Extracellular vesicles (EVs) mediate critical intercellular interactions, yet their special functions in anaerobic denitrification process remain poorly understood. Utilizing the model denitrifier Pseudomonas aeruginosa PAO1, we investigated how environmental oxygen and nitrate gradients reprogram vesicular cargo and functionality. Proteomic profiling revealed a clear condition-specific cargo: aerobically generated EVs selectively packaged virulence factors and nutrient acquisition proteins. In contrast, imposing severe bioenergetic stress via anaerobic denitrification forced a radical compositional inversion, driving a targeted accumulation of with peptidoglycan-degrading enzymes. Functionally, these EVs exhibited a dose-dependent duality. At physiological EV-to-cell ratios, they facilitated transient denitrification activity and early growth in recipient cohorts. Crossing a critical density threshold, however, they transitioned to cytotoxic effectors, suppressing culture density through a cytotoxic activity that emerged post-denitrification. Notably, despite carrying a full suite of denitrification enzymes, purified EVs displayed no intrinsic activity, indicating their primary roles is that of effector carriers rather than independent catalytic units. Furthermore, a substantial fraction of these bioactive nanoparticulates survived sequential multi-barrier municipal drinking water purification processes. Together, these findings redefine EVs not merely as condition-regulated ecological regulators during severe bioenergetic stress, but also as structurally recalcitrant colloidal entities within engineered aquatic environments.

Viruses are ubiquitous biological entities with important roles in microbial mortality, genetic exchange and biogeochemical cycles. Clays, the most abundant mineral constituents on Earth, function as highly effective adsorbents for viruses, critically modulating their environmental persistence and ecological impact. However, the mechanisms and environmental drivers governing virus-clay interactions remain poorly understood. This study systematically investigates the adsorption kinetics of nine marine viruses onto a clay mineral (smectite, NAu-2) under environmentally representative conditions. Viruses isolated from distinct hosts (heterotrophic bacteria, cyanobacteria and eukaryotic algae) and exhibiting diverse morphological features (myovirus, siphovirus, podovirus and tailless virus) were evaluated across gradients of NAu-2/virus concentrations, pH, temperature and ionic strength. Elevated NAu-2 concentrations (> 50 mg/L) induced rapid and near-complete virus adsorption (90%-99%) within 1 min, while low concentrations (0.1-10 mg/L) yielded efficiencies from negligible to > 50%. Adsorption dynamics showed minimal correlation with viral morphology and instead depended on physicochemical properties. Electron microscopy revealed preferential viral localization at edge sites of NAu-2, driven by charge heterogeneity from protonated alumina-oxygen octahedra. These findings provide quantitative information on virus-clay interactions and offer key insights into the availability, transport and fate of viruses in the environment, paving the way for more accurate parameterization of biogeochemical cycling across ecosystems.

Giant viruses have fundamentally expanded our understanding of virology by challenging the conventional boundaries of both virion size and genome complexity. However, the scarcity of isolates has left many of their unique biological features unexplored. Here, we report the isolation and characterization of four new giant virus species belonging to the subfamily Megamimivirinae, sampled from distinct environments across China. Among these, Megavirus daqingense is the first giant virus isolated from an oil reservoir; it exhibits virion stability under high salinity, chloroform exposure, and elevated temperatures, suggesting fitness adaptations to subsurface conditions. Using a hybrid sequencing approach that integrates short- and long-read technologies, we assembled complete linear genomes for all four isolates, each flanked by long terminal inverted repeats (TIRs). Comparative genomic and synteny analyses identified 29 distinct TIRs from 46 megamimivirus genomes. Gene content within these TIRs was highly diverse, with no orthologous proteins conserved across all repeats. Furthermore, TIR genes experienced weaker purifying selection than those in non-TIR regions (i.e., the genomic regions excluding the TIRs), consistent with their role as drivers of genome plasticity. Notably, we discovered for the first time that identical tRNA genes are shared between TIRs and non-TIR regions of eukaryotic viruses. Collectively, our work provides insights into the structural and evolutionary complexity of megamimiviruses, revealing TIRs as reservoirs of genetic diversity and hotspots for gene transfer, thereby playing a pivotal role in shaping the dynamic architecture of giant virus genomes. IMPORTANCE:Terminal inverted repeats (TIRs) are critical structural elements at the termini of linear genomes essential for fundamental processes such as recombination, replication, and integration across diverse organisms. However, the inherent limitations of short-read sequencing technologies have left the complete structure, diversity, and evolutionary significance of long TIRs in giant viruses unexplored. In this study, we leverage hybrid sequencing and comparative genomic analyses to unveil the complexity of TIRs across the subfamily Megamimivirinae. We demonstrate that TIRs are dynamic genomic hotspots characterized by remarkable gene diversity and unexpected conservation of specific tRNA genes. These findings establish TIRs as key drivers of genome plasticity, serving as hotspots for horizontal gene transfer and genetic innovation. By resolving the long-hidden terminal structures of megamimivirus genomes, this work provides a foundational framework for understanding how TIRs shape the evolution of giant viruses and, more broadly, advances our understanding of genome architecture in large DNA viruses.

