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Virus-like particle (VLP) is a term that has been in use for about 80 years. Usually, VLP has meant a particle that is like a virus, generally by appearance, but without either proven or actual virus functionality. Initially VLP referred to particles seen in electron microscope images of tissues. More recently, VLP has come to mean other things to other researchers. A key divergence has been use of VLP in association with vaccine and biotechnology applications versus use of VLP in enumeration of viruses in environmental samples. To these viral ecologists, a VLP is a particle that is virus sized, has nucleic acid, and could be a functional virus. But to vaccine developers and biotechnology researchers a VLP instead is a viral structure that intentionally lacks a viral genome. In this study, we look at the history of use of VLP, following changes in meaning as the technology to study VLPs changed.

Biological wastewater treatment (BWT) is currently the most widely applied approach for treating wastewater. The performance of BWT systems depends on the complex microbial communities they support. Although bacteriophages (phages), which are the viruses that infect prokaryotes, are recognized as the most abundant life entities, understanding of their ecological roles in BWT systems remains limited. Here, we review recent progress in phage-associated researches in BWT systems, including the interactions between phage and host, polyvalent phages, the influence of phage activity on BWT performance, and the potential applications of phage-based control for sludge bulking/foaming and pathogens. The challenges and perspectives of phage ecology are also outlined, which are expected to provide implications for future research and applications.Key points• Phage-host interactions in BWT systems are summarized• Impacts of phage activities on BWT performance• Potential applications of phages in BWT systems.

Temperate phages play important roles in bacterial communities but have been largely overlooked, particularly in non-pathogenic bacteria. In rhizobia the presence of temperate phages has the potential to have significant ecological impacts but few examples have been described. Here we characterize a novel group of 5 Rhizobium leguminosarum prophages, capable of sustaining infections across a broad host range within their host genus. Genome comparisons identified further putative prophages infecting multiple Rhizobium species isolated globally, revealing a wider family of 10 temperate phages including one previously described lytic phage, RHEph01, which appears to have lost the ability to form lysogens. Phylogenetic discordance between prophage and host phylogenies suggests a history of active mobilization between Rhizobium lineages. Genome comparisons revealed conservation of gene content and order, with the notable exception of an approximately 5 kb region of hypervariability, containing almost exclusively hypothetical genes. Additionally, several horizontally acquired genes are present across the group, including a putative antirepressor present only in the RHEph01 genome, which may explain its apparent inability to form lysogens. In summary, both phenotypic and genomic comparisons between members of this group of phages reveals a clade of viruses with a long history of mobilization within and between Rhizobium species.

Phage bacteria interactions can affect structure, dynamics, and function of microbial communities. In the context of biological wastewater treatment (BWT), the presence of phages can alter the efficiency of the treatment process and influence the quality of the treated effluent. The active role of phages in BWT has been demonstrated, but many questions remain unanswered regarding the diversity of phages in these engineered environments, the dynamics of infection, the determination of bacterial hosts, and the impact of their activity in full-scale processes. A deeper understanding of the phage ecology in BWT can lead the improvement of process monitoring and control, promote higher influent quality, and potentiate the use of phages as biocontrol agents. In this review, we highlight suitable methods for studying phages in wastewater adapted from other research fields, provide a critical overview on the current state of knowledge on the effect of phages on structure and function of BWT bacterial communities, and highlight gaps, opportunities, and priority questions to be addressed in future research.

Food fermentations rely on the application of robust bacterial starter cultures, the majority of which are represented by members of the lactic acid bacteria including Lactococcus lactis and Streptococcus thermophilus. Bacteriophage (or phage) proliferation remains one of the most significant threats to the fermentation industry. Therefore, it is imperative to define the phage ecology of fermented foods and to elucidate the mechanisms by which they recognize and bind to their bacterial hosts. Through a combination of functional and comparative genomics and structural analysis of the phage-host interactome, it is now possible to link the genotypes of strains of certain bacterial species to the chemical composition/structure of the associated cell wall polysaccharides (CWPS). In this paper, I discuss how the identification of common host recognition and binding strategies facilitates the development of rational starter culture systems and the implications of these findings in the context of sustainable food production systems.

The bacteriophages (phages) in the watery kimchis (Baek-kimchi and Dongchimi) were characterized to determine the phage ecology of lactic acid bacteria (LAB). Kimchi obtained from the Seoul markets had an average of 2.1 log phage particles/mL, corresponding to 28% of the bacterial counts on a log scale. High counts of 5.5–6.5 log particles/mL of phages were noted in the early phase of fermentation (reaching pH 4), and 2.1–3.0 log phage particles/mL were found in the later phase, with some fluctuation in numbers. The LAB hosts changed from Weissella and Leuconostoc to Lactobacillus during Dongchimi fermentation. Fifteen phages, except for those of Lactobacillus, were isolated from diverse strains in the early phase. Five Weissella phages were Podoviridae, and all 10 Leuconostoc phages were Myoviridae. Phages had narrow and different host infection spectra to strains of the same species and high acidic stability. Therefore, the mortality and diversity of LAB during natural kimchi fermentation may be related to the specific phages of the hosts. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10068-021-00930-y.

