Ruijie Ma

While tailed bacteriophages dominate contemporary models of phage biology, non-tailed phages-particularly filamentous inoviruses and double jelly-roll (DJR) capsid phages-remain poorly understood, despite their environmental ubiquity and ecological importance. Here, we identify 515 inoviruses and 258 DJR prophages from 688 Vibrio spp. genomes and precisely annotate prophage-bacterium junctions. This curated dataset enables us to systematically classify genomic variations and characterize lysogeny-related elements for each prophage subtype. We discover a conserved lysogeny module shared by specific inoviral and DJR subtypes, which represents an evolutionary strategy where phylogenetically distinct phages use horizontal gene transfer to co-opt host mechanisms for integration. Comparative genomics reveal that phage-encoded hypervariable regions (pHVRs) are hotspots for weaponized genetic innovation, such as anti-phage systems and virulence factors. The significantly higher prevalence of pHVR-encoding prophages compared to their pHVR-deficient counterparts across Vibrio species indicates that acquiring these adaptive genes promotes prophage persistence through mutualistic fitness benefits. Because these non-tailed elements frequently form tandem arrays and existing tools struggle with their accurate characterization, we establish a refined methodology to enhance predictive accuracy.

Vibrio harveyi , a pathogenic bacterium, contains prophages that significantly influence its pathogenesis and evolutionary traits. Investigating the prevalence, evolution, and ecological roles of these prophages is of great importance as V. harveyi is responsible for luminous bacteriosis in aquatic organisms. In this study, 13 tailed prophages were identified from 55 globally sourced V. harveyi genomes, with prophage–bacterium junctions precisely annotated. These prophages exhibited distinct parasitic mechanisms, including Mu-type transposition, site-specific recombination, and a plasmid-like non-integrated state, reflecting their adaptive plasticity. Proteome-based phylogenetic analysis classified these prophages into eight subfamilies and nine genera, with half representing novel taxonomic singletons. Network analysis of V. harveyi prophages and a large set of prophages across Vibrio species revealed distinct prophage distribution patterns, including broad cross-species dissemination and clade-specific or strain-specific colonization. Further genomic analysis identified homologs of experimentally validated virulence factors associated with motility and biofilm formation, suggesting a potential role of these prophages in enhancing bacterial pathogenicity and adaptive fitness. CRISPR spacer matching provided the intra-species lytic history for 7 out of 13 identified prophages, underscoring their involvement in horizontal transfer of virulence traits. In summary, this study established a comprehensive genomic database of V. harveyi prophages, shedding light on their diversity, prevalence, and parasitic strategies. IMPORTANCE: Understanding how prophages parasitize Vibrio harveyi holds significant commercial implications, given the pathogen’s notoriety for inducing vibriosis across diverse aquatic species and causing substantial economic losses in the global aquaculture industry. We report here 13 well-curated prophage genomes identified from 55 globally collected V. harveyi genomes. Notably, these prophages exhibited previously unrecognized genomic diversity, along with distinct parasitic strategies and hierarchical distribution patterns. In-depth analysis of their genetic profiles identified multiple homologs of experimentally validated virulence determinants involved in regulating bacterial motility and biofilm formation. Lytic history was detected for over half of these prophages, suggesting their role in driving the dissemination of virulence traits within the species.

