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1. Gene Co-occurrence Networks Reflect Bacteriophage Ecology and Evolution

   https://doi.org/10.1128/mBio.01870-17  

   Shapiro, Jason W.; Putonti, Catherine; Keim, Paul (May 2018, mBio)

   ABSTRACT 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. 

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2. Flagellotropic Bacteriophages: Opportunities and Challenges for Antimicrobial Applications

   https://doi.org/10.3390/ijms23137084  

   Esteves, Nathaniel C.; Scharf, Birgit E. (July 2022, International Journal of Molecular Sciences)

   Bacteriophages (phages) are the most abundant biological entities in the biosphere. As viruses that solely infect bacteria, phages have myriad healthcare and agricultural applications including phage therapy and antibacterial treatments in the foodservice industry. Phage therapy has been explored since the turn of the twentieth century but was no longer prioritized following the invention of antibiotics. As we approach a post-antibiotic society, phage therapy research has experienced a significant resurgence for the use of phages against antibiotic-resistant bacteria, a growing concern in modern medicine. Phages are extraordinarily diverse, as are their host receptor targets. Flagellotropic (flagellum-dependent) phages begin their infection cycle by attaching to the flagellum of their motile host, although the later stages of the infection process of most of these phages remain elusive. Flagella are helical appendages required for swimming and swarming motility and are also of great importance for virulence in many pathogenic bacteria of clinical relevance. Not only is bacterial motility itself frequently important for virulence, as it allows pathogenic bacteria to move toward their host and find nutrients more effectively, but flagella can also serve additional functions including mediating bacterial adhesion to surfaces. Flagella are also a potent antigen recognized by the human immune system. Phages utilizing the flagellum for infections are of particular interest due to the unique evolutionary tradeoff they force upon their hosts: by downregulating or abolishing motility to escape infection by a flagellotropic phage, a pathogenic bacterium would also likely attenuate its virulence. This factor may lead to flagellotropic phages becoming especially potent antibacterial agents. This review outlines past, present, and future research of flagellotropic phages, including their molecular mechanisms of infection and potential future applications. 

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3. Mutant and Recombinant Phages Selected from In Vitro Coevolution Conditions Overcome Phage-Resistant Listeria monocytogenes

   https://doi.org/10.1128/AEM.02138-20  

   Peters, Tracey Lee; Song, Yaxiong; Bryan, Daniel W.; Hudson, Lauren K.; Denes, Thomas G. (October 2020, Applied and Environmental Microbiology)

   Dudley, Edward G. (Ed.)

   ABSTRACT Bacteriophages (phages) are currently available for use by the food industry to control the foodborne pathogen Listeria monocytogenes . Although phage biocontrols are effective under specific conditions, their use can select for phage-resistant bacteria that repopulate phage-treated environments. Here, we performed short-term coevolution experiments to investigate the impact of single phages and a two-phage cocktail on the regrowth of phage-resistant L. monocytogenes and the adaptation of the phages to overcome this resistance. We used whole-genome sequencing to identify mutations in the target host that confer phage resistance and in the phages that alter host range. We found that infections with Listeria phages LP-048, LP-125, or a combination of both select for different populations of phage-resistant L. monocytogenes bacteria with different regrowth times. Phages isolated from the end of the coevolution experiments were found to have gained the ability to infect phage-resistant mutants of L. monocytogenes and L. monocytogenes strains previously found to be broadly resistant to phage infection. Phages isolated from coinfected cultures were identified as recombinants of LP-048 and LP-125. Interestingly, recombination events occurred twice independently in a locus encoding two proteins putatively involved in DNA binding. We show that short-term coevolution of phages and their hosts can be utilized to obtain mutant and recombinant phages with adapted host ranges. These laboratory-evolved phages may be useful for limiting the emergence of phage resistance and for targeting strains that show general resistance to wild-type (WT) phages. IMPORTANCE Listeria monocytogenes is a life-threatening bacterial foodborne pathogen that can persist in food processing facilities for years. Phages can be used to control L. monocytogenes in food production, but phage-resistant bacterial subpopulations can regrow in phage-treated environments. Coevolution experiments were conducted on a Listeria phage-host system to provide insight into the genetic variation that emerges in both the phage and bacterial host under reciprocal selective pressure. As expected, mutations were identified in both phage and host, but additionally, recombination events were shown to have repeatedly occurred between closely related phages that coinfected L. monocytogenes . This study demonstrates that in vitro evolution of phages can be utilized to expand the host range and improve the long-term efficacy of phage-based control of L. monocytogenes . This approach may also be applied to other phage-host systems for applications in biocontrol, detection, and phage therapy. 

