suggesting that their tail fibers target this glycan as host receptor (see also the dedicated section below). This is reminiscent of their Pbunavirus relatives which are known to target the LPS of P. aeruginosa as host receptor [86,89,90]. All our isolates could only be obtained using E. coli K-12 without cryptic prophages as host and their plaque formation is severely impaired in their presence (Fig 9D), though a quantitative reduction of plaque counts was only observed for VerenaGrendelmeier (Bas101; Fig 9E). All isolates are not sensitive to any tested bacterial defense system including RM systems despite the lack of any known genome protection mechanism (Fig 9E and S2 Table). One notable exception is the EcoP1_I type III RM system, to which they are very sensitive even though they have similar numbers of recognition sites for this system as for the EcoCFT_II type III RM system to which they are highly resistant (Fig 9E and S2 Table).

Properties of Autographiviridae: Studiervirinae genera Teseptimavirus and Kayfunavirus Podoviruses of Autographiviridae subfamily Studiervirinae are well-known from classical representatives T3 (Teetrevirus genus) and T7 (Teseptimavirus genus) and have been frequently isolated against diverse enterobacterial hosts. In our previous work, we characterized T3 and T7 together with several new isolates from different genera of this subfamily [10]. As also suggested by the literature, we found that all these phages depended on the LPS core of E. coli K-12 as a host receptor when infecting this host, though they were invariably unable to infect this host when O-antigen expression was restored [10,91]. These phages exhibited considerable flexibility in their glycan recognition in a way that truncations of the LPS core reduced infectivity but that usually no individual truncation fully abolished it [10,91]. Given the ubiquity of O-antigen expression among wild E. coli strains and its barrier function, it seems likely that these phages have evolved to target these or other surface glycans as host receptor and more accidentally recognize also the LPS core of E. coli K-12. Previous work has already well established that diverse representatives of Autographiviridae target specific types of O-antigen as host receptor which determines the host range of these phages [29,33,92–94]. In the current study, we indeed isolated several different Teseptimavirus phages that depend on restored O16-type O-antigen expression of E. coli K-12 and included three of these in the expanded BASEL collection (Bas102–104; Fig 10). Furthermore, we isolated ElsieAttenhofer (Bas105), an O16-dependent phage of the distantly related Kayfunavirus genus that also contains various representatives known to target host glycans such as phage K1F targeting the K1 capsule [95] or CLB_P1 targeting the O104-type O-antigen of E. coli strain 55989 [96] (Fig 10B). Our four new isolates encode a single tailspike or tail fiber in their genomes analogous to other Autographiviridae phages and likely use it to target O16-type O-antigen (see in the dedicated section below). Like their relatives, they have genomes of ca. 40kb size and are sensitive particularly to type II and type III RM systems if they have a considerable number of recognition sites (Fig 10E and S2 Table) [10]. Beyond restriction, studies on model phages T3 and T7 as well as our previous work suggested that Autographiviridae phages are specifically targeted by the PifA defense protein dependent on the allelic variant of the gp1.2 dGTPase inhibitor gene [10,97,98]. Of the three new Teseptimavirus phages, only MeretOppenheim (Bas102) encodes a gp1.2 allele that is similar to those of sensitive phages T7 and JacobBurckhardt (Bas65), and it is indeed significantly inhibited by PifA (Figs 10E and S6A). However, also the Kayfunavirus ElsieAttenhofer (Bas105) is sensitive to PifA (Fig 10E), but it carries a distinct variant of the gp1.2 gene (S6A Fig). These results suggest that Autographiviridae sensitivity to PifA is more complex than two allelic variants of one locus and/ or that additional factors such as the major capsid protein may play a role (as indicated by previous work [98]). PPT PowerPoint slide