Giant viruses challenge the traditional definition of viruses due to their large virion sizes, intricate genomes, and enigmatic mechanisms of host modulation. So far, only three giant viruses belonging to the mollivirus group have been identified, resulting in a significant gap in our understanding of the biological properties of mollivirus. In this study, we present the isolation and in-depth characterization of the fourth strain of molliviruses, Mollivirus R33X, which was isolated from a soil sample in the Chinese subtropical zone. Our results demonstrate that the replication cycle of R33X spans approximately 24 h, yielding a burst size of about 266 viral particles per infected cell. R33X particles exhibit tolerance to acidic and high-salinity conditions yet show sensitivity to thermal variations. Its 645-kb genome, featuring inverted terminal repeats and comprising 557 genes, shares an average nucleotide identity of 95.8

Solar radiation plays a pivotal role in shaping viral ecology, fundamentally influencing ecological processes and biogeochemical cycles in aquatic environments. However, the dynamics of viruses in high-altitude lakes with intense solar radiation remain largely unexplored, hindering our understanding of their ecological significance. This study investigates diel and vertical variations in viral abundance, activity, diversity, and community in Lake Nam Co at an altitude of 4700 meters. We assess the effects of solar radiation on viral dynamics in the field through parallel light-transmitting and dark incubation experiments. Our findings reveal that intense solar radiation alters viral life cycles by extending latent periods, promoting lysogenic cycles, and accelerating degradation. Diel variations in viral dynamics are shaped by physicochemical shifts, particularly turbidity and pH changes driven by stream inflows, which buffer the effects of solar radiation and obscure clear diel patterns. Conversely, vertical variations in viral dynamics and community composition are predominantly dictated by solar exposure. This research represents the first comprehensive exploration of viral ecology in a high-altitude lake, significantly advancing our understanding of virus-mediated processes in biogeochemical cycling across alpine lake systems globally.

Marine bacteriophages, the most abundant biological entities in marine ecosystems, are essential in biogeochemical cycling. Despite extensive genomic data, many phage genes remain uncharacterised, creating a gap between genomic diversity and gene function knowledge. This gap limits our understanding of phage life cycles, assembly, and host interactions. In this study, we used mass spectrometry to profile the proteomes of 13 marine phages from diverse lifestyles and hosts. The analysis accurately annotated hypothetical genes, mapped virion protein arrangements, and revealed structural similarities among phages infecting the same host, particularly in tail fibre proteins. Protein structure comparisons showed conservation and variability in head and tail proteins, particularly in key domains involved in virion stabilisation and host recognition. For the first time, we identified post-translational modifications (PTMs) in marine phage proteins, which may enhance phage adaptability and help evade host immune systems. These findings suggest that phages optimise their infection strategies through structural variations and PTM modifications, improving their adaptability and host interactions.

Solar radiation plays a pivotal role in shaping viral ecology, fundamentally influencing ecological processes and biogeochemical cycles in aquatic environments. However, the dynamics of viruses in high-altitude lakes with intense solar radiation remain largely unexplored, hindering our understanding of their ecological significance. This study investigates diel and vertical variations in viral abundance, activity, diversity, and community in Lake Nam Co at an altitude of 4700 meters. We assess the effects of solar radiation on viral dynamics in the field through parallel light-transmitting and dark incubation experiments. Our findings reveal that intense solar radiation alters viral life cycles by extending latent periods, promoting lysogenic cycles, and accelerating degradation. Diel variations in viral dynamics are shaped by physicochemical shifts, particularly turbidity and pH changes driven by stream inflows, which buffer the effects of solar radiation and obscure clear diel patterns. Conversely, vertical variations in viral dynamics and community composition are predominantly dictated by solar exposure. This research represents the first comprehensive exploration of viral ecology in a high-altitude lake, significantly advancing our understanding of virus-mediated processes in biogeochemical cycling across alpine lake systems globally.