Bacteria-phage symbioses are ubiquitous in nature and serve as valuable biological models. Historically, the ecology and evolution of bacteria-phage systems have been studied in either very simple or very complex communities. Although both approaches provide insight, their shortcomings limit our understanding of bacteria and phages in multispecies contexts. To address this gap, here we synthesize the emerging body of bacteria-phage experiments in medium-complexity communities, specifically those that manipulate bacterial community presence. Generally, community presence suppresses both focal bacterial (phage host) and phage densities, while sometimes altering bacteria-phage ecological interactions in diverse ways. Simultaneously, community presence can have an array of evolutionary effects. Sometimes community presence has no effect on the coevolutionary dynamics of bacteria and their associated phages, whereas other times the presence of additional bacterial species constrains bacteria-phage coevolution. At the same time, community context can alter mechanisms of adaptation and interact with the pleiotropic consequences of (co)evolution. Ultimately, these experiments show that community context can have important ecological and evolutionary effects on bacteria-phage systems, but many questions still remain unanswered and ripe for additional investigation.

Bacteriophages or phages, the viruses of bacteria, are abundant components of most ecosystems, including those where bacteria predominantly occupy biofilm niches. Understanding the phage impact on bacterial biofilms therefore can be crucial toward understanding both phage and bacterial ecology. Here, we take a critical look at the study of bacteriophage interactions with bacterial biofilms as carried out in vitro, since these studies serve as bases of our ecological and therapeutic understanding of phage impacts on biofilms. We suggest that phage-biofilm in vitro experiments often may be improved in terms of both design and interpretation. Specific issues discussed include (a) not distinguishing control of new biofilm growth from removal of existing biofilm, (b) inadequate descriptions of phage titers, (c) artificially small overlying fluid volumes, (d) limited explorations of treatment dosing and duration, (e) only end-point rather than kinetic analyses, (f) importance of distinguishing phage enzymatic from phage bacteriolytic anti-biofilm activities, (g) limitations of biofilm biomass determinations, (h) free-phage interference with viable-count determinations, and (i) importance of experimental conditions. Toward bettering understanding of the ecology of bacteriophage-biofilm interactions, and of phage-mediated biofilm disruption, we discuss here these various issues as well as provide tips toward improving experiments and their reporting.

The majority of viruses within the human gut are obligate bacterial viruses known as bacteriophages (phages)1. Their bacteriotropism underscores the study of phage ecology in the gut, where they sustain top–down control2—4 and co–evolve5 with gut bacterial communities. Traditionally, these were investigated empirically via in vitro experimental evolution6—8 and more recently, in vivo models were adopted to account for gut niche effects4,9. Here, we probed beyond conventional phage–bacteria co–evolution to investigate the potential evolutionary interactions between phages and the mammalian ″host″. To capture the role of the mammalian host, we recapitulated a life–like mammalian gut mucosa using in vitro lab–on–a–chip devices (to wit, the gut–on–a–chip) and showed that the mucosal environment supports stable phage–bacteria co–existence. Next, we experimentally evolved phage populations within the gut–on–a–chip devices and discovered that phages adapt by de novo mutations and genetic recombination. We found that a single mutation in the phage capsid protein Hoc — known to facilitate phage adherence to mucus10 — caused altered phage binding to fucosylated mucin glycans. We demonstrated that the altered glycan–binding phenotype provided the evolved mutant phage a competitive fitness advantage over their ancestral wildtype phage in the gut–on–a–chip mucosal environment. Collectively, our findings revealed that phages — in addition to their evolutionary relationship with bacteria — are also able to engage in evolution with the mammalian host.

7 páginas, 2 figuras, Phage satellites are genetic elements that depend on helper phages for induction, packaging and transfer. To promote their lifestyles, they have evolved elegant and sophisticated strategies to inhibit phage reproduction, which will be reviewed here. We will principally focus on the convergent interference mechanisms used by phage-inducible chromosomal islands (PICIs), which are a family of satellite phages present in both Gram-positive and Gram-negative bacteria. While some PICI elements have been extensively studied for their roles in virulence and antibiotic resistance, recent studies have highlighted their relevance in controlling phage ecology and diversity. In many cases, these interference mechanisms are complemented by additional strategies that promote the preferential PICI packaging and dissemination of these elements in nature. Since the PICI-encoded mechanisms target conserved phage mechanisms, we propose here that the PICIs form part of the initial innate immune system that phages must overcome to infect their bacterial host., This work was supported by grants MR/M003876/1 and MR/S00940X/1 from the Medical Research Council (UK), BB/N002873/1 and BB/S003835/1 from the Biotechnology and Biological Sciences Research Council (BBSRC, UK), Wellcome Trust201531/Z/16/Z, and ERC-ADG-2014 Proposal n 670932 Dut-signal from EU to J.R.P. L.M-R is the recipient of a Spanish postdoctoral fellowship from Fundacio´n Ramo´n Areces. J.R.P. is thankful to the Royal Society and the Wolfson Foundation for providing him support through a Royal Society Wolfson Fellowship.