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TIFF original image Download: Fig 10. Overview of Autographiviridae: Studiervirinae and Lederbergvirus phages. (A) Schematic model of host recognition by podoviruses with enzymatic tailspikes targeting O-antigen (e.g., Kayfunavirus and Lederbergvirus) based on previous work on phage T7 and P22 [71,101,203]. (B) Maximum-likelihood phylogeny of the Studiervirinae subfamily of Autographiviridae based on a curated whole-genome alignment with bootstrap support of branches shown if > 70/100. The phylogeny was midpoint-rooted between distantly related genus Kayfunavirus and the other genera. Phages of the BASEL collection are highlighted by violet (new) or gray (old) phage icons. (C) Representative TEM micrograph of Kayfunavirus ElsieAttenhofer (Bas105). (D) Maximum-Likelihood phylogeny of the Lederbergvirus genus based on an alignment of their structural genes with bootstrap support of branches shown if >70/100. This phylogeny was midpoint-rooted between two major clades of this genus. Phage CurroJimenez (Bas106) is highlighted by a violet phage icon while phages Huey, Dewey, and Louie are highlighted by gray phage icons. (E) The results of quantitative phenotyping experiments with Autographiviridae phages regarding sensitivity to altered surface glycans and bacterial immunity systems are presented as efficiency of plating (EOP). Small notes of “ΔRM” or “Δ9CP” indicate on which host strain the respective phage has been characterized (see Materials and methods). Data points and error bars represent average and standard deviation of at least three independent experiments. Raw data and calculations are available in S1 Data. https://doi.org/10.1371/journal.pbio.3003063.g010

Properties of podoviruses in genus Lederbergvirus Phages in the genus Lederbergvirus are among the most well-known phages that use tailspikes to target enterobacterial surface glycans and particularly the O-antigen as host receptors (Fig 10A) [33,99,100]. These phages are podoviruses with genomes of around 40 kb size and a temperate lifestyle typically governed by lambda-like lysogeny control elements comprising a cI-like repressor, cII, and Cro [99]. The prototypic Lederbergvirus is phage P22 that has become a key model for viral host interaction. This phage targets the O:4 O-antigen of its Salmonella Typhimurium host with its tailspike before a tail needle penetrates the outer membrane and eventually a trans-envelope channel for DNA injection is formed (Fig 10A) [101,102]. Similarly, well-known Lederbergvirus phages like E. coli phage HK620 and Shigella phage Sf6 bind specific O-antigen variants of their respective hosts, while others target different glycans like the K1 capsule in the case of E. coli phage CUS-3 [103,104]. Its potency in the transduction of genes between Salmonella Typhimurium strains has made P22 a crucial tool for research on this organism [105], and the propensity of this phage for lateral transduction due to the initiation of viral replication and packaging before excision from the host genome can result in extremely high rates of gene transfer [106]. Beyond these classical models, Lederbergvirus phages infecting enterobacterial hosts are known to be common and diverse [107]. It was thus unsurprising that we isolated three different phages of this genus—Huey, Dewey, and Louie—which showed homogeneous and highly turbid plaques indicative of lysogen formation and self-immunity (S6B Fig; NCBI GenBank accessions PQ850600, PQ850609, and PQ850616) [108]. However, we also isolated one representative called CurroJimenez (Bas106) that did not show temperate behavior when infecting E. coli K-12 with restored O-antigen and was included in the BASEL collection that focuses on virulent phages (S6B Fig). Since it does not have obvious defects in its lysogeny control genes, we speculate that E. coli K-12 may lack the specific attB site which would be targeted by the integrase of this phage. The phylogeny of Lederbergvirus phages shows different distinct clades among which CurroJimenez, Dewey, and Louie are closely related in a sister group of P22 itself, while Huey is found in a more distant clade closer to CUS-3 and also HK620 (Fig 10D). However, the high genomic mosaicism of this viral genus makes it difficult to draw conclusions about the relationships of individual phages beyond the specific genes that have been used to calculate the phylogeny [33,109]. Phage CurroJimenez totally depends on the expression of restored O16-type O-antigen for the infection of E. coli K-12, which identifies this glycan as the host receptor targeted by its tailspike (Fig 10E). The phage is also highly sensitive to restriction-modification systems, which is not unexpected given the absence of any predictable anti-restriction mechanisms (Fig 10E and S2 Table) and seems to be common among temperate phages [10].

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