BackgroundCyanophages, viruses that infect cyanobacteria, are diverse and ubiquitous in the marine environment and play important roles in regulating the host community's structure, dynamics, and metabolism. Isolation is an efficient method to explore the genetic diversity of cyanophages and their interactions with hosts. However, our understanding of these interactions is still limited, and further in-depth research is needed to address this gap.ResultsIn this study, we report a novel marine T4-like cyanophage, S-SCSM2R, that is able to cross-infect eight picocyanobacterial phylogenetic clades, including three Prochlorococcus clades of both high-light and low-light ecotypes and eight Synechococcus subclades across all five Synechococcus clades of 5.1, 5.2, 5.3, Subalpine II and Bornholm Sea. S-SCSM2R contains novel auxiliary metabolic genes involved in photosynthesis, alleviation of oxidative stress, cell wall synthesis and modification, and antibiotic synthesis. Alongside the receptor-binding protein gene, a set of counter-defense genes related to DNA methylation and NAD+ synthesis provide clues about the broad cross-infectivity of S-SCSM2R.ConclusionsS-SCSM2R has an extremely broad host range and novel genetic features related to phage-host interactions. The discovery and characterization of S-SCSM2R broaden our knowledge of cyanophage cross-infectivity and reveal new ways in which cyanophages manipulate host metabolism in marine ecosystems.

Vibrio alginolyticus poses a common threat to seafood, resulting in substantial losses in aquaculture and increasing the risks to human health through seafood-borne infections. With the emergence of multidrug-resistant strains, more attention has been paid to phages as a potential alternative or supplement to antibiotics for biocontrol. In this study, a siphovirus, named vB_ValS_R23Z (R23Z), was isolated from shrimp aquatic water and showed specific infectivity against V. alginolyticus ATCC 17749T, with a latent period of approximately 60 min and a burst size of 58 ± 16 plaque-forming units per cell. Genome analysis revealed that phage R23Z possesses two lysis genes, with no lysogenic, antibiotic resistance or virulence genes detected, underscoring its high lytic capacity and genetic safety. Notably, phage R23Z had a broad host range, efficiently lysing over 16 Vibrio strains across four species. It performed well across a wide temperature range (4°C-45 °C) and pH range (4-9), and displayed high lytic efficiency against V. alginolyticus ATCC 17749T at all tested multiplicity of infections (0.01, 0.1, 1, 10 and 100), suggesting adaptability and effectiveness to diverse aquaculture conditions. Additionally, in vivo trials revealed that R23Z application as a single phage therapy significantly enhanced shrimp survival rates (>16 %) during V. alginolyticus infection period, underscoring its practical efficacy in aquaculture. These findings position R23Z as a safe, effective, and environmentally adaptable biocontrol agent against Vibrio infections in aquaculture, with significant potential for applications in disease prevention and food safety, either as a standalone treatment or as part of phage cocktails.

Bacteria belonging to the Roseobacter clade are key players in marine ecosystems, contributing significantly to carbon and sulfur cycles. Marine viruses, particularly those targeting Roseobacter, play crucial roles in regulating microbial communities and biogeochemical processes. Despite their importance, phages infecting organisms of the Roseobacter clade remain poorly understood. In this study, a novel roseophage, vB_DshS-R26L (R26L), infecting Dinoroseobacter shibae DFL12T, was isolated and characterized in terms of physiological and genomic properties. R26L has siphovirus morphology with an elongated head and a long, non-flexible tail. The phage has a narrow host range and demonstrates a long infection cycle with a latent period of 3.5 h and a burst size of 22 plaque-forming units (PFU cell- 1). R26L possesses a circular, double-stranded DNA genome of 79,534 bp with a G + C content of 62.6%, encoding a total of 116 open reading frames. Notably, seven auxiliary metabolic genes (AMGs), including those related to phosphate metabolism and queuosine biosynthesis, were identified. Phylogenetic and taxonomic analyses revealed that R26L represents a new genus, with its highest intergenomic similarities being 54.7% to another roseophage (R5C). By elucidating the unique characteristics of R26L, this study highlights the complexity of phage infections and the genomic diversity of roseophages, offering valuable insights into the ecological significance of Roseobacter-phage interactions in marine environments.

