Search

Abstract Background Bacteriophage genomes have mosaic architectures and are replete with small open reading frames of unknown function, presenting challenges in their annotation, comparative analysis, and representation. Results We describe here a bioinformatic tool, Phamerator, that assorts protein-coding genes into phamilies of related sequences using pairwise comparisons to generate a database of gene relationships. This database is used to generate genome maps of multiple phages that incorporate nucleotide and amino acid sequence relationships, as well as genes containing conserved domains. Phamerator also generates phamily circle representations of gene phamilies, facilitating analysis of the different evolutionary histories of individual genes that migrate through phage populations by horizontal genetic exchange. Conclusions Phamerator represents a useful tool for comparative genomic analysis and comparative representations of bacteriophage genomes.

LBerry, Pembroke, and Zolita are newly isolated bacteriophages that infect Mycobacterium smegmatis mc²155. Based on gene content similarity, LBerry and Pembroke are assigned to cluster A3, and Zolita is assigned to cluster A5. LBerry and Pembroke are 99% identical to Anaysia and Caviar, and Zolita is 99% identical to SydNat., Competing Interests: The authors declare no conflict of interest.

Bacteriophages are ubiquitous biological entities which can be found in a variety of habitats. Here, we describe protocols for the isolation of bacteriophages on a variety of Actinobacterial genera. Two approaches to phage isolation, direct isolation and enriched isolation, are described, which can be performed individually or in parallel. The protocols described can be adapted to isolate a wide array of bacteriophages., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Frankenweenie is a newly isolated bacteriophage that infects Streptomyces scabiei RL-34. Frankenweenie was discovered in Gaithersburg, MD, and has 366 genes comprising a 200,048-bp genome. Frankenweenie is grouped in cluster BM and is predicted to possess a unique tailspike protein that potentially widens its host range., Competing Interests: The authors declare no conflict of interest.

Importance: Mycobacterium species include several human pathogens and mycobacteriophages show potential for therapeutic use to control Mycobacterium infections. However, phage infection profiles vary greatly among Mycobacterium abscessus clinical isolates and phage therapies must be personalized for individual patients. Mycobacterium phage susceptibility is likely determined primarily by accessory parts of bacterial genomes, and we have identified the prophage and phage-related genomic regions across sequenced Mycobacterium strains. The prophages are numerous and diverse, especially in M. abscessus genomes, and provide a potentially rich reservoir of new viruses that can be propagated lytically and used to expand the repertoire of therapeutically useful phages., Competing Interests: G.F.H. had a Collaborative Research Agreement with Janssen Inc. between March 2021 and March 2023. All other authors have no conflicts to declare.

Bacteriophage PineapplePizza is a podovirus infecting Microbacterium foliorum NRRL B-24224. The genome is 16,662 bp long and contains 23 predicted protein-coding genes. Interestingly, PineapplePizza shows amino acid similarities to well-studied Bacillus subtilis phage phi29., Competing Interests: The authors declare no conflict of interest.

