Marine Bacteriophages Research Articles

Marine bacteriophages disturb the associated microbiota of Aurelia aurita with a recoverable effect on host morphology.

The concept of the metaorganism describes a multicellular host and its diverse microbial community, which form one biological unit with a combined genetic repertoire that significantly influences health and survival of the host. The present study delved into the emerging field of bacteriophage research within metaorganisms, focusing on the moon jellyfish Aurelia aurita as a model organism. The previously isolated Pseudomonas phage BSwM KMM1 and Citrobacter phages BSwM KMM2 - KMM4 demonstrated potent infectivity on bacteria present in the A. aurita-associated microbiota. In a host-fitness experiment, Baltic Sea subpopulation polyps were exposed to individual phages and a phage cocktail, monitoring polyp survival and morphology, as well as microbiome changes. The following effects were obtained. First, phage exposure in general led to recoverable malformations in polyps without affecting their survival. Second, analyses of the community structure, using 16S rRNA amplicon sequencing, revealed alterations in the associated microbial community in response to phage exposure. Third, the native microbiota is dominated by an uncultured likely novel Mycoplasma species, potentially specific to A. aurita. Notably, this main colonizer showed resilience through the recovery after initial declines, which aligned with abundance changes in Bacteroidota and Proteobacteria, suggesting a dynamic and adaptable microbial community. Overall, this study demonstrates the resilience of the A. aurita metaorganism facing phage-induced perturbations, emphasizing the importance of understanding host-phage interactions in metaorganism biology. These findings have implications for ecological adaptation and conservation in the rapidly changing marine environment, particularly regarding the regulation of blooming species and the health of marine ecosystems during ongoing environmental changes.

Understanding Viral Impacts in Soil Microbial Ecology Through the Persistence and Decay of Infectious Bacteriophages.

Marine bacteriophages have been well characterized in terms of decay rates, population dynamics in relation to their hosts, and their impacts on biogeochemical cycles in the global ocean. Knowledge in soil bacteriophage ecology lags considerably behind, with few studies documenting population dynamics with hosts and even fewer reporting phage decay rates. By using sterile soil or aquatic microcosms inoculated with single bacteriophage isolates, phage decay rates (loss of infectivity over time) were determined, independent of host interactions, for 5 model phage isolates. Decay rates varied by phage from 0.11-2.07% h-1 in soils to 0.07-0.28% h-1 in aquatic microcosms. For phages incubated in both soil and aquatic microcosms, the observed decay rate was consistently higher in soil microcosms than in aquatic microcosms by at least a factor of two. However, when decay rates for soil phage isolates in the present study were compared to those reported for marine and freshwater phage isolates from previous studies, the decay constants for soil phages were, on average, 4 times lower than those for aquatic phages. Slower rates of phage decay in soils indicate a lower turnover rate, which may have subsequent and potentially far-reaching impacts on virus-mediated mortality and bacterial activity. The wide range of decay rates observed in the present study and the lack of information on this critical aspect of virus-host dynamics in soil emphasizes the need for continued research in this field.

Structural Study of the Cobetia marina Bacteriophage 1 (Carin-1) by Cryo-EM.

Most of studied bacteriophages (phages) are terrestrial viruses. However, marine phages are shown to be highly involved in all levels of oceanic regulation. They are, however, still largely overlooked by the scientific community. By inducing cell lysis on half of the bacterial population daily, their role and influence on the bacterial biomass and evolution, as well as their impact in the global biogeochemical cycles, is undeniable. Cobetia marina virus 1 (Carin-1) is a member of the Podoviridae family infecting the γ-protoabacteria C. marina. Here, we present the almost complete, nearly-atomic resolution structure of Carin-1 comprising capsid, portal, and tail machineries at 3.5 Å, 3.8 Å and 3.9 Å, respectively, determined by cryo-electron microscopy (cryo-EM). Our experimental results, combined with AlphaFold2 (AF), allowed us to obtain the nearly-atomic structure of Carin-1 by fitting and refining the AF atomic models in the high resolution cryo-EM map, skipping the bottleneck of de-novo manual building and speeding up the structure determination process. Our structural results highlighted the T7-like nature of Carin1, as well as several novel structural features like the presence of short spikes on the capsid, reminiscent those described for Rhodobacter capsulatus gene transfer agent (RcGTA). This is, to our knowledge, the first time such assembly is described for a bacteriophage, shedding light into the common evolution and shared mechanisms between gene transfer agents and phages. This first full structure determined for a marine podophage allowed to propose an infection mechanism different than the one proposed for the archetypal podophage T7. IMPORTANCE Oceans play a central role in the carbon cycle on Earth and on the climate regulation (half of the planet's CO2 is absorbed by phytoplankton photosynthesis in the oceans and just as much O2 is liberated). The understanding of the biochemical equilibriums of marine biology represents a major goal for our future. By lysing half of the bacterial population every day, marine bacteriophages are key actors of these equilibriums. Despite their importance, these marine phages have, so far, only been studied a little and, in particular, structural insights are currently lacking, even though they are fundamental for the understanding of the molecular mechanisms of their mode of infection. The structures described in our manuscript allow us to propose an infection mechanism that differs from the one proposed for the terrestrial T7 virus, and might also allow us to, in the future, better understand the way bacteriophages shape the global ecosystem.

