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The host recognition modules encoding the injection machinery and receptor binding proteins (RBPs) of bacteriophages are predisposed to mutation and recombination to maintain infectivity towards co-evolving bacterial hosts. In this study, we reveal how Alteromonas mediterranea schitovirus A5 shares its host recognition module, including tail fiber and cognate chaperone, with phages from distantly related families including Alteromonas myovirus V22. While the V22 chaperone is essential for producing active tail fibers, here we demonstrate production of functional A5 tail fibers regardless of chaperone co-expression. AlphaFold-generated models of tail fiber and chaperone pairs from phages A5, V22, and other Alteromonas phages reveal how amino acid insertions within both A5-like proteins results in a knob domain duplication in the tail fiber and a chaperone β-hairpin "tentacle" extension. These structural modifications are linked to differences in chaperone dependency between the A5 and V22 tail fibers. Structural similarity between the chaperones and intramolecular chaperone domains of other phage RBPs suggests an additional function of these chaperones as transient fiber "caps". Finally, our identification of homologous host recognition modules from morphologically distinct phages implies that horizontal gene transfer and recombination events between unrelated phages may be a more common process than previously thought among Caudoviricetes phages., (© 2023. Springer Nature Limited.)

Bacteriophages (phages) are widely used as biocontrol agents in food and as antibacterial agents for treatment of food production plant surfaces. An important feature of such phages is broad infectivity towards a given pathogenic species. Phages attach to the surfaces of bacterial cells using receptor binding proteins (RBPs), namely tail fibers or tailspikes (TSPs). The binding range of RBPs is the primary determinant of phage host range and infectivity, and therefore dictates a phage's suitability as an antibacterial agent. Phages EP75 and EP335 broadly infect strains of E. coli serotype O157. To better understand host recognition by both phages, here we focused on characterizing the structures and functions of their RBPs. We identified two distinct tail fibers in the genome of the podovirus EP335: gp12 and gp13. Using fluorescence microscopy, we reveal how gp13 recognizes strains of E. coli serotypes O157 and O26. Phage EP75 belongs to the Kuttervirus genus within the Ackermannviridae family and features a four TSP complex (TSPs 1-4) that is universal among such phages. We demonstrate enzymatic activity of TSP1 (gp167) and TSP2 (gp168) toward the O18A and O157 O-antigens of E. coli, respectively, as well as TSP3 activity (gp169.1) against O4, O7, and O9 Salmonella O-antigens. TSPs of EP75 present high similarity to TSPs from E. coli phages CBA120 (TSP2) and HK620 (TSP1) and Salmonella myovirus Det7 (TSP3), which helps explain the cross-genus infectivity observed for EP75., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Marine phages play a variety of critical roles in regulating the microbial composition of our oceans. Despite constituting the majority of genetic diversity within these environments, there are relatively few isolates with complete genome sequences or in-depth analyses of their host interaction mechanisms, such as characterization of their receptor binding proteins (RBPs). Here, we present the 92,760-bp genome of the Alteromonas- targeting phage V22. Genomic and morphological analyses identify V22 as a myovirus; however, due to a lack of sequence similarity to any other known myoviruses, we propose that V22 be classified as the type phage of a new Myoalterovirus genus within the Myoviridae family. V22 shows gene homology and synteny with two different subfamilies of phages infecting enterobacteria, specifically within the structural region of its genome. To improve our understanding of the V22 adsorption process, we identified putative RBPs (gp23, gp24, and gp26) and tested their ability to decorate the V22 propagation strain, Alteromonas mediterranea PT11, as recombinant green fluorescent protein (GFP)-tagged constructs. Only GFP-gp26 was capable of bacterial recognition and identified as the V22 RBP. Interestingly, production of functional GFP-gp26 required coexpression with the downstream protein gp27. GFP-gp26 could be expressed alone but was incapable of host recognition. By combining size-exclusion chromatography with fluorescence microscopy, we reveal how gp27 is not a component of the final RBP complex but instead is identified as a new type of phage-encoded intermolecular chaperone that is essential for maturation of the gp26 RBP. IMPORTANCE Host recognition by phage-encoded receptor binding proteins (RBPs) constitutes the first step in all phage infections and the most critical determinant of host specificity. By characterizing new types of RBPs and identifying their essential chaperones, we hope to expand the repertoire of known phage-host recognition machineries. Due to their genetic plasticity, studying RBPs and their associated chaperones can shed new light onto viral evolution affecting phage-host interactions, which is essential for fields such as phage therapy or biotechnology. In addition, since marine phages constitute one of the most important reservoirs of noncharacterized genetic diversity on the planet, their genomic and functional characterization may be of paramount importance for the discovery of novel genes with potential applications., (Copyright © 2020 Gonzalez-Serrano et al.)

Marine viruses play a critical role not only in the global geochemical cycles but also in the biology and evolution of their hosts. Despite their importance, viral diversity remains underexplored mostly due to sampling and cultivation challenges. Direct sequencing approaches such as viromics has provided new insights into the marine viral world. As a complementary approach, we analysed 24 microbial metagenomes (>0.2 μm size range) obtained from six sites in the Mediterranean Sea that vary by depth, season and filter used to retrieve the fraction. Filter-size comparison showed a significant number of viral sequences that were retained on the larger-pore filters and were different from those found in the viral fraction from the same sample, indicating that some important viral information is missing using only assembly from viromes. Besides, we were able to describe 1,323 viral genomic fragments that were more than 10Kb in length, of which 36 represented complete viral genomes including some of them retrieved from a cross-assembly from different metagenomes. Host prediction based on sequence methods revealed new phage groups belonging to marine prokaryotes like SAR11, Cyanobacteria or SAR116. We also identified the first complete virophage from deep seawater and a new endemic clade of the recently discovered Marine group II Euryarchaeota virus. Furthermore, analysis of viral distribution using metagenomes and viromes indicated that most of the new phages were found exclusively in the Mediterranean Sea and some of them, mostly the ones recovered from deep metagenomes, do not recruit in any database probably indicating higher variability and endemicity in Mediterranean bathypelagic waters. Together these data provide the first detailed picture of genomic diversity, spatial and depth variations of viral communities within the Mediterranean Sea using metagenome assembly.