Vibrio parahaemolyticus, a major foodborne pathogen, contaminates aquatic products and causes significant economic losses in aquaculture while threatening food security and public health. Given the rising prevalence of multidrug-resistant V. parahaemolyticus strains, phage therapy has emerged as an effective and environmentally sustainable alternative for controlling vibriosis in aquaculture. In this study, a virulent bacteriophage, vB_VpaP_R28Z (R28Z), which infects V. parahaemolyticus ATCC 17802T, was isolated and comprehensively characterized. R28Z was isolated from sewage collected at a seafood market in southern China and exhibits pronounced lytic properties against pathogenic Vibrio species prevalent in aquaculture environments, including Vibrio parahaemolyticus, Vibrio diabolicus, and Vibrio alginolyticus. Transmission electron microscopy (TEM) analysis revealed that R28Z belongs to the podophage morphotype. Genomic analysis indicated that R28Z is a double-stranded DNA phage with a genome size of 43,284 bp and a G + C content of 49.34%. Notably, no genes associated with lysogeny, virulence, or antibiotic resistance were detected in R28Z, suggesting its suitability for therapeutic applications. R28Z demonstrated high efficacy in lysing host cells, with a short latent period of less than 10 min and a large burst size of 113 ± 19 PFU/cell (average ± s.d.). Additionally, R28Z exhibited stability over a broad pH range of 4–10 and at temperatures ranging from 4 °C to 50 °C. Taxonomic assessments validated the novelty of R28Z, suggesting that it represents a new species of the Maculvirus genus and enriches the diversity of vibriophage candidates for therapeutic interventions against V. parahaemolyticus infections. The newly discovered phage R28Z, characterized by its high lytic efficiency and environmental stability, represents a novel species as a potential alternative biocontrol agent for managing V. parahaemolyticus infections in aquaculture.

Lysogeny was frequently detected in marine ecosystems, while how temperate phage genomes (prophages) impact marine microbial population or individual dynamics remained poorly understood. Using marine Citromicrobium strain collection as a model system, we revealed that 58% (22/38) were lysogens harboring 31 prophages that can be grouped into five novel genera (φA–φE). Prophage-encoded genes constituted 9% of host accessory genome, significantly expanding the microdiversity among citromicrobial clonal strains. Metagenomic abundance correlations indirectly supported the “Piggyback-the-Winner” dynamics for φA/φE, evidenced by their sublinear growth pattern with increasing host abundance. Most prophages were capable of spontaneous induction and exhibited high lytic activity when triggered by mitomycin C. Importantly, host-range profiling revealed these prophages deployed a dual “Kill-the-Relatives” and “Colonize-the-Relatives” strategy, and meanwhile, they protected parental host strains through superinfection immunity and enhanced phage resistance with greater prophage carriage. Sequencing data showed the dominance of Mu-like phages over non-Mu-like partners upon induced from the double lysogens. Our analysis further hinted at a unique Mu-type within-host competitive strategy: selectively targeting genes of co-resident prophages and host hypothetical genes, while avoiding self-damage and host metabolic genes potentially essential for phage lytic growth or progeny release. Collectively, this work establishes prophages as key architects of bacterial adaptation and provides new perspectives for prophage-driven evolution in marine bacterial hosts.

Caulobacter phage CbK has been extensively studied as a model system in virology and bacteriology. Lysogeny‐related genes have been found in each CbK‐like isolate, suggesting a life strategy of both lytic and lysogenic cycles. However, whether CbK‐related phages can enter lysogeny is still undetermined. This study identified new CbK‐like sequences and expanded the collection of CbK‐related phages. A common ancestry with a temperate lifestyle was predicted for the group, however, which subsequently evolved into two clades of different genome sizes and host associations. Through the examination of phage recombinase genes, alignment of attachment sites on the phage and bacterial genomes ( attP – attB pairing), and the experimental validation, different lifestyles were found among the different members. A majority of clade II members retain a lysogenic lifestyle, whereas all clade I members have evolved into an obligate lytic lifestyle via a loss of the gene encoding Cre‐like recombinase and the coupled attP fragment. We postulated that the loss of lysogeny may be a by‐product of the increase in phage genome size, and vice versa. Clade I is likely to overcome the costs through maintaining more auxiliary metabolic genes (AMGs), particularly for those involved in protein metabolism, to strengthen host takeover and further benefit virion production.

The marine bacterium Alteromonas is prevalent in the global ocean with crucial ecological significance; however, little is known about the diversity and evolution of its bacteriophages that profoundly affect the bacterial communities. Our study characterized a novel genus of three newly isolated Alteromonas phages that exhibited a distinct infection strategy of broad host spectrum and small burst size.