Auxiliary Metabolic Gene Functions in Pelagic and Benthic Viruses of the Baltic Sea

Marine microbial communities are facing various ecosystem fluctuations (e.g., temperature, organic matter concentration, salinity, or redox regimes) and thus have to be highly adaptive. This might be supported by the acquisition of auxiliary metabolic genes (AMGs) originating from virus infections. Marine bacteriophages frequently contain AMGs, which allow them to augment their host’s metabolism or enhance virus fitness. These genes encode proteins for the same metabolic functions as their highly similar host homologs. In the present study, we analyzed the diversity, distribution, and composition of marine viruses, focusing on AMGs to identify their putative ecologic role. We analyzed viruses and assemblies of 212 publicly available metagenomes obtained from sediment and water samples across the Baltic Sea. In general, the virus composition in both compartments differed compositionally. While the predominant viral lifestyle was found to be lytic, lysogeny was more prevalent in sediments than in the pelagic samples. The highest proportion of AMGs was identified in the genomes of Myoviridae. Overall, the most abundantly occurring AMGs are encoded for functions that protect viruses from degradation by their hosts, such as methylases. Additionally, some detected AMGs are known to be involved in photosynthesis, 7-cyano-7-deazaguanine synthesis, and cobalamin biosynthesis among other functions. Several AMGs that were identified in this study were previously detected in a large-scale analysis including metagenomes from various origins, i.e., different marine sites, wastewater, and the human gut. This supports the theory of globally conserved core AMGs that are spread over virus genomes, regardless of host or environment.

Characterization and Genomic Analysis of Marinobacter Phage vB_MalS-PS3, Representing a New Lambda-Like Temperate Siphoviral Genus Infecting Algae-Associated Bacteria.

Marinobacter is the abundant and important algal-associated and hydrocarbon biodegradation bacteria in the ocean. However, little knowledge about their phages has been reported. Here, a novel siphovirus, vB_MalS-PS3, infecting Marinobacter algicola DG893(T), was isolated from the surface waters of the western Pacific Ocean. Transmission electron microscopy (TEM) indicated that vB_MalS-PS3 has the morphology of siphoviruses. VB_MalS-PS3 was stable from −20 to 55°C, and with the latent and rise periods of about 80 and 10 min, respectively. The genome sequence of VB_MalS-PS3 contains a linear, double-strand 42,168-bp DNA molecule with a G + C content of 56.23% and 54 putative open reading frames (ORFs). Nineteen conserved domains were predicted by BLASTp in NCBI. We found that vB_MalS-PS3 represent an understudied viral group with only one known isolate. The phylogenetic tree based on the amino acid sequences of whole genomes revealed that vB_MalS-PS3 has a distant evolutionary relationship with other siphoviruses, and can be grouped into a novel viral genus cluster with six uncultured assembled viral genomes from metagenomics, named here as Marinovirus. This study of the Marinobacter phage vB_MalS-PS3 genome enriched the genetic database of marine bacteriophages, in addition, will provide useful information for further research on the interaction between Marinobacter phages and their hosts, and their relationship with algal blooms and hydrocarbon biodegradation in the ocean.

The genomic content and context of auxiliary metabolic genes in roseophages.