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4. Phage-Encoded Sigma Factors Alter Bacterial Dormancy

   https://doi.org/10.1128/msphere.00297-22  

   Schwartz, D. A.; Lehmkuhl, B. K.; Lennon, J. T. (August 2022, mSphere)

   Imperiale, Michael J. (Ed.)

   ABSTRACT By entering a reversible state of reduced metabolic activity, dormant microorganisms are able to tolerate suboptimal conditions that would otherwise reduce their fitness. Dormancy may also benefit bacteria by serving as a refuge from parasitic infections. Here, we focus on dormancy in the Bacillota , where endospore development is transcriptionally regulated by the expression of sigma factors. A disruption of this process could influence the survivorship or reproduction of phages that infect spore-forming hosts with implications for coevolutionary dynamics. We characterized the distribution of sigma factors in over 4,000 genomes of diverse phages capable of infecting hosts that span the bacterial domain. From this, we identified homologs of sporulation-specific sigma factors in phages that infect spore-forming hosts. Unlike sigma factors required for phage reproduction, we provide evidence that sporulation-like sigma factors are nonessential for lytic infection. However, when expressed in the spore-forming Bacillus subtilis , some of these phage-derived sigma factors can activate the bacterial sporulation gene network and lead to a reduction in spore yield. Our findings suggest that the acquisition of host-like transcriptional regulators may allow phages to manipulate a complex and ancient trait in one of the most abundant cell types on Earth. IMPORTANCE As obligate parasites, phages exert strong top-down pressure on host populations with eco-evolutionary implications for community dynamics and ecosystem functioning. The process of phage infection, however, is constrained by bottom-up processes that influence the energetic and nutritional status of susceptible hosts. Many phages have acquired auxiliary genes from bacteria, which can be used to exploit host metabolism with consequences for phage fitness. In this study, we demonstrate that phages infecting spore-forming bacteria carry homologs of sigma factors, which their hosts use to orchestrate gene expression during spore development. By tapping into regulatory gene networks, phages may manipulate the physiology and survival strategies of nongrowing bacteria in ways that influence host-parasite coevolution. 

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5. Diverse and abundant phages exploit conjugative plasmids

   https://doi.org/10.1038/s41467-024-47416-z  

   Quinones-Olvera, Natalia; Owen, Siân V.; McCully, Lucy M.; Marin, Maximillian G.; Rand, Eleanor A.; Fan, Alice C.; Martins Dosumu, Oluremi J.; Paul, Kay; Sanchez Castaño, Cleotilde E.; Petherbridge, Rachel; et al (April 2024, Nature Communications)

   Abstract Phages exert profound evolutionary pressure on bacteria by interacting with receptors on the cell surface to initiate infection. While the majority of phages use chromosomally encoded cell surface structures as receptors, plasmid-dependent phages exploit plasmid-encoded conjugation proteins, making their host range dependent on horizontal transfer of the plasmid. Despite their unique biology and biotechnological significance, only a small number of plasmid-dependent phages have been characterized. Here we systematically search for new plasmid-dependent phages targeting IncP and IncF plasmids using a targeted discovery platform, and find that they are common and abundant in wastewater, and largely unexplored in terms of their genetic diversity. Plasmid-dependent phages are enriched in non-canonical types of phages, and all but one of the 65 phages we isolated were non-tailed, and members of the lipid-containing tectiviruses, ssDNA filamentous phages or ssRNA phages. We show that plasmid-dependent tectiviruses exhibit profound differences in their host range which is associated with variation in the phage holin protein. Despite their relatively high abundance in wastewater, plasmid-dependent tectiviruses are missed by metaviromic analyses, underscoring the continued importance of culture-based phage discovery. Finally, we identify a tailed phage dependent on the IncF plasmid, and find related structural genes in phages that use the orthogonal type 4 pilus as a receptor, highlighting the evolutionarily promiscuous use of these distinct contractile structures by multiple groups of phages. Taken together, these results indicate plasmid-dependent phages play an under-appreciated evolutionary role in constraining horizontal gene transfer via conjugative plasmids. 

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