Phages can reshape the metabolic network of hosts to support specific requirements for replication during infection. However, metabolomic profiling of phage-elicited host global metabolic alterations and the linkage of phage-encoded auxiliary metabolic genes to these alterations are understudied. In this study, the dynamics of intracellular metabolites of Dinoroseobacter shibae DFL12, a member of marine environmentally and biogeochemically relevant Roseobacter clade, in response to four distinct lytic roseophage infections were investigated. Metabolomic profiling indicated that roseophage infections significantly altered host metabolism in a phage-specific manner. Pathway enrichment analyses showed that the central carbon pathway and DNA, amino acid, and coenzyme metabolism were commonly altered by roseophages, revealing a central role of these pathways in phage replication. Furthermore, clear infection stage-specific host responses were observed, corresponding to different metabolic demands of phage replication in the early and late infection stages. Interestingly, the content of host vitamin B1, which is the essential nutrient provided by D. shibae to its symbiotic microalgae, increased in the early infection stage for most roseophages, implying that phage infection may impact the symbiosis of D. shibae with microalgae. Finally, combined metabolomic and phage genomics analyses showed that roseophages adopt different strategies to expand the host pyrimidine pool (recycling or de novo synthesis of pyrimidine nucleotides), and this difference was likely related to variation in the GC content between phage and host genomes. Collectively, these results highlight the potential importance of phage-specific and infection stage-specific host metabolic reprogramming in marine phage-host interactions, bacteria-microalgae symbiosis, and biogeochemical cycles.

Denitrification is a crucial process in the global nitrogen cycle, in which two functionally equivalent genes, nirS and nirK, catalyse the critical reaction and are usually used as marker genes. The nirK gene can function independently, whereas nirS requires additional genes to encode nitrite reductase and is more sensitive to environmental factors than nirK. However, the ecological differentiation mechanisms of those denitrifying microbial communities and their adaptation strategies to environmental stresses remain unclear. Here, we conducted metagenomic analysis for sediments and bioreactor samples from Lake Donghu, China. We found that nirS-type denitrifying communities had a significantly lower horizontal gene transfer frequency than that of nirK-type denitrifying communities, and nirS gene phylogeny was more congruent with taxonomy than that of nirK gene. Metabolic reconstruction of metagenome-assembled genomes further revealed that nirS-type denitrifying communities have robust metabolic systems for energy conservation, enabling them to survive under environmental stresses. Nevertheless, nirK-type denitrifying communities seemed to adapt to oxygen-limited environments with the ability to utilize various carbon and nitrogen compounds. Thus, this study provides novel insights into the ecological differentiation mechanism of nirS and nirK-type denitrifying communities, as well as the regulation of the global nitrogen cycle and greenhouse gas emissions.

ABSTRACTMarine ecosystems contain an immense diversity of phages, many of which infect cyanobacteria (cyanophage) that are largely responsible for primary productivity. To characterize the genetic diversity and biogeographic distribution of the marine T4-like cyanophage community in the northern South China Sea, the T4-like cyanophage portal protein gene (g20) was amplified. Phylogenetic analysis revealed that marine T4-like cyanophages were highly diverse, with g20 operational taxonomic units being affiliated with five defined clades (Clusters I–V). Cluster II had a wide geographic distribution, Cluster IV was the most abundant in the open sea, and Cluster I was dominant in coastal shelf environments. Our results showed T4-like cyanophages (based on g20) community was generally shaped via heterogeneous selection. Highly variable environmental factors (such as salinity and temperature) can heterogeneously select different cyanophage communities. Nevertheless, the dominant drivers of the T4-like cyanophage community based on the g20 and g23 (T4-like phage major capsid protein gene) were different, probably due to different coverages by the primer sets. Furthermore, the community assembly processes of T4-like cyanophages were affected by host traits (abundance and distribution), viral traits (latent period, burst size, and host range), and environmental properties (temperature and salinity).IMPORTANCECyanophages are abundant and ubiquitous in the oceans, altering population structures and evolution of cyanobacteria, which account for a large portion of global carbon fixation, through host mortality, horizontal gene transfer, and the modulation of host metabolism. However, little is known about the biogeography and ecological drivers that shape the cyanophage community. Here, we use g20 and g23 genes to examine the biogeographic patterns and the assembly mechanisms of T4-like cyanophage community in the northern part of the South China Sea. The different coverages of primer sets might lead to the different dominant drivers of T4-like cyanophage community based on g20 and g23 genes. Our results showed that characteristics of viral traits (latent period, burst size, and host range) and host traits (abundance and distribution) were found to either limit or enhance the biogeographic distribution of T4-like cyanophages. Overall, both virus and host properties are critical to consider when determining rules of community assembly for viruses.