Virus-host dynamics is poorly understood in natural systems, despite known viral influences on host mortality and community structure, food web dynamics, and biogeochemical cycles. Without a universal marker gene for viruses, assessments of viral diversity and dynamics have previously relied upon models and/or low-resolution data, like viral counts or gel electrophoretic estimates of genome size. Here, de novo assemblies were created from 6.4 Gb of metagenomic sequence from eight community viral concentrate samples, collected from 12 hours to three years apart from hypersaline Lake Tyrrell (LT), Victoria, Australia. Seven complete and 133 partial, novel viral genomes were reconstructed, and new methods for assessing the diversity and dynamics of full viral assemblages were developed. This dissertation provides the first constraints on the timescales over which viral populations and assemblages tend to be stable (days) or dynamic (months-to-years). Comparisons to and reanalyses of previously reported haloviral metagenomes confirm that similar dynamics exist in other hypersaline systems and suggest that most haloviral populations have a limited or temporally variable global distribution. To place the LT viral metagenomes (viromes) in context, metagenomic data were assembled from 63 previously reported viromes from diverse environments, and genetic composition was compared across viromes. Despite spatial and temporal variation within LT, LT viral assemblages were most similar to each other and grouped with other hypersaline viromes. The 71 viromes (including eight from LT) generally clustered by ecosystem, and salinity is inferred to be a major determinant of the genetic composition of viral assemblages.To further investigate LT microbial ecology, virus-host dynamics were assessed across 17 LT samples, including the eight summer samples from which viral concentrates were sequenced, five additional summer samples, and four winter samples. Contrary to previous reports of microbial population stability in hypersaline systems, dynamics were observed in host populations on similar spatial and temporal scales as in viral populations. An analysis of clustered regularly interspaced short palindromic repeat (CRISPR) regions, which confer host immunity to viruses, indicates that both rare and highly abundant LT viruses were targeted, primarily by lower abundance host organisms. Although very few CRISPR spacers had hits to the NCBI nr database and to the 140 complete and near-complete LT viral genomes, 21% had hits to unassembled LT viral concentrate reads, indicating adaptation to the LT system and successful CRISPR maintenance of viral populations at low enough abundance to preclude metagenomic assembly. In addition to using model systems like Lake Tyrrell to understand fundamental aspects of microbial ecology, it is important to characterize understudied ecosystems, including subsurface systems, which are likely to harbor much of the carbon on Earth and have the potential to play important roles in solutions to human-induced climate change. In order to characterize the community structure and metabolic potential of a high-CO2 subsurface ecosystem, water was collected from iron-rich, CO2-driven Crystal Geyser (Green River, Utah), an established natural analog for geologic carbon sequestration (i.e., the proposed storage of anthropogenic CO2 in subsurface aquifers to mitigate climate change). Metagenomic sequences (~1.3 Gb) were generated from the 0.2 - 3.0 µm size fraction, and metagenomic assembly and binning resulted in the reconstruction of near-complete genomes of neutrophilic, iron-oxidizing Mariprofundus sp. and sulfur-oxidizing Thiomicrospira crunogena. Significant assembly was also achieved for a number of other organisms, including novel Bacterial phyla and both aerobic and anaerobic respirers predicted to oxidize iron, sulfur, and/or complex carbon. At least one-third of the microorganisms sampled were likely chemoautotrophs, demonstrating that microbial carbon fixation is an important component of the carbon cycle in this system. These results suggest that the biogeochemistry and carbon storage potential of subsurface carbon sequestration reservoirs could be significantly influenced by microorganisms of diverse phylogeny and metabolic potential.This dissertation demonstrates that next-generation sequencing, de novo metagenomic assembly, and novel analytical techniques can provide answers to important and fundamental questions in the field of microbial ecology. Specifically, this dissertation demonstrates the application of these techniques to: 1) assess viral diversity and dynamics at the highest resolution to date, 2) place those dynamics in the context of a natural ecosystem, and 3) characterize the community structure and metabolic potential of an understudied subsurface ecosystem with relevance to anthropogenic climate change.

Just as the expansion in genome sequencing has revealed and permitted the exploitation of phylogenetic signals embedded in bacterial genomes, the application of metagenomics has begun to provide similar insights at the ecosystem level for microbial communities. However, little is known regarding this aspect of bacteriophage associated with microbial ecosystems, and if phage encode discernible habitat-associated signals diagnostic of underlying microbiomes. Here we demonstrate that individual phage can encode clear habitat-related 'ecogenomic signatures', based on relative representation of phage-encoded gene homologues in metagenomic data sets. Furthermore, we show the ecogenomic signature encoded by the gut-associated ɸB124-14 can be used to segregate metagenomes according to environmental origin, and distinguish 'contaminated' environmental metagenomes (subject to simulated in silico human faecal pollution) from uncontaminated data sets. This indicates phage-encoded ecological signals likely possess sufficient discriminatory power for use in biotechnological applications, such as development of microbial source tracking tools for monitoring water quality.