Glycosylation of eukaryotic virus particles is common and influences their uptake, trafficking, and immune recognition. In contrast, glycosylation of bacteriophage particles has not been reported; phage virions typically do not enter the cytoplasm upon infection, and they do not generally inhabit eukaryotic systems. We show here that several genomically distinct phages of Mycobacteria are modified with glycans attached to the C terminus of capsid and tail tube protein subunits. These O-linked glycans influence antibody production and recognition, shielding viral particles from antibody binding and reducing production of neutralizing antibodies. Glycosylation is mediated by phage-encoded glycosyltransferases, and genomic analysis suggests that they are relatively common among mycobacteriophages. Putative glycosyltransferases are also encoded by some Gordonia and Streptomyces phages, but there is little evidence of glycosylation among the broader phage population. The immune response to glycosylated phage virions in mice suggests that glycosylation may be an advantageous property for phage therapy of Mycobacterium infections., Competing Interests: Declaration of interests G.F.H. received support from Janssen Inc. through a Collaborative Research Agreement, which did not fund work in this report. J.V.W. serves on the Scientific Advisory Board of Quidel and an Independent Data Monitoring Committee for GlaxoSmithKline, neither involved in the present work., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Many double-stranded DNA viruses, including tailed bacteriophages (phages) and herpesviruses, use the HK97-fold in their major capsid protein to make the capsomers of the icosahedral viral capsid. After the genome packaging at near-crystalline densities, the capsid is subjected to a major expansion and stabilization step that allows it to withstand environmental stresses and internal high pressure. Several different mechanisms for stabilizing the capsid have been structurally characterized, but how these mechanisms have evolved is still not understood. Using cryo-EM structure determination of 10 capsids, structural comparisons, phylogenetic analyses, and Alphafold predictions, we have constructed a detailed structural dendrogram describing the evolution of capsid structural stability within the actinobacteriophages. We show that the actinobacteriophage major capsid proteins can be classified into 15 groups based upon their HK97-fold., Competing Interests: Declaration of interests G.F.H. is a compensated consultant for Tessera and for Janssen Inc. The remaining authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Mycobacterium abscessus infections are relatively common in patients with cystic fibrosis and are clinically challenging, with frequent intrinsic resistance to antibiotics. Therapeutic treatment with bacteriophages offers some promise but faces many challenges including substantial variation in phage susceptibilities among clinical isolates, and the need to personalize therapies for individual patients. Many strains are not susceptible to any phages or are not efficiently killed by lytic phages, including all smooth colony morphotype strains tested to-date. Here, we analyze a set of new M. abscessus isolates for the genomic relationships, prophage content, spontaneous phage release, and phage susceptibilities. We find that prophages are common in these M. abscessus genomes, but some have unusual arrangements, including tandemly integrated prophages, internal duplications, and they participate in active exchange of polymorphic toxin-immunity cassettes secreted by ESX systems. Relatively few strains are efficiently infected by any mycobacteriophages, and the infection patterns do not reflect the overall phylogenetic relationships of the strains. Characterization of these strains and their phage susceptibility profiles will help to advance the broader application of phage therapies for NTM infections., Competing Interests: GFH receives research support through a Collaborative Research Agreement with Janssen Inc., but this did not fund or influence the work reported here., (Copyright: © 2023 Amarh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Nontuberculous Mycobacterium infections, particularly Mycobacterium abscessus, are increasingly common among patients with cystic fibrosis and chronic bronchiectatic lung diseases. Treatment is challenging due to intrinsic antibiotic resistance. Bacteriophage therapy represents a potentially novel approach. Relatively few active lytic phages are available and there is great variation in phage susceptibilities among M. abscessus isolates, requiring personalized phage identification., Methods: Mycobacterium isolates from 200 culture-positive patients with symptomatic disease were screened for phage susceptibilities. One or more lytic phages were identified for 55 isolates. Phages were administered intravenously, by aerosolization, or both to 20 patients on a compassionate use basis and patients were monitored for adverse reactions, clinical and microbiologic responses, the emergence of phage resistance, and phage neutralization in serum, sputum, or bronchoalveolar lavage fluid., Results: No adverse reactions attributed to therapy were seen in any patient regardless of the pathogen, phages administered, or the route of delivery. Favorable clinical or microbiological responses were observed in 11 patients. Neutralizing antibodies were identified in serum after initiation of phage delivery intravenously in 8 patients, potentially contributing to lack of treatment response in 4 cases, but were not consistently associated with unfavorable responses in others. Eleven patients were treated with only a single phage, and no phage resistance was observed in any of these., Conclusions: Phage treatment of Mycobacterium infections is challenging due to the limited repertoire of therapeutically useful phages, but favorable clinical outcomes in patients lacking any other treatment options support continued development of adjunctive phage therapy for some mycobacterial infections., Competing Interests: Potential conflicts of interest. G. F. H. is a consultant for and receives grant support not directly related to this work from Janssen Pharmaceuticals (Collaborative Research Agreement); reports consulting fees from Janssen Inc and Tessera Inc; and reports presentation honoraria from the Pittsburgh Foundation