The metabolism of methane in anoxic ecosystems is mainly mediated by methanogens and methane-oxidizing archaea (MMA), key players in global carbon cycling. Viruses are vital in regulating their host fate and ecological function. However, our knowledge about the distribution and diversity of MMA viruses and their interactions with hosts is rather limited. Here, by searching metagenomes containing mcrA (the gene coding for the α-subunit of methyl-coenzyme M reductase) from a wide variety of environments, 140 viral operational taxonomic units (vOTUs) that potentially infect methanogens or methane-oxidizing archaea were retrieved. Four MMA vOTUs (three infecting the order Methanobacteriales and one infecting the order Methanococcales) were predicted to cross-domain infect sulfate-reducing bacteria. By facilitating assimilatory sulfur reduction, MMA viruses may increase the fitness of their hosts in sulfate-depleted anoxic ecosystems and benefit from synthesis of the sulfur-containing amino acid cysteine. Moreover, cell-cell aggregation promoted by MMA viruses may be beneficial for both the viruses and their hosts by improving infectivity and environmental stress resistance, respectively. Our results suggest a potential role of viruses in the ecological and environmental adaptation of methanogens and methane-oxidizing archaea.

This study isolated and characterized a new phage infecting the marine photoheterotrophic bacterium Citromicrobium bathyomarinum, which fills the gap in research on phages targeting this ecologically important species. The phage vB_CbaS-RXM (RXM) has a dsDNA genome with a length of 104,206 bp and G+C content of 61.64%. The taxonomic analysis found a close evolutionary relationship between RXM, Erythrobacter phage vB_EliS-L02, and Sphingobium phage Lacusarx, and we propose that RXM represents a new species of the Lacusarxvirus genus. A one-step growth curve revealed a burst size of 75 plaque-forming units (PFUs) per cell in a 3-hour infection cycle. The lysis profile of RXM showed an intraspecific lethal rate of 26.3% against 38 citromicrobial strains. RXM contains 15 auxiliary metabolic genes (AMGs) related to diverse cellular processes, such as putative metabolic innovation and hijacking of host nucleotide metabolism to enhance its biosynthetic capacity. An in-depth analysis showed that phage functional genes strongly rely on the host for translation, while the translation of unique phage genes with less host dependency may be complemented by phage tRNA. Overall, our study investigated the infection kinetics, genetic traits, taxonomy, and predicted roles of AMGs and tRNA genes of this new phage, which contributes to a better understanding of phage diversity and phage–bacterium interactions.

Bacteriophages substantially contribute to bacterial mortality in the ocean and play critical roles in global biogeochemical processes. Alteromonas is a ubiquitous bacterial genus in global tropical and temperate waters, which can cross-protect marine cyanobacteria and thus has important ecological benefits. However, little is known about the biological and ecological features of Alteromonas phages (alterophages). Here, we describe a novel alterophage vB_AmeP-R8W (R8W), which belongs to the Autographiviridae family and infects the deep-clade Alteromonas mediterranea. R8W has an equidistant and icosahedral head (65 ± 1 nm in diameter) and a short tail (12 ± 2 nm in length). The genome size of R8W is 48,825 bp, with a G + C content of 40.55%. R8W possesses three putative auxiliary metabolic genes encoding proteins involved in nucleotide metabolism and DNA binding: thymidylate synthase, nucleoside triphosphate pyrophosphohydrolase, and PhoB. R8W has a rapid lytic cycle with a burst size of 88 plaque-forming units/cell. Notably, R8W has a wide host range, such that it can infect 35 Alteromonas strains; it exhibits a strong specificity for strains isolated from deep waters. R8W has two specific receptor binding proteins and a compatible holin–endolysin system, which contribute to its wide host range. The isolation of R8W will contribute to the understanding of alterophage evolution, as well as the phage–host interactions and ecological importance of alterophages.