Viruses are major players in the biosphere, yet little is known about their dynamics and life strategies in alpine lakes, particularly those on the Tibetan Plateau. We investigated microbial abundance, viral dynamics, and viral life strategies in 10 high-altitude Tibetan lakes and found that they harbor high levels of active viruses. Salinity was identified as a crucial factor influencing viral abundance, dynamics, as well as viral life strategies. Lytic and lysogenic viral productions in moderate- and high-salinity lakes were significantly higher than those in freshwater lakes. A trade-off between viral life strategies resulted in a switch from lysis to lysogeny in high-salinity lakes. Virus-mediated cell lysis and virus decay in sampled Tibetan lakes could release about 162.72 and 2.84 mu g C L-1 d-1, respectively. These findings revealed the crucial role of viruses in the carbon cycle of Tibetan lakes, and a switch in viral life strategies may impact their contribution to the carbon cycle. The potential impact of salinity changes triggered by climate change on the carbon cycle in alpine lakes worldwide is highlighted.

ABSTRACT Through particles sinking as well as the movement of water masses, a fraction of bacterioplankton and virioplankton can be transported vertically from the surface to the deep oceans, and display significant changes in viral–bacterial interactions. The survival and activity of the sinking prokaryotes and viruses in the deep-sea environment is crucial for our understanding of deep-sea ecosystems and biogeochemical cycles. However, due to the substantial challenge involved in situ deep-sea incubation, the effect of the deep-sea environments on the ecology of surface prokaryotes and viruses is poorly studied. To fill this knowledge gap, we used an in situ deep-sea long-term incubation device to examine the effect of the natural deep-sea environment on the stability and activity of four viruses and their hosts (Prochlorococcus, Synechococcus, and heterotrophic bacteria) isolated from the surface ocean. Our results showed that viral particles had still not decayed completely after in situ incubation for 1 year, with an average retention rate of 5.69% ± 5.13% (ranging from 1.25% to 13.06%) for infectivity and 68.11% ± 40.50% (ranging from 8.55% to 99.08%) for particles. This suggests that surface viruses probably retain long-term infectivity after sinking and may influence deep-sea microbial populations in terms of activity, function, diversity, and community structure through viral–bacterial interactions and ultimately affect deep-sea biogeochemical cycles. IMPORTANCE The survival of the sinking prokaryotes and viruses in the deep-sea environment is crucial for deep-sea ecosystems and biogeochemical cycles. Through an in situ deep-sea long-term incubation device, our results showed that viral particles and infectivity had still not decayed completely after in situ incubation for 1 year. This suggests that, via infection and lysis, surface viruses with long-term infectious activity in situ deep-sea environments may influence deep-sea microbial populations in terms of activity, function, diversity, and community structure and ultimately affect deep-sea biogeochemical cycles, highlighting the need for additional research in this area.

It is commonly recognized that viruses control the composition, metabolism, and evolutionary trajectories of prokaryotic communities, with resulting vital feedback on ecosystem functioning and nutrient cycling in a wide range of ecosystems. Although the deep biosphere has been estimated to be the largest reservoir for viruses and their prokaryotic hosts, the biology and ecology of viruses therein remain poorly understood.The deep virosphere is an enigmatic field of study in which many critical questions are still to be answered.Is the deep virosphere simply a repository for deeply preserved, non-functioning virus particles? Or are deep viruses infectious agents that can readily infect suitable hosts and subsequently shape microbial populations and nutrient cycling? Can the cellular content released by viral lysis, and even the organic structures of virions themselves, serve as the source of bioavailable nutrients for microbial activity in the deep biosphere as in other ecosystems? In this review, we synthesize our current knowledge of viruses in the deep biosphere and seek to identify topics with the potential for substantial discoveries in the future.