and a leadership or fiduciary role with the Charles E. Kaufman Foundation scientific advisory board. R. M. De. and G. F. H. are co-inventors on patent applications related to the use of phages for treating nontuberculous mycobacterial (NTM) infections filed by the University of Pittsburgh of the Commonwealth System of Higher Education. D. v. D. is a consultant for Actavis, Tetraphase, Sanofi Pasteur, MedImmune, Astellas, Merck, Allergan, T2Biosystems, Roche, Achaogen, Neumedicine, Shionogi, Pfizer, Entasis, QPex, Wellspring, Karius, Melinta, and Utility; receives an editor’s stipend from British Society for Antimicrobial Chemotherapy; has received funding for unrelated projects from NIH, Merck, and Shionogi; reports payments for lectures, presentations, speaker’s bureaus, manuscript writing, or educational events from Pfizer and Entasis; reports paid participation on a data and safety monitoring board (DSMB) or advisory board for Utility, Union, Entasis, and Merck; and reports a paid leadership or fiduciary role with the British Society for Antimicrobial Chemotherapy. K. A. C. has received consulting fees from Insmed (clinical trial site), Hillrom (clinical trial site), Paratek, Microbion, and AN2, and reports honoraria for a presentation from Insmed. G. H. receives grant support unrelated to this study from Karius, Allovir, and AstraZeneca, and reports participation on a DSMB or advisory board with Karius. R. T. S. is a paid consultant to Vir Biotechnology and to LysNtech; holds stock options in Antiva Biosciences and CytoDyn and stock or stock options with NoniGenex and Arcturus; previously served as an uncompensated member of the AmpliPhi scientific advisory board; reports grants or contracts paid to institution from the National Institute of Allergy and Infectious Diseases; reports consulting fees from Pfizer, Sempra Energy, and Nurix; has patents planned, issued, or pending for orally bioavailable anti-coronavirus compounds; reports paid participation on DSMBs or advisory boards for Merck, VIr Biosciences, SNIPR Biome, and Pardes Biosciences; and holds leadership or fiduciary roles with the International Antiviral Society (IAS)–USA and Specialists in Global Health. C. A. B. reports contracts to institution for clinical trials from Gilead and DNAe; payment to author for educational lectures from IAS-USA, Practice Point Communications (Optimal Management of HIV Disease and Hepatitis Clinical Conference [OPMAN] conference), and University of Arizona; has received payment for travel to the OPMAN conference from Practice Point Communications; served on a DSMB for ViiV/GlaxoSmith Kline; has held unpaid volunteer roles with IAS-USA and the Conference on Retroviruses and Opportunistic Infections Foundation Board; and has served as Deputy Editor/Associate Editor for the Infectious Diseases Society of America. A. K. reports the following grants or contracts unrelated to this work: National Health and Medical Research Council (NHMRC, Australia) Investigator Grant at Emerging Leadership 1 level, Conquer CF, Innovation Grant from Cystic Fibrosis Australia, Research Establishment Fellowship from the Royal Australasian College of Physicians and Research Award from the Australasian Society for Infectious Diseases (all paid to institution); payment to institution for grant application review for the Italian Cystic Fibrosis Research Foundation; unpaid role as member of DSMB for FluBubs (Safety and Immunogenicity of Early Quadrivalent Influenza Vaccine); unpaid leadership or fiduciary roles as Deputy Director (Clinical) of Phage Australia, pediatric infectious diseases research representative on the Australian Society for Infectious Diseases Clinical Research Network Steering Committee and the Australia and New Zealand Paediatric Infectious Diseases Group Executive Committee, member of the Sydney Children’s Hospitals Network Human Research Ethics Committee Scientific Advisory Committee, and member of the Sydney Children’s Hospitals Network Advanced Therapeutics Steering Committee. A. W. B. reports a role as a part-time employee of the Cystic Fibrosis Foundation, which provides some grant support to G. F. H.’s laboratory and, for the purposes of this manuscript, is the treating physician of one of the NTM patients in the cystic fibrosis clinic at Inova Fairfax Hospital. C. B. G. reports consulting fees from Advisory Janssen. B. J. M. reports an NHMRC Investigator Grant and philanthropic grant from the Curing Homesickness Foundation, both paid to institution and unrelated to this work; unpaid participation as member of the DSMB for the PATRIC trial; and unpaid position as board director of the Australasian Society for Infectious Diseases. M. G. T. O. reports a Cystic Fibrosis Foundation Adult Center Award and Cystic Fibrosis Foundation Therapeutic Development Center Award, unrelated to this work; support for attending meetings and/or travel, paid to University of Miami, from the Cystic Fibrosis Foundation Adult Center Award for attending North American Cystic Fibrosis Conference and a Cystic Fibrosis Foundation Therapeutic Development Center Award for attending the Therapeutics Development Network spring meeting; and an unpaid position as Cystic Fibrosis Lifestyle Foundation board member. J. I. reports an Investigator Grant (personal support) unrelated to this work from NHMRC. A. M. reports consulting fees paid to author as a member of the Air Liquide Advisory Board. J. A. N. reports contracts or grants unrelated to this work from the Cystic Fibrosis Foundation. M. S. reports funding on a project to set up a phage therapy laboratory in Finland, unrelated to this work, from the Jane and Aatos Erkko Foundation. P. W. reports consulting fees from AstraZeneca, GlaxoSmithKline, Pfizer, Sanofi Regeneron, and Vertex; payment or honoraria for lectures, presentations, speaker’s bureaus, manuscript writing, or educational events from AstraZeneca, GlaxoSmithKline, Pfizer, Boehringer Ingelheim, and Vertex; and a leadership or fiduciary role with the Cystic Fibrosis Australia National Asthma Council of Australia. D. E. M. reports stock or stock options (no payments) with Moderna (1 share) and Pfizer (5 shares). R. M. Da. reports grants from the NIH unrelated to this work (grant number K01-AI125726 [principal investigator]). All other authors report no potential conflicts. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Infectious Diseases Society of America.)