If there is something we have learned from the antibiotic era, it is that indiscriminate use of a therapeutic agent without a clear understanding of its long-term evolutionary impact can have enormous health repercussions. This knowledge is particularly relevant when the therapeutic agents are remarkably adaptable and diverse biological entities capable of a plethora of interactions, most of which remain largely unexplored. Although phage therapy (PT) undoubtedly holds the potential to save lives, its current efficacy in case studies recalls the golden era of antibiotics, when these compounds were highly effective and the possibility of them becoming ineffective seemed remote. Safe PT schemes depend on our understanding of how phages interact with, and evolve in, highly complex environments. Here, we summarize and review emerging evidence in a commonly overlooked theme in PT: bacteria-phage interactions. In particular, we discuss the influence of quorum sensing (QS) on phage susceptibility, the consequent role of phages in modulating bacterial cooperation, and the potential implications of this relationship in PT, including how we can use this knowledge to inform PT strategies. We highlight that the influence of QS on phage susceptibility seems to be widespread but can have contrasting outcomes depending on the bacterial host, underscoring the need to thoroughly characterize this link in various bacterial models. Furthermore, we encourage researchers to exploit competition experiments, experimental evolution, and mathematical modeling to explore this relationship further in relevant infection models. Finally, we emphasize that long-term PT success requires research on phage ecology and evolution to inform the design of optimal therapeutic schemes.

Huanglongbing (HLB) disease is threatening citrus production worldwide. The causative agent is “Candidatus Liberibacter asiaticus.” Prior work using mapping-based approaches identified prophage-like sequences in some “Ca. Liberibacter asiaticus” genomes but not all. Here, we utilized a de novo approach that expands the number of prophage-like elements found in “Ca. Liberibacter asiaticus” from 16 to 33 and identified at least one prophage-like sequence in all “Ca. Liberibacter asiaticus” strains. Furthermore, we identified a prophage-like sequence type that is a remnant of an integrated prophage—expanding the number of prophage types in “Ca. Liberibacter asiaticus” from 3 to 4. Overall, the findings will help researchers investigate the role of prophage in the ecology, evolution, and pathogenicity of “Ca. Liberibacter asiaticus.”, Huanglongbing disease (HLB; yellow shoot disease) is a severe worldwide infectious disease for citrus family plants. The pathogen “Candidatus Liberibacter asiaticus” is an alphaproteobacterium of the Rhizobiaceae family that has been identified as the causative agent of HLB. The virulence of “Ca. Liberibacter asiaticus” has been attributed, in part, to prophage-carried genes. Prophage and prophage-like elements have been identified in 12 of the 15 available “Ca. Liberibacter asiaticus” genomes and are classified into three prophage types. Here, we reexamined all 15 “Ca. Liberibacter asiaticus” genomes using a de novo prediction approach and expanded the number of prophage-like elements from 16 to 33. Further, we found that all of the “Ca. Liberibacter asiaticus” genomes contained at least one prophage-like sequence. Comparative analysis revealed a prevalent, albeit previously unknown, prophage-like sequence type that is a remnant of an integrated prophage. Notably, this remnant prophage is found in the Ishi-1 “Ca. Liberibacter asiaticus” strain that had previously been reported as lacking prophages. Our findings provide both a resource for data and new insights into the evolutionary relationship between phage and “Ca. Liberibacter asiaticus” pathogenicity. IMPORTANCE Huanglongbing (HLB) disease is threatening citrus production worldwide. The causative agent is “Candidatus Liberibacter asiaticus.” Prior work using mapping-based approaches identified prophage-like sequences in some “Ca. Liberibacter asiaticus” genomes but not all. Here, we utilized a de novo approach that expands the number of prophage-like elements found in “Ca. Liberibacter asiaticus” from 16 to 33 and identified at least one prophage-like sequence in all “Ca. Liberibacter asiaticus” strains. Furthermore, we identified a prophage-like sequence type that is a remnant of an integrated prophage—expanding the number of prophage types in “Ca. Liberibacter asiaticus” from 3 to 4. Overall, the findings will help researchers investigate the role of prophage in the ecology, evolution, and pathogenicity of “Ca. Liberibacter asiaticus.”

Contains fulltext : 202676.pdf (Publisher’s version ) (Closed access) The host range of a bacteriophage is the taxonomic diversity of hosts it can successfully infect. Host range, one of the central traits to understand in phages, is determined by a range of molecular interactions between phage and host throughout the infection cycle. While many well studied model phages seem to exhibit a narrow host range, recent ecological and metagenomics studies indicate that phages may have specificities that range from narrow to broad. There is a growing body of studies on the molecular mechanisms that enable phages to infect multiple hosts. These mechanisms, and their evolution, are of considerable importance to understanding phage ecology and the various clinical, industrial, and biotechnological applications of phage. Here we review knowledge of the molecular mechanisms that determine host range, provide a framework defining broad host range in an evolutionary context, and highlight areas for additional research.