Tailed bacteriophages (order, Caudovirales) account for the majority of all phages. However, the long flexible tail of siphophages hinders comprehensive investigation of the mechanism of viral gene delivery. Here, we report the atomic capsid and in-situ structures of the tail machine of the marine siphophage, vB_DshS-R4C (R4C), which infects Roseobacter. The R4C virion, comprising 12 distinct structural protein components, has a unique five-fold vertex of the icosahedral capsid that allows genome delivery. The specific position and interaction pattern of the tail tube proteins determine the atypical long rigid tail of R4C, and further provide negative charge distribution within the tail tube. A ratchet mechanism assists in DNA transmission, which is initiated by an absorption device that structurally resembles the phage-like particle, RcGTA. Overall, these results provide in-depth knowledge into the intact structure and underlining DNA delivery mechanism for the ecologically important siphophages.

The unicellular picocyanobacterium Prochlorococcus is the most abundant photoautotroph and contributes substantially to global CO2 fixation. In the vast euphotic zones of the open ocean, Prochlorococcus converts CO2 into organic compounds and supports diverse organisms, forming an intricate network of interactions that regulate the magnitude of carbon cycling and storage in the ocean. An understanding of the biological interactions with Prochlorococcus is critical for accurately estimating the contributions of Prochlorococcus and interacting organisms to the marine carbon cycle. This review synthesizes the primary production contributed by Prochlorococcus in the global ocean. We outline recent progress on the interactions of Prochlorococcus with heterotrophic bacteria, phages, and grazers that multifacetedly determine Prochlorococcus carbon production and fate. We discuss that climate change might affect the biological interactions with Prochlorococcus and thus the marine carbon cycle.

Vibriosis is one of the most common bacterial diseases that cause high rates of mortality and considerable economic losses in aquaculture. Phage therapy has been considered as a promising alternative method to antibiotics in the biocontrol of infectious diseases. Genome sequencing and characterization of the phage candidates are prerequisites before field applications to ensure environmental safety. In this study, a lytic phage, named vB_VhaS-R18L (R18L), was isolated from the coastal seawater of Dongshan Island, China. The phage was characterized in terms of morphology, genetic content, infection kinetics, lytic profile, and virion stability. Transmission electronic microscopy indicated that R18L is siphovirus-like, comprising an icosahedral head (diameter 88.6 +/- 2.2 nm) and a long noncontractile tail (225 x 11 nm). Genome analysis indicated R18L to be a double-stranded DNA virus with a genome size of 80,965 bp and a G + C content of 44.96%. No genes that encode known toxins or genes implicated in lysogeny control were found in R18L. A one-step growth experiment showed that R18L had a latent period of approximately 40 min and a burst size of 54 phage particles per infected cell. R18L showed lytic activity against a wide range of at least five Vibrio species (V. alginolyticus, V. cholerae, V. harveyi, V. parahemolyticus, and V. proteolyticus). R18L was relatively stable at pH 6-11 and at temperatures ranging from 4 degrees C to 50 degrees C. The broad lytic activity across Vibrio species and the stability in the environment make R18L a potential candidate for phage therapy in controlling vibriosis in aquaculture systems.

Cyanophages affect the abundance, diversity, metabolism, and evolution of picocyanobacteria in marine ecosystems. Here we report an estuarine Synechococcus phage, S-CREM2, which represents a novel viral genus and leads to the establishment of a new T4-like cyanophage clade named cluster C. S-CREM2 possesses the longest tail (~418 nm) among isolated cyanomyoviruses and encodes six tail-related proteins that are exclusively homologous to those predicted in the cluster C cyanophages. Furthermore, S-CREM2 may carry three regulatory proteins in the virion, which may play a crucial role in optimizing the host intracellular environment for viral replication at the initial stage of infection. The cluster C cyanophages lack auxiliary metabolic genes (AMGs) that are commonly found in cyanophages of the T4-like clusters A and B and encode unique AMGs like an S-type phycobilin lyase gene. A variation in the composition of tRNA and cis-regulatory RNA genes was observed between the marine and freshwater phage strains in cluster C, reflecting their different modes of coping with hosts and habitats. The cluster C cyanophages are widespread in estuarine and coastal regions and exhibit equivalent or even higher relative abundance compared to those of clusters A and B cyanophages in certain estuarine regions. The isolation of cyanophage S-CREM2 provides new insights into the phage–host interactions mediated by both newly discovered AMGs and virion-associated proteins and emphasizes the ecological significance of cluster C cyanophages in estuarine environments.