Broadening access to science, technology, engineering, and mathematics (STEM) professions through the provision of early-career research experiences for a wide range of demographic groups is important for the diversification of the STEM workforce. The size and diversity of the community college system make it a prime educational site for achieving this aim. However, some evidence shows that women and Black, Latinx, and Native American student groups have been hindered in STEM at the community college level. One option for enhancing persistence in STEM is to incorporate the course-based research experiences (CREs) into the curriculum as a replacement for the prevalent traditional laboratory. This can be achieved through the integration of community colleges within extant, multi-institutional CREs such as the SEA-PHAGES program. Using a propensity score-matching technique, students in a CRE and traditional laboratory were compared on a range of psychosocial variables (project ownership, self-efficacy, science identity, scientific community values, and networking). Results revealed higher ratings for women and persons excluded because of their ethnicity or race (PEERs) in the SEA-PHAGES program on important predictors of persistence such as project ownership and science identity. This suggests that the usage of CREs at community colleges could have positive effects in addressing the gender gap for women and enhance inclusiveness for PEER students in STEM.

The course-based research experience (CRE) with its documented educational benefits is increasingly being implemented in science, technology, engineering, and mathematics education. This article reports on a study that was done over a period of 3 years to explicate the instructional processes involved in teaching an undergraduate CRE. One hundred and two instructors from the established and large multi-institutional SEA-PHAGES program were surveyed for their understanding of the aims and practices of CRE teaching. This was followed by large-scale feedback sessions with the cohort of instructors at the annual SEA Faculty Meeting and subsequently with a small focus group of expert CRE instructors. Using a qualitative content analysis approach, the survey data were analyzed for the aims of inquiry instruction and pedagogical practices used to achieve these goals. The results characterize CRE inquiry teaching as involving three instructional models: 1) being a scientist and generating data; 2) teaching procedural knowledge; and 3) fostering project ownership. Each of these models is explicated and visualized in terms of the specific pedagogical practices and their relationships. The models present a complex picture of the ways in which CRE instruction is conducted on a daily basis and can inform instructors and institutions new to CRE teaching.

Bacteriophage EasyJones is a myovirus infecting Mycobacterium smegmatis mc 2 155, with a genome length and gene content similar to those of phages grouped in subcluster C1. Interestingly, EasyJones contains a gene found in a subset of C1 genomes that is similar to the well-characterized immunity repressor of subcluster A1 mycobacteriophage Bxb1.

The explosion of SARS-CoV-2 infections in 2020 prompted a flurry of activity in vaccine development and exploration of various vaccine platforms, some well-established and some new. Phage-based vaccines were described previously, and we explored the possibility of using mycobacteriophages as a platform for displaying antigens of SARS-CoV-2 or other infectious agents. The potential advantages of using mycobacteriophages are that a large and diverse variety of them have been described and genomically characterized, engineering tools are available, and there is the capacity to display up to 700 antigen copies on a single particle approximately 100 nm in size. The phage body may itself be a good adjuvant, and the phages can be propagated easily, cheaply, and to high purity. Furthermore, the recent use of these phages therapeutically, including by intravenous administration, suggests an excellent safety profile, although efficacy can be restricted by neutralizing antibodies. We describe here the potent immunogenicity of mycobacteriophage Bxb1, and Bxb1 recombinants displaying SARS-CoV-2 Spike protein antigens.