Despite the abundance, ubiquity and impact of environmental viruses, their inherent genomic plasticity and extreme diversity pose significant challenges for the examination of bacteriophages on Earth. Viral metagenomic studies have offered insight into broader aspects of phage ecology and repeatedly uncover genes to which we are currently unable to assign function. A combined effort of phage isolation and metagenomic survey of Chicago&rsquo, s nearshore waters of Lake Michigan revealed the presence of Pbunaviruses, relatives of the Pseudomonas phage PB1. This prompted our expansive investigation of PB1-like phages. Genomic signatures of PB1-like phages and Pbunaviruses were identified, permitting the unambiguous distinction between the presence/absence of these phages in soils, freshwater and wastewater samples, as well as publicly available viral metagenomic datasets. This bioinformatic analysis led to the de novo assembly of nine novel PB1-like phage genomes from a metagenomic survey of samples collected from Lake Michigan. While this study finds that Pbunaviruses are abundant in various environments of Northern Illinois, genomic variation also exists to a considerable extent within individual communities.

Bacteriophages are the most abundant and diverse biological entities on the planet, and new phage genomes are being discovered at a rapid pace. As more phage genomes are published, new methods are needed for placing these genomes in an ecological and evolutionary context. Phages are difficult to study by phylogenetic methods, because they exchange genes regularly, and no single gene is conserved across all phages. Here, we demonstrate how gene-level networks can provide a high-resolution view of phage genetic diversity and offer a novel perspective on virus ecology. We focus our analyses on virus host range and show how network topology corresponds to host relatedness, how to find groups of genes with the strongest host-specific signatures, and how this perspective can complement phage host prediction tools. We discuss extensions of gene network analysis to predicting the emergence of phages on new hosts, as well as applications to features of phage biology beyond host range., IMPORTANCE Bacteriophages (phages) are viruses that infect bacteria, and they are critical drivers of bacterial evolution and community structure. It is generally difficult to study phages by using tree-based methods, because gene exchange is common, and no single gene is shared among all phages. Instead, networks offer a means to compare phages while placing them in a broader ecological and evolutionary context. In this work, we build a network that summarizes gene sharing across phages and test how a key constraint on phage ecology, host range, corresponds to the structure of the network. We find that the network reflects the relatedness among phage hosts, and phages with genes that are closer in the network are likelier to infect similar hosts. This approach can also be used to identify genes that affect host range, and we discuss possible extensions to analyze other aspects of viral ecology.

Background Rumen microbes play a greater role in host energy acquisition than that of gut-associated microbes in monogastric animals. Although genome-enabled advancements are providing access to the vast diversity of uncultivated microbes, our understanding of variables shaping rumen microbial communities is in its infancy. Viruses have been shown to impact microbial populations through a myriad of processes, including cell lysis and reprogramming of host metabolism. However, little is known about the processes shaping the distribution of rumen viruses or how viruses may modulate microbial-driven processes in the rumen. To this end, we investigated how rumen bacterial and viral community structure and function responded in five steers fed four randomized dietary treatments in a crossover design. Results Total digestible nutrients (TDN), a measure of dietary energy, best explained the variation in bacterial and viral communities. Additional ecological drivers of viral communities included dietary zinc content and microbial functional diversity. Using partial least squares regression, we demonstrate significant associations between the abundances of 267 viral populations and variables driving the variation in rumen viral communities. While rumen viruses were dynamic, 14 near ubiquitous viral populations were identified, suggesting the presence of a core rumen virome largely comprised of novel viruses. Moreover, analysis of virally encoded auxiliary metabolic genes (AMGs) indicates rumen viruses have glycosidic hydrolases to potentially augment the breakdown of complex carbohydrates to increase energy production. Other AMGs identified have a role in redirecting carbon to the pentose phosphate pathway and one carbon pools by folate to boost viral replication. Conclusions We demonstrate that rumen bacteria and viruses have differing responses and ecological drivers to dietary perturbation. Our results show that rumen viruses have implications for understanding the structuring of the previously identified core rumen microbiota and impacting microbial metabolism through a vast array of AMGs. AMGs in the rumen appear to have consequences for microbial metabolism that are largely in congruence with the current paradigm established in marine systems. This study provides a foundation for future hypotheses regarding the dynamics of viral-mediated processes in the rumen. Electronic supplementary material The online version of this article (10.1186/s40168-017-0374-3) contains supplementary material, which is available to authorized users.

