Community context matters for bacteria-phage ecology and evolution

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.

Bacteriophage adaptation to a mammalian mucosa reveals a trans–domain evolutionary axis

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.

Lifestyle of sponge symbiont phages by host prediction and correlative microscopy

Bacteriophages (phages) are ubiquitous elements in nature, but their ecology and role in animals remains little understood. Sponges represent the oldest known extant animal-microbe symbiosis and are associated with dense and diverse microbial consortia. Here we investigate the tripartite interaction between phages, bacterial symbionts, and the sponge host. We combined imaging and bioinformatics to tackle important questions on who the phage hosts are and what the replication mode and spatial distribution within the animal is. This approach led to the discovery of distinct phage-microbe infection networks in sponge versus seawater microbiomes. A new correlative in situ imaging approach (‘PhageFISH-CLEM‘) localised phages within bacterial symbiont cells, but also within phagocytotically active sponge cells. We postulate that the phagocytosis of free virions by sponge cells modulates phage-bacteria ratios and ultimately controls infection dynamics. Prediction of phage replication strategies indicated a distinct pattern, where lysogeny dominates the sponge microbiome, likely fostered by sponge host-mediated virion clearance, while lysis dominates in seawater. Collectively, this work provides new insights into phage ecology within sponges, highlighting the importance of tripartite animal-phage-bacterium interplay in holobiont functioning. We anticipate that our imaging approach will be instrumental to further understanding of viral distribution and cellular association in animal hosts.

Phase variation-based biosensors for bacteriophage detection and phage receptor discrimination

Environmental monitoring of bacteria using phage-based biosensors has been widely developed for many different species. However, there are only a few available methods to detect specific bacteriophages in raw environmental samples. In this work, we developed a simple and efficient assay to rapidly monitor the phage content of a given sample. The assay is based on the bistable expression of the Salmonella enterica opvAB operon. Under regular growth conditions, opvAB is only expressed by a small fraction of the bacterial subpopulation. In the OpvABON subpopulation, synthesis of the OpvA and OpvB products shortens the O-antigen in the lipopolysaccharide and confers resistance to phages that use LPS as a receptor. As a consequence, the OpvABON subpopulation is selected in the presence of such phages. Using an opvAB::gfp fusion, we could monitor LPS-binding phages in various media, including raw water samples. To enlarge our phage-biosensor panoply, we also developed several coliphage biosensors that proved efficient to detect LPS- as well as protein-binding coliphages. Moreover, the combination of these tools allows to identify what is the bacterial receptor triggering phage infection. The opvAB::gfp biosensor thus comes in different flavours to efficiently detect a wide range of bacteriophages and identify the type of receptor they recognize.ImportanceDetection and accurate counting of bacteriophages, the viruses that specifically infect bacteria, from environmental samples still constitutes a challenge for those interested in isolating and characterizing bacteriophages for ecological or biotechnological purposes. This work provides a simple and accurate method based on the bi-stable expression of genes that confer resistance to certain classes of bacteriophages in different bacterial models. It paves the way for future development of highly efficient phage biosensors that can discriminate among several receptor-binding phages and that could be declined in many more versions. In a context where phage ecology, research, and therapy are flourishing again, it becomes essential to possess simple and efficient tools for phage detection.

Gene Co-occurrence Networks Reflect Bacteriophage Ecology and Evolution

ABSTRACTBacteriophages 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.IMPORTANCEBacteriophages (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.

Assessment of a metaviromic dataset generated from nearshore Lake Michigan

Bacteriophages are powerful ecosystem engineers. They drive bacterial mortality rates and genetic diversity, and affect microbially mediated biogeochemical processes on a global scale. This has been demonstrated in marine environments; however, phage communities have been less studied in freshwaters, despite representing a potentially more diverse environment. Lake Michigan is one of the largest bodies of freshwater on the planet, yet to date the diversity of its phages has yet to be examined. Here, we present a composite survey of viral ecology in the nearshore waters of Lake Michigan. Sequence analysis was performed using a web server previously used to analyse similar data. Our results revealed a diverse community of DNA phages, largely comprising the order Caudovirales. Within the scope of the current study, the Lake Michigan virome demonstrates a distinct community. Although several phages appeared to hold dominance, further examination highlighted the importance of interrogating metagenomic data at the genome level. We present our study as baseline information for further examination of the ecology of the lake. In the current study we discuss our results and highlight issues of data analysis which may be important for freshwater studies particularly, in light of the complexities associated with examining phage ecology generally.