Bacteriophage genomes harbor the broadest chemical diversity of nucleobases across all life forms. Certain DNA viruses that infect hosts as diverse as cyanobacteria, proteobacteria, and actinobacteria exhibit wholesale substitution of aminoadenine for adenine, thereby forming three hydrogen bonds with thymine and violating Watson-Crick pairing rules. Aminoadenine-encoded DNA polymerases, homologous to the Klenow fragment of bacterial DNA polymerase I that includes 3'-exonuclease but lacks 5'-exonuclease, were found to preferentially select for aminoadenine instead of adenine in deoxynucleoside triphosphate incorporation templated by thymine. Polymerase genes occur in synteny with genes for a biosynthesis enzyme that produces aminoadenine deoxynucleotides in a wide array of Siphoviridae bacteriophages. Congruent phylogenetic clustering of the polymerases and biosynthesis enzymes suggests that aminoadenine has propagated in DNA alongside adenine since archaic stages of evolution., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Mycobacterium abscessus is an opportunistic pathogen whose treatment is confounded by widespread multidrug resistance. The therapeutic use of bacteriophages against Mycobacterium abscessus infections offers a potential alternative approach, although the spectrum of phage susceptibilities among M. abscessus isolates is not known. We determined the phage infection profiles of 82 M. abscessus recent clinical isolates and find that colony morphotype-rough or smooth-is a key indicator of phage susceptibility. None of the smooth strains are efficiently killed by any phages, whereas 80% of rough strains are infected and efficiently killed by at least one phage. The repertoire of phages available for potential therapy of rough morphotype infections includes those with relatively broad host ranges, host range mutants of Mycobacterium smegmatis phages, and lytically propagated viruses derived from integrated prophages. The rough colony morphotype results from indels in the glycopeptidolipid synthesis genes mps1 and mps2 , negating reversion to smooth as a common route to phage resistance. Resistance is thus rare, and although mutations in polyketide synthesis, uvrD2 , and rpoZ can confer resistance, these likely also impair survival in vivo The expanded therapeutic repertoire and the resistance profiles show that small cocktails or single phages could be suitable for controlling infections with rough strains. IMPORTANCE Mycobacterium abscessus infections in cystic fibrosis patients are challenging to treat due to widespread antibiotic resistance. The therapeutic use of lytic bacteriophages presents a new potential strategy, but the great variation among clinical M. abscessus isolates demands determination of phage susceptibility prior to therapy. Elucidation of the variation in phage infection and factors determining it, expansion of the suite of therapeutic phage candidates, and a greater understanding of phage resistance mechanisms substantially advances the potential for broad implementation of new therapeutic options for M. abscessus infections., (Copyright © 2021 Dedrick et al.)

Mycobacterium abscessus is an emerging pathogen that is often refractory to antibiotic control. Treatment is further complicated by considerable variation among clinical isolates in both their genetic constitution and their clinical manifestations. Here, we show that the prophage and plasmid mobilome is a likely contributor to this variation. Prophages and plasmids are common, abundant, and highly diverse, and code for large repertoires of genes influencing virulence, antibiotic susceptibility, and defense against viral infection. At least 85% of the strains we describe carry one or more prophages, representing at least 17 distinct and diverse sequence "clusters," integrated at 18 different attB locations. The prophages code for 19 distinct configurations of polymorphic toxin and toxin-immunity systems, each with WXG-100 motifs for export through type VII secretion systems. These are located adjacent to attachment junctions, are lysogenically expressed, and are implicated in promoting growth in infected host cells. Although the plethora of prophages and plasmids confounds the understanding of M. abscessus pathogenicity, they also provide an abundance of tools for M. abscessus engineering. IMPORTANCE Mycobacterium abscessus is an important emerging pathogen that is challenging to treat with current antibiotic regimens. There is substantial genomic variation in M. abscessus clinical isolates, but little is known about how this influences pathogenicity and in vivo growth. Much of the genomic variation is likely due to the large and varied mobilome, especially a large and diverse array of prophages and plasmids. The prophages are unrelated to previously characterized phages of mycobacteria and code for a diverse array of genes implicated in both viral defense and in vivo growth. Prophage-encoded polymorphic toxin proteins secreted via the type VII secretion system are common and highly varied and likely contribute to strain-specific pathogenesis., (Copyright © 2021 Dedrick et al.)

Mycobacteriophage phiT45-1 is a newly isolated bacteriophage spontaneously released from Mycobacterium abscessus strain Taiwan-45 that lytically infects M. abscessus strain BWH-C; phiT45-1 also infects M. abscessus ATCC 19977 but not Mycobacterium smegmatis Phage phiT45-1 has a 43,407-bp genome and carries a polymorphic toxin-immunity cassette associated with type VII secretion systems., (Copyright © 2021 Amarh et al.)

The bacteriophage population is vast, dynamic, old, and genetically diverse. The genomics of phages that infect bacterial hosts in the phylum Actinobacteria show them to not only be diverse but also pervasively mosaic, and replete with genes of unknown function. To further explore this broad group of bacteriophages, we describe here the isolation and genomic characterization of 116 phages that infect Microbacterium spp. Most of the phages are lytic, and can be grouped into twelve clusters according to their overall relatedness; seven of the phages are singletons with no close relatives. Genome sizes vary from 17.3 kbp to 97.7 kbp, and their G+C% content ranges from 51.4% to 71.4%, compared to ~67% for their Microbacterium hosts. The phages were isolated on five different Microbacterium species, but typically do not efficiently infect strains beyond the one on which they were isolated. These Microbacterium phages contain many novel features, including very large viral genes (13.5 kbp) and unusual fusions of structural proteins, including a fusion of VIP2 toxin and a MuF-like protein into a single gene. These phages and their genetic components such as integration systems, recombineering tools, and phage-mediated delivery systems, will be useful resources for advancing Microbacterium genetics., Competing Interests: The authors have declared that no competing interests exist.