Advances in phage therapy and novel applications of phages in biotechnology encourage interest in phage impact on human and animal immunity. Here we present comparative studies of immunogenic properties of T4 phage head surface proteins gp23*, gp24*, Hoc, and Soc, both as elements of the phage capsid and as isolated agents. Studies comprise evaluation of specific antibodies in the human population, analysis of the proteins' impact on the primary and secondary responses in mice, and the effect of specific antibodies on phage antibacterial activity in vitro and in vivo in mice. In humans, natural antibodies specific to T4-like phages were abundant (81% of investigated sera). Among those, significantly elevated levels of IgG antibodies only against major head protein (gp23*) were found, which probably reflected cross-reactions of T4 with antibodies induced by other T4-like phages. Both IgM and IgG antibodies were induced mostly by gp23* and Hoc, while weak (gp24*) and very weak (Soc) reactivities of other head proteins were noticed. Thus, T4 head proteins that markedly contribute to immunological memory to the phage are highly antigenic outer capsid protein (Hoc) and major capsid protein (gp23*). Specific anti-gp23* and anti-Hoc antibodies substantially decreased T4 phage activity in vitro and to some extent in vivo . Cooperating with antibodies, the immune complement system also contributed to annihilating phages. IMPORTANCE Current descriptions of phage immunogenicity and its biological consequences are still vague and incomplete; thus, the central problem of this work is timely and may have strong practical implications. Here is presented the very first description of the contribution of bacteriophage proteins to immunological memory of the phage. Understanding of interactions between phages and mammalian immunology may help in biotechnological adaptations of phages for therapeutic requirements as well as for better appreciation of phage ecology and their role in the biosphere.

To reduce high-salt waste from cucumber fermentations, low-salt fermentations are under development. These fermentations may require the use of starter cultures to ensure normal fermentations. Because potential phage infection can cause starter culture failure, it is important to understand phage ecology in the fermentations. This study investigated the phage ecology in a commercial cucumber fermentation. Brine samples taken from a fermentation tank over a 90-day period were plated onto deMan-Rogosa-Sharpe agar plates. A total of 576 lactic acid bacterial isolates were randomly selected to serve as potential hosts for phage isolation. Filtered brine served as a phage source. Fifty-seven independent phage isolates were obtained, indicating that 10% of the bacterial isolates were sensitive to phage attack. Phage hosts include Lactobacillus brevis (67% of all hosts), Lactobacillus plantarum (21%), Weissella paramesenteroides, Weissella cibaria, and Pediococcus ethanolidurans. Nearly 50% of phages were isolated on day 14, and the majority of them attacked L. brevis. Some phages had a broad host range and were capable of infecting multiple hosts in two genera. Other phages were species specific or strain specific. About 30% of phage isolates produced turbid pinpoint plaques or only caused reduced cell growth on the bacterial lawns. Six phages with distinct host ranges were characterized. The data from this study showed that abundant and diverse phages were present in the commercial cucumber fermentation, which could cause significant mortality to the lactic acid bacteria population. Therefore, a phage control strategy may be needed in low-salt cucumber fermentations. L ike most other vegetable fermentations, cucumber fermentations are driven by a variety of lactic acid bacteria (LAB) naturally present on vegetables. The metabolic activities of LAB determine the quality and safety of the final fermentation products (30). Vegetable fermentation is a dynamic biochemical system in which the chemical composition and microbial ecology are continuously changing. Those changes can be influenced by many physical, chemical, and biological factors such as temperature, pH, salt concentration, and the microbiota (bacteria, fungi, bacteriophages, etc.) present on vegetables (5, 12). The presence of diverse phages against LAB in vegetable fermentations can potentially cause significant mortality to LAB, thereby influencing the number, type, and succession of LAB populations during the fermentation, as well as the type, rate, and extent of carbohydrate metabolism. Phages are ubiquitous in nature. Several studies showed that a diverse phage community was present in vegetable fermentations (2, 19, 20, 36, 37). We previously reported the phage ecology in commercial sauerkraut fermentations (20), and the genome sequences of two LAB phages (JL-1 and 1-A4, infecting Lactobacillus plantarum and Leuconostoc mesenteroides, respectively) isolated from vegetable fermentations (17, 18). Although several LAB phages active againstL. plantarumandPediococcussp. have been isolated from commercial cucumber fermentations (19, 36), phage ecology in commercial cucumber fermentations has not been investigated. In the pickle industry, cucumbers are typically fermented in a brinecontaining6%NaClinopen-toptanksthatareabout40,000 liters in volume (5). In cold climates additional salt, up to 12%, is

Bacteriophage are voracious predators of bacteria and a major determinant in shaping bacterial life strategies. Many phage species are virulent, meaning that infection leads to certain death of the host and immediate release of a large batch of phage progeny. Despite this apparent voraciousness, bacteria have stably coexisted with virulent phages for eons. Here, using individual-based stochastic spatial models, we study the conditions for achieving coexistence on the edge between two habitats, one of which is a bacterial refuge with conditions hostile to phage whereas the other is phage friendly. We show how bacterial density-dependent, or quorum-sensing, mechanisms such as the formation of biofilm can produce such refuges and edges in a self-organized manner. Coexistence on these edges exhibits the following properties, all of which are observed in real phage–bacteria ecosystems but difficult to achieve together in nonspatial ecosystem models: ( i ) highly efficient virulent phage with relatively long lifetimes, high infection rates and large burst sizes; ( ii ) large, stable, and high-density populations of phage and bacteria; ( iii ) a fast turnover of both phage and bacteria; and ( iv ) stability over evolutionary timescales despite imbalances in the rates of phage vs. bacterial evolution.

In this commentary I consider use of the term "lysis from without" (LO) along with the phenomenon's biological relevance. LO originally described an early bacterial lysis induced by high-multiplicity virion adsorption and that occurs without phage production (here indicated as LO(V)). Notably, this is more than just high phage multiplicities of adsorption leading to bacterial killing. The action on bacteria of exogenously supplied phage lysin, too, has been described as a form of LO (here, LO(L)). LO(V) has been somewhat worked out mechanistically for T4 phages, has been used to elucidate various phage-associated phenomena including discovery of the phage eclipse, may be relevant to phage ecology, and, with resistance to LO (LO(R)), is blocked by certain phage gene products. Speculation as to the impact of LO(V) on phage therapy also is fairly common. Since LO(V) assays are relatively easily performed and not all phages are able to induce LO(V), a phage's potential to lyse bacteria without first infecting should be subject to at least in vitro experimental confirmation before the LO(V) label is applied. The term "abortive infection" may be used more generally to describe non-productive phage infections that kill bacteria.

SUMMARY Over the last few decades, advances in cultivation-independent methods have significantly contributed to our understanding of microbial diversity and community composition in the environment. At the same time, cultivation-dependent methods have thrived, and the growing number of organisms obtained thereby have allowed for detailed studies of their physiology and genetics. Still, most microorganisms are recalcitrant to cultivation. This review not only conveys current knowledge about different isolation and cultivation strategies but also discusses what implications can be drawn from pure culture work for studies in microbial ecology. Specifically, in the light of single-cell individuality and genome heterogeneity, it becomes important to evaluate population-wide measurements carefully. An overview of various approaches in microbial ecology is given, and the cell as a central unit for understanding processes on a community level is discussed.

Summary Oceanic phages are critical components of the global ecosystem, where they play a role in microbial mortality and evolution. Our understanding of phage diversity is greatly limited by the lack of useful genetic diversity measures. Previous studies, focus- ing on myophages that infect the marine cyano- bacterium Synechococcus, have used the coliphage T4 portal-protein-encoding homologue, gene 20 (g20), as a diversity marker. These studies revealed 10 sequence clusters, 9 oceanic and 1 freshwater, where only 3 contained cultured representatives. We sequenced g20 from 38 marine myophages isolated using a diversity of Synechococcus and Prochloro- coccus hosts to see if any would fall into the clusters that lacked cultured representatives. On the contrary, all fell into the three clusters that already contained sequences from cultured phages. Further, there was no obvious relationship between host of isolation, or host range, and g20 sequence similarity. We next expanded our analyses to all available g20 sequences (769 sequences), which include PCR amplicons from wild uncultured phages, non-PCR amplified sequences identified in the Global Ocean Survey (GOS) metagenomic database, as well as sequences from cultured phages, to evaluate the relationship between g20 sequence clusters and habitat features from which the phage sequences were isolated. Even in this meta-data set, very few sequences fell into the sequence clusters without cultured representatives, suggesting that the latter are very rare, or sequencing artefacts. In contrast, sequences most similar to the culture-containing clusters, the freshwater cluster and two novel clusters, were more highly repre- sented, with one particular culture-containing cluster representing the dominant g20 genotype in the un- amplified GOS sequence data. Finally, while some g20 sequences were non-randomly distributed with respect to habitat, there were always numerous exceptions to general patterns, indicating that phage portal proteins are not good predictors of a phage's host or the habitat in which a particular phage may thrive.

Molecular ecology and metagenomics applied to the study of microbial biodiversity are changing our comprehension of the biosphere. An impressive diversity of archaea, bacteria and, more recently, protists has been uncovered by molecular tools. Efforts to couple function to the phylogenetic diversity observed in natural environments are leading to the discovery of novel metabolisms and to a re-evaluation of the global ecological impact of known ones. Interesting questions relating to mechanisms of speciation and evolutionary trends at the smallest and largest phylogenetic scales are emerging.

Phages are the most abundant biological entities on Earth and play major ecological roles, yet the current sequenced phage genomes do not adequately represent their diversity, and little is known about the abundance and distribution of these sequenced genomes in nature. Although the study of phage ecology has benefited tremendously from the emergence of metagenomic sequencing, a systematic survey of phage genes and genomes in various ecosystems is still lacking, and fundamental questions about phage biology, lifestyle, and ecology remain unanswered. To address these questions and improve comparative analysis of phages in different metagenomes, we screened a core set of publicly available metagenomic samples for sequences related to completely sequenced phages using the web tool, Phage Eco-Locator. We then adopted and deployed an array of mathematical and statistical metrics for a multidimensional estimation of the abundance and distribution of phage genes and genomes in various ecosystems. Experiments using those metrics individually showed their usefulness in emphasizing the pervasive, yet uneven, distribution of known phage sequences in environmental metagenomes. Using these metrics in combination allowed us to resolve phage genomes into clusters that correlated with their genotypes and taxonomic classes as well as their ecological properties. We propose adding this set of metrics to current metaviromic analysis pipelines, where they can provide insight regarding phage mosaicism, habitat specificity, and evolution.

Knowledge of bacteriophage ecology in vegetable fermentations is essential for developing phage control strategies for consistent and high quality of fermented vegetable products. The ecology of phages infecting lactic acid bacteria (LAB) in commercial sauerkraut fermentations was investigated. Brine samples were taken from four commercial sauerkraut fermentation tanks over a 60- or 100-day period in 2000 and 2001. A total of 171 phage isolates, including at least 26 distinct phages, were obtained. In addition, 28 distinct host strains were isolated and identified as LAB by restriction analysis of the intergenic transcribed spacer region and 16S rRNA sequence analysis. These host strains included Leuconostoc , Weissella , and Lactobacillus species. It was found that there were two phage-host systems in the fermentations corresponding to the population shift from heterofermentative to homofermentative LAB between 3 and 7 days after the start of the fermentations. The data suggested that phages may play an important role in the microbial ecology and succession of LAB species in vegetable fermentations. Eight phage isolates, which were independently obtained two or more times, were further characterized. They belonged to the family Myoviridae or Siphoviridae and showed distinct host ranges and DNA fingerprints. Two of the phage isolates were found to be capable of infecting two Lactobacillus species. The results from this study demonstrated for the first time the complex phage ecology present in commercial sauerkraut fermentations, providing new insights into the bioprocess of vegetable fermentations.

Viruses of microorganisms (VoMs)—the encapsidated, acelluluar parasites of archaea, bacteria, and microbial eukaryotes (Hyman and Abedon, 2012)—can be both responsive to and the source of change in their environments. Defining the diverse VoMs in an environment has become easier with the advent of various new technologies, so analyzing VoM populations or communities involved with disturbances could be the next step to drive research forward. Contributions assembled in this Research Topic explore the effects of disturbances on one type of VoM, the bacteriophages (phages), as well as disturbances caused by phages. The collection presented in this ebook opens with an editorial describing the importance of change to life on Earth, and by defining the multiple levels at which change can occur: genetic, organismal, and environmental (Allen and Abedon, 2013). We have arranged and introduced the manuscripts in this collection based on which of these levels they address.

Background Bacteriophages (phages) have been used extensively as analytical tools to type bacterial cultures and recently for control of zoonotic foodborne pathogens in foods and in animal reservoirs. Methods We examined the host range, morphology, genome and proteome of the lytic E. coli O157 phage rV5, derived from phage V5, which is a member of an Escherichia coli O157:H7 phage typing set. Results Phage rV5 is a member of the Myoviridae family possessing an icosahedral head of 91 nm between opposite apices. The extended tail measures 121 x 17 nm and has a sheath of 44 x 20 nm and a 7 nm-wide core in the contracted state. It possesses a 137,947 bp genome (43.6 mol%GC) which encodes 233 ORFs and six tRNAs. Until recently this virus appeared to be phylogenetically isolated with almost 70% of its gene products ORFans. rV5 is closely related to coliphages Delta and vB-EcoM-FY3, and more distantly related to Salmonella phages PVP-SE1 and SSE-121, Cronobacter sakazakii phage vB_CsaM_GAP31, and coliphages phAPEC8 and phi92. A complete shotgun proteomic analysis was carried out on rV5, extending what had been gleaned from the genomic analyses. Host range studies revealed that rV5 is active against several other E. coli.

Background One of the most effective targets for control of zoonotic foodborne pathogens in the farm to fork continuum is their elimination in food animals destined for market. Phage therapy for Escherichia coli O157:H7 in ruminants, the main animal reservoir of this pathogen, is a popular research topic. Since phages active against this pathogen may be endemic in host animals and their environment, they may emerge during trials of phage therapy or other interventions, rendering interpretation of trials problematic. Methods During separate phage therapy trials, sheep and cattle inoculated with 109 to 1010 CFU of E. coli O157:H7 soon began shedding phages dissimilar in plaque morphology to the administered therapeutic phages. None of the former was previously identified in the animals or in their environment. The dissimilar “rogue” phage was isolated and characterized by host range, ultrastructure, and genomic and proteomic analyses. Results The “rogue” phage (Phage vB_EcoS_Rogue1) is distinctly different from the administered therapeutic Myoviridae phages, being a member of the Siphoviridae (head: 53 nm; striated tail: 152 x 8 nm). It has a 45.8 kb genome which is most closely related to coliphage JK06, a member of the “T1-like viruses” isolated in Israel. Detailed bioinformatic analysis reveals that the tail of these phages is related to the tail genes of coliphage lambda. The presence of “rogue” phages resulting from natural enrichments can pose problems in the interpretation of phage therapeutic studies. Similarly, evaluation of any interventions for foodborne or other bacterial pathogens in animals may be compromised unless tests for such phages are included to identify their presence and potential impact.