Marine bacteriophages frequently possess auxiliary metabolic genes (AMGs) that accelerate host metabolism during phage infection. The significance of AMGs in phage infecting the ecologically important Roseobacter clade, found predominantly in marine environments, remains to be determined. Here, we analysed the distribution and genomic context of 180 AMGs, annotated into 20 types, across 50 roseophage genomes. Roseophages share seven high-frequency AMGs (trx, grx, RNR, thyX, DCD, phoH, and mazG)， most of them involved in the nucleotide biosynthesis pathway that represent conserved intra and inter operational taxonomic units (OTUs), and share ≥97% full-length DNA sequence similarity. Sporadic AMGs (dUTPase, lexA, degS, Que, NAPRT, AHL, pcnB, ctrA, RTX, RNR-nrdA, RNR-nrdE, wclP, and flgJ), present in only one or two OTUs, show high functional diversity. The roseophage AMG repertoire weakly correlates with environmental factors, while host range partially explains the sporadic AMG distribution. Locally co-linear blocks distribution index (LDI) analysis indicated that high-frequency roseopodovirus AMGs are restricted to particular genomic islands, possibly originating from limited historical acquisition events. Low-frequency roseopodovirus AMGs and all roseosiphovirus AMGs have high LDI values, implying multiple historical acquisition events. In summary, roseophages have acquired a range of AMGs through horizontal gene transfer, and the forces shaping the evolution of roseophages are described.

Alteromonas Myovirus V22 Represents a New Genus of Marine Bacteriophages Requiring a Tail Fiber Chaperone for Host Recognition.

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.

Genomic and ecological attributes of marine bacteriophages encoding bacterial virulence genes

BackgroundBacteriophages encode genes that modify bacterial functions during infection. The acquisition of phage-encoded virulence genes is a major mechanism for the rise of bacterial pathogens. In coral reefs, high bacterial density and lysogeny has been proposed to exacerbate reef decline through the transfer of phage-encoded virulence genes. However, the functions and distribution of these genes in phage virions on the reef remain unknown.ResultsHere, over 28,000 assembled viral genomes from the free viral community in Atlantic and Pacific Ocean coral reefs were queried against a curated database of virulence genes. The diversity of virulence genes encoded in the viral genomes was tested for relationships with host taxonomy and bacterial density in the environment. These analyses showed that bacterial density predicted the profile of virulence genes encoded by phages. The Shannon diversity of virulence-encoding phages was negatively related with bacterial density, leading to dominance of fewer genes at high bacterial abundances. A statistical learning analysis showed that reefs with high microbial density were enriched in viruses encoding genes enabling bacterial recognition and invasion of metazoan epithelium. Over 60% of phages could not have their hosts identified due to limitations of host prediction tools; for those which hosts were identified, host taxonomy was not an indicator of the presence of virulence genes.ConclusionsThis study described bacterial virulence factors encoded in the genomes of bacteriophages at the community level. The results showed that the increase in microbial densities that occurs during coral reef degradation is associated with a change in the genomic repertoire of bacteriophages, specifically in the diversity and distribution of bacterial virulence genes. This suggests that phages are implicated in the rise of pathogens in disturbed marine ecosystems.

Isolation and Complete Genome of the Marine Pseudoalteromonas Phage C7 from Coastal Seawater of Yellow Sea, China.

A novel Pseudoalteromonas atlantica phage C7 was isolated from the coastal surface water in the Yellow Sea of China using the agar overlay method. Transmission electron microscopy analysis reveals that it belongs to Siphoviridae with a head of 56nm in diameter and a tail of 102nm in length. Microbiological characterization suggests that the phage is stable at a range of temperatures (from - 20 to 65°C) and the optimal pH range is 6-12. One-step growth curve shows a 45-min latent period and a 105-min rise period. Genomic analysis shows that C7 has a linear, double-stranded 42,261-bp DNA molecule with 40.6% GC content and 76 putative open reading frames (ORFs) with one tRNA. The ORFs are classified into six functional groups as follows: hypothetical protein, phage structure, phage packaging, DNA replication and regulation, transcription, and some additional functions. There are many obviously similarities between C7 and a previously published Pseudoalteromonas phage vB_PspS-H40/1 by genomic comparison. This study provides an important data for further research on the interaction between marine bacteriophages and their hosts.

Order reduction for a model of marine bacteriophage evolution

A typical mechanistic model of viral evolution necessary includes several time scales which can differ by orders of magnitude. Such a diversity of time scales makes analysis of these models difficult. Reducing the order of a model is highly desirable when handling such a model. A typical approach applied to such slow-fast (or singularly perturbed) systems is the time scales separation technique. Constructing the so-called quasi-steady-state approximation is the usual first step in applying the technique. While this technique is commonly applied, in some cases its straightforward application can lead to unsatisfactory results.In this paper we construct the quasi-steady-state approximation for a model of evolution of marine bacteriophages based on the Beretta-Kuang model. We show that for this particular model the quasi-steady-state approximation is able to produce only qualitative but not quantitative fit.

Marine phage genomics: the tip of the iceberg.

Marine viruses are the most abundant biological entity in the oceans, the majority of which infect bacteria and are known as bacteriophages. Yet, the bulk of bacteriophages form part of the vast uncultured dark matter of the microbial biosphere. In spite of the paucity of cultured marine bacteriophages, it is known that marine bacteriophages have major impacts on microbial population structure and the biogeochemical cycling of key elements. Despite the ecological relevance of marine bacteriophages, there are relatively few isolates with complete genome sequences. This minireview focuses on knowledge gathered from these genomes put in the context of viral metagenomic data and highlights key advances in the field, particularly focusing on genome structure and auxiliary metabolic genes.

Complete Genome of a Novel Pseudoalteromonas Phage PHq0.

We isolated and purified a novel virulent Pseudoalteromonas bacteriophage PHq0 and the host bacterium Pseudoalteromonas BQ0 from seawater collected in a coastal area of the Yellow Sea of China. (36°06′N, 120°32′E). Transmission electron microscopy revealed that the phage had an icosahedral head of 50 nm in diameter with a long tail of 100 nm. The one-step growth curve showed the latent period of about 15 min, a rise period of 15 min, and a burst size of about 363 virions. The genome of phage PHq0 was found to consist of a linear, double-stranded 33,399-bp DNA molecule with a GC content of 40.29 % and 56 putative open reading frames (ORFs). Among these genes, 23 conserved domains were detected by BLASTP, 17 were functionally known, leaving 39 unknown putative genes, BLASTP results show that 57.14 % of the 56 predicted ORFs were not found to have any matches of putative functions or conserved domains in the BLASTP database which should be classified as a new member of the Siphoviridae family. The phage PHq0 genome adds a new Siphoviridae-family phage genome for marine bacteriophages which will provide useful basic information for further molecular research on interaction mechanism between bacteriophages and their hosts.

Optimal Foraging Predicts the Ecology but Not the Evolution of Host Specialization in Bacteriophages

We explore the ability of optimal foraging theory to explain the observation among marine bacteriophages that host range appears to be negatively correlated with host abundance in the local marine environment. We modified Charnov's classic diet composition model to describe the ecological dynamics of the related generalist and specialist bacteriophages φX174 and G4, and confirmed that specialist phages are ecologically favored only at high host densities. Our modified model accurately predicted the ecological dynamics of phage populations in laboratory microcosms, but had only limited success predicting evolutionary dynamics. We monitored evolution of attachment rate, the phenotype that governs diet breadth, in phage populations adapting to both low and high host density microcosms. Although generalist φX174 populations evolved even broader diets at low host density, they did not show a tendency to evolve the predicted specialist foraging strategy at high host density. Similarly, specialist G4 populations were unable to evolve the predicted generalist foraging strategy at low host density. These results demonstrate that optimal foraging models developed to explain the behaviorally determined diets of predators may have only limited success predicting the genetically determined diets of bacteriophage, and that optimal foraging probably plays a smaller role than genetic constraints in the evolution of host specialization in bacteriophages.

TEM of Bacteriophages Found in Marine Sources

Abstract We describe here a project to illustrate the diversity and abundance of marine bacteriophages undertaken by two high school students participating in The Scripps Research Institute's High School Student Research Education Program. The students were interns in the Automated Molecular Imaging group over the summer of 2007, during which time they acquired high magnification transmission electron microscopy images of bacteriophage filtered from samples collected from nearby marine waters. The basic protocols for sample collection, grid preparation, and electron microscopy imaging are described in this manuscript along with some of the images of the bacteriophages.

Characterizing Water Circulation and Contaminant Transport in Lake Geneva Using Bacteriophage Tracer Experiments and Limnological Methods

Multi-tracer tests with three types of marine bacteriophages (H4/4, H6/1, and H40/1), together with various limnological methods, including physicochemical depth profiling, surface drifters, deep current measurements, and fecal indicator bacteria analyses, have been applied to characterize water circulation and pathogen transport in the Bay of Vidy (Lake Geneva, Switzerland). The experimental program was carried out twice, first in November 2005, when the lake was stratified, and a second time during holomixis in February 2006. The bacteriophages were injected at three points at different depths, where contaminated waters enter the lake, including the outlet pipe of a wastewater treatment plant, a river, and a stormwater outlet. Thereafter, water samples were collected in the lake at 2 m depth during a 48 h sampling campaign. The results demonstrate that (i) contaminated river water spreads rapidly in the bay; (ii) a well-developed thermocline is highly effective in preventing contamination from the depth to rise up to the surface; (iii) rapid vertical mixing and pathogen transport occur under thermally homogeneous conditions; and (iv) repeated multi-tracertests with bacteriophages are a powerful technique to assess water circulation and contaminant transport in lakes where high dilution occurs.

Metagenomics Offers a Big-Picture View of the Diversity and Distribution of Marine Viruses

Metagenomics Offers a Big-Picture View of the Diversity and Distribution of Marine Viruses

Actinophages as indicators of actinomycete taxa in marine environments

It is necessary to continue to screen for new metabolites and evaluate the potential of less known and new bacterial taxa so that new and improved compounds for future use against drug-resistant bacteria or for chemical modification may be developed. There has been considerable interest in the detection and identification of marine microorganisms since they have been reported to produce bioactive compounds ranging from antitumour to antibacterial and antiviral agents. In this study, an improved technique that involves the exploitation of marine actinophages as indicators of the marine actinomycete taxa and uses marine bacteriophages as tools to reduce the numbers of common marine bacteria, which impedes the growth of rare actinomycetes on isolation plates, has been applied. This technique reduced the numbers of colony forming units of unwanted bacteria on isolation plates and hence increased the chances of detecting novel marine actinomycete genera for isolation and subsequent screening for antiviral activity.

Optimization of procedures for counting viruses by flow cytometry.

The development of sensitive nucleic acid stains, in combination with flow cytometric techniques, has allowed the identification and enumeration of viruses in aquatic systems. However, the methods used in flow cytometric analyses of viruses have not been consistent to date. A detailed evaluation of a broad range of sample preparations to optimize counts and to promote the consistency of methods used is presented here. The types and concentrations of dyes, fixatives, dilution media, and additives, as well as temperature and length of incubation, dilution factor, and storage conditions were tested. A variety of different viruses, including representatives of phytoplankton viruses, cyanobacteriophages, coliphages, marine bacteriophages, and natural mixed marine virus communities were examined. The conditions that produced optimal counting results were fixation with glutaraldehyde (0.5% final concentration, 15 to 30 min), freezing in liquid nitrogen, and storage at -80 degrees C. Upon thawing, samples should be diluted in Tris-EDTA buffer (pH 8), stained with SYBR Green I (a 5 x 10(-5) dilution of commercial stock), incubated for 10 min in the dark at 80 degrees C, and cooled for 5 min prior to analysis. The results from examinations of storage conditions clearly demonstrated the importance of low storage temperatures (at least -80 degrees C) to prevent strong decreases (occasionally 50 to 80% of the total) in measured total virus abundance with time.

Characterization of marine bacteriophages isolated from the Alboran Sea (Western Mediterranean)

It has been shown recently that viruses are important agents for the bacterial mortality in marine ecosystems. In this study, 26 bacteriophages were isolated from the Alboran Sea and subsequently characterized in order to determine the diversity of the viral community in this marine environment. All the host bacteria were Gram-negative motile rods, and were unable to grow in media without NaCl. Bacteriophages contained DNA, were not sensitive to chloroform (except phage 16), and showed a close and narrow host range. Morphological studies revealed that most of them were included in two of the three tailed families of the Caudovirales order (viruses belonging to the Podoviridae family were not observed). All bacteriophages had icosahedral heads, the most abundant size class being those ranging from 30 to 60 nm in diameter. The protein profiles of viral clones showed higher variability than that observed by morphological analyses. Phages were grouped in 11 classes on the basis of the protein patterns. In short, phage-host systems analysed in this study are a diverse group and representative of the marine viral community. For this reason, they can be considered as models in studies on the interaction between viral and bacterial communities.

Degree of ultraviolet radiation damage and repair capabilities are related to G+C content in marine vibriophages

A key issue in the ecology of viruses in the marine environment is the rate of viral production and decay. The ultraviolet (UV) radiation in sunlight has been found to cause loss of infec- tivity in marine bacteriophages at rates nearly equal to all other decay mechanisms combined. There are 2 main host-mediated mechanisms that can repair UV-damaged phage DNA: photoreactivation and excision repair. Both these mechanisms were investigated in 2 marine Vibrio parahaemolyticus hosts as they catalyzed the reactivation of 7 phages. Photoreactivation was the dominant repair mode in all but one case. A significant correlation was found between G+C content of the phage DNAs (16 to 70%) and degree of DNA damage (r = 0.955), indicating a strong relationship between the number of thymine dimer targets and the capability to photoreactivate DNA damage. Evolution of high G+C content may be a strategy for protection from UV damage in marine phages.