Here, we describe the structure of three actinobacteriophage capsids that infect Mycobacterium smegmatis . The capsid structures were resolved to approximately six angstroms, which allowed confirmation that each bacteriophage uses the HK97-fold to form their capsid. One bacteriophage, Rosebush, may have a novel variation of the HK97-fold. Four novel accessory proteins that form the capsid head along with the major capsid protein were identified. Two of the accessory proteins were minor capsid proteins and showed some homology, based on bioinformatic analysis, to the TW1 bacteriophage. The remaining two accessory proteins are decoration proteins that are located on the outside of the capsid and do not resemble any previously described bacteriophage decoration protein. SDS-PAGE and mass spectrometry was used to identify the accessory proteins and bioinformatic analysis of the accessory proteins suggest they are used in many actinobacteriophage capsids.

We report here the sequences of 20 bacteriophages isolated on Gordonia terrae 3612. These phages span considerable sequence diversity, represent 12 clusters and a singleton genome, and range in genome length from 16.2 kbp to 151.3 kbp. Phages Pupper and SCentae are the first reported Myoviridae phages of Gordonia spp., (Copyright © 2020 Pope et al.)

Genome modifications are central components of the continuous arms race between viruses and their hosts. The archaeosine base (G + ), which was thought to be found only in archaeal tRNAs, was recently detected in genomic DNA of Enterobacteria phage 9g and was proposed to protect phage DNA from a wide variety of restriction enzymes. In this study, we identify three additional 2'-deoxy-7-deazaguanine modifications, which are all intermediates of the same pathway, in viruses: 2'-deoxy-7-amido-7-deazaguanine (dADG), 2'-deoxy-7-cyano-7-deazaguanine (dPreQ 0 ) and 2'-deoxy-7- aminomethyl-7-deazaguanine (dPreQ 1 ). We identify 180 phages or archaeal viruses that encode at least one of the enzymes of this pathway with an overrepresentation (60%) of viruses potentially infecting pathogenic microbial hosts. Genetic studies with the Escherichia phage CAjan show that DpdA is essential to insert the 7-deazaguanine base in phage genomic DNA and that 2'-deoxy-7-deazaguanine modifications protect phage DNA from host restriction enzymes.

A 15-year-old patient with cystic fibrosis with a disseminated Mycobacterium abscessus infection was treated with a three-phage cocktail following bilateral lung transplantation. Effective lytic phage derivatives that efficiently kill the infectious M. abscessus strain were developed by genome engineering and forward genetics. Intravenous phage treatment was well tolerated and associated with objective clinical improvement, including sternal wound closure, improved liver function, and substantial resolution of infected skin nodules.

Temperate phages play important roles in the physiology of their bacterial hosts and establish a lysogenic relationship with the host through which prophage-expressed genes confer new phenotypes. A key phenotype is prophage-mediated defense against heterotypic viral attack, in which temperate phages collude with their bacterial host to prevent other phages from attacking, sometimes with exquisite specificity. Such defense systems have been described in Pseudomonas and Mycobacterium phages but are likely widespread throughout the microbial community. Here, we describe a novel prophage-mediated defense system encoded by Gordonia phage CarolAnn, which defends against infection by unrelated phages grouped in cluster CZ. CarolAnn genes 43 and 44 are coexpressed with the repressor and are necessary and sufficient to confer defense against phage Kita and its close relatives. Kita and these relatives are targeted through Kita gene 53 , a gene that is of unknown function but which is the location of defense escape mutations that overcome CarolAnn defense. Expression of Kita gene 53 is toxic to Gordonia terrae in the presence of CarolAnn genes 43 and 44 , suggesting that defense may be mediated by an abortive infection type of mechanism. CarolAnn genes 43 and 44 are distant relatives of mycobacteriophage Sbash genes 31 and 30 , respectively, which also confer viral defense but use a different targeting system. IMPORTANCE Prophage-mediated viral defense systems play a key role in microbial dynamics, as lysogeny is established relatively efficiently, and prophage-expressed genes can strongly inhibit lytic infection of other, unrelated phages. Demonstrating such defense systems in Gordonia terrae suggests that these systems are widespread and that there are a multitude of different systems with different specificities for the attacking phages., (Copyright © 2019 Montgomery et al.)

The arms race between bacteria and their bacteriophages profoundly influences microbial evolution. With an estimated 10 23 phage infections occurring per second, there is strong selection for both bacterial survival and phage coevolution for continued propagation. Many phage resistance systems, including restriction-modification systems, clustered regularly interspaced short palindromic repeat-Cas (CRISPR-Cas) systems, a variety of abortive infection systems, and many others that are not yet mechanistically defined, have been described. Temperate bacteriophages are common and form stable lysogens that are immune to superinfection by the same or closely related phages. However, temperate phages collude with their hosts to confer defense against genomically distinct phages, to the mutual benefit of the bacterial host and the prophage. Prophage-mediated viral systems have been described in Mycobacterium phages and Pseudomonas phages but are predicted to be widespread throughout the microbial world. Here we describe a new viral defense system in which the mycobacteriophage Sbash prophage colludes with its Mycobacterium smegmatis host to confer highly specific defense against infection by the unrelated mycobacteriophage Crossroads. Sbash genes 30 and 31 are lysogenically expressed and are necessary and sufficient to confer defense against Crossroads but do not defend against any of the closely related phages grouped in subcluster L2. The mapping of Crossroads defense escape mutants shows that genes 132 and 141 are involved in recognition by the Sbash defense system and are proposed to activate a loss in membrane potential mediated by Sbash gp30 and gp31. IMPORTANCE Viral infection is an ongoing challenge to bacterial survival, and there is strong selection for development or acquisition of defense systems that promote survival when bacteria are attacked by bacteriophages. Temperate phages play central roles in these dynamics through lysogenic expression of genes that defend against phage attack, including those unrelated to the prophage. Few prophage-mediated viral defense systems have been characterized, but they are likely widespread both in phage genomes and in the prophages integrated in bacterial chromosomes., (Copyright © 2019 Gentile et al.)

Bacteriophages Balsa, Golden, and Lucky3 are cluster EA phages isolated from flowers and infect Microbacterium foliorum NRRL B-24224. The genomes of Golden and Lucky3 (subcluster EA1) are closely related, whereas Balsa (subcluster EA4) is a more distant relative.

Modern agriculture is expected to face an increasing global demand for food while also needing to comply with higher sustainability standards. Therefore, control of crop pathogens requires new, green alternatives to current methods. Potatoes are susceptible to several bacterial diseases, with infections by soft rot Enterobacteriaceae (SRE) being a significant contributor to the major annual losses. As there are currently no efficient ways of combating SRE, we sought to develop an approach that could easily be incorporated into the potato production pipeline. To this end, 46 phages infecting the emerging potato pathogen Dickeya solani were isolated and thoroughly characterized. The 46 isolated phages were grouped into three different groups based on DNA similarity, representing two distinct clusters and a singleton. One cluster showed similarity to phages previously used to successfully treat soft rot in potatoes, whereas the remaining phages were novel and showed only very limited similarity to previously isolated phages. We selected six diverse phages in order to create the hereto most complex phage cocktail against SRE. The cocktail was applied in a proof-of-principle experiment to treat soft rot in potatoes under simulated storage conditions. We show that the phage cocktail was able to significantly reduce the incidence of soft rot as well as disease severity after 5 days of storage post-infection with Dickeya solani . This confirms results from previous studies that phages represent promising biocontrol agents against SRE infection in potato.

Background: A remarkable exception to the large genetic diversity often observed for bacteriophages infecting a specific bacterial host was found for the Cutibacterium acnes (formerly Propionibacterium acnes) phages, which are highly homogeneous. Phages infecting the related species, which is also a member of the Propionibacteriaceae family, Propionibacterium freudenreichii, a bacterium used in production of Swiss-type cheeses, have also been described and are common contaminants of the cheese manufacturing process. However, little is known about their genetic composition and diversity., Results: We obtained seven independently isolated bacteriophages that infect P. freudenreichii from Swiss-type cheese samples, and determined their complete genome sequences. These data revealed that all seven phage isolates are of similar genomic length and GC% content, but their genomes are highly diverse, including genes encoding the capsid, tape measure, and tail proteins. In contrast to C. acnes phages, all P. freudenreichii phage genomes encode a putative integrase protein, suggesting they are capable of lysogenic growth. This is supported by the finding of related prophages in some P. freudenreichii strains. The seven phages could further be distinguished as belonging to two distinct genomic types, or 'clusters', based on nucleotide sequences, and host range analyses conducted on a collection of P. freudenreichii strains show a higher degree of host specificity than is observed for the C. acnes phages., Conclusions: Overall, our data demonstrate P. freudenreichii bacteriophages are distinct from C. acnes phages, as evidenced by their higher genetic diversity, potential for lysogenic growth, and more restricted host ranges. This suggests substantial differences in the evolution of these related species from the Propionibacteriaceae family and their phages, which is potentially related to their distinct environmental niches.

We report here the complete genome sequences of 44 phages infecting Arthrobacter sp. strain ATCC 21022. These phages have double-stranded DNA genomes with sizes ranging from 15,680 to 70,707 bp and G+C contents from 45.1% to 68.5%. All three tail types (belonging to the families Siphoviridae , Myoviridae , and Podoviridae ) are represented., (Copyright © 2018 Klyczek et al.)

Four bacteriophages infecting Mycobacterium smegmatis mc 2 155 (three belonging to subcluster P1 and one belonging to subcluster P2) were isolated from soil and sequenced. All four phages are similar in the left arm of their genomes, but the P2 phage differs in the right arm. All four genomes contain features of temperate phages., (Copyright © 2018 Doyle et al.)

Cluster BE1 Streptomyces bacteriophages belong to the Siphoviridae , with genome sizes over 130 kbp, and they contain direct terminal repeats of approximately 11 kbp. Eight newly isolated closely related cluster BE1 phages contain 43 to 48 tRNAs, one transfer-messenger RNA (tmRNA), and 216 to 236 predicted open reading frames (ORFs), but few of their genes are shared with other phages, including those infecting Streptomyces species., (Copyright © 2018 Hughes et al.)

Current sequencing technologies allow for the rapid and inexpensive sequencing of complete bacteriophage genomes, using small quantities of nucleic acid as starting material. Determination of the location and function of the gene features within the genome sequence, or annotation, is a necessary next step prior to submission to a public database, publication in a scientific journal, or advanced comparative genomic and proteomic studies. Gene prediction can be largely accomplished through the use of several freely available programs. However, manual inspection and refinement is essential to the production of the most accurate genome annotations. Here, we describe an overview of the annotation of a bacteriophage genome sequence using the freely available program DNA Master.

Engaging undergraduate students in scientific research promises substantial benefits, but it is not accessible to all students and is rarely implemented early in college education, when it will have the greatest impact. An inclusive Research Education Community (iREC) provides a centralized scientific and administrative infrastructure enabling engagement of large numbers of students at different types of institutions. The Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) is an iREC that promotes engagement and continued involvement in science among beginning undergraduate students. The SEA-PHAGES students show strong gains correlated with persistence relative to those in traditional laboratory courses regardless of academic, ethnic, gender, and socioeconomic profiles. This persistent involvement in science is reflected in key measures, including project ownership, scientific community values, science identity, and scientific networking., Competing Interests: The authors declare no conflict of interest., (Copyright © 2017 the Author(s). Published by PNAS.)

We report the genome sequences of 14 cluster K mycobacteriophages isolated using Mycobacterium smegmatis mc²155 as host. Four are closely related to subcluster K1 phages, and 10 are members of subcluster K6. The phage genomes span considerable sequence diversity, including multiple types of integrases and integration sites., (Copyright © 2017 Anders et al.)

We report the complete genome sequences of 19 cluster CA bacteriophages isolated from environmental samples using Rhodococcus erythropolis as a host. All of the phages are Siphoviridae , have similar genome lengths (46,314 to 46,985 bp) and G+C contents (58.5 to 58.8%), and share nucleotide sequence similarity., (Copyright © 2017 Bonilla et al.)

Four subcluster L2 mycobacteriophages, Finemlucis, Miley16, Wilder, and Zakai, that infect Mycobacterium smegmatis mc 2 155 were isolated. The four phages are closely related to each other and code for 12 to 14 tRNAs and 130 to 132 putative protein-coding genes, including tyrosine integrases, cro , immunity repressors, and excise genes involved in the establishment of lysogeny., (Copyright © 2017 Herren et al.)

Twelve siphoviral phages isolated using Arthrobacter sp. strain ATCC 21022 were sequenced. The phages all have relatively small genomes, ranging from 15,319 to 15,556 bp. All 12 phages are closely related to previously described cluster AN Arthrobacter phages., (Copyright © 2017 Lee-Soety et al.)

We report here the complete genome sequences of four subcluster L3 mycobacteriophages newly isolated from soil samples, using Mycobacterium smegmatis mc 2 155 as the host. Comparative genomic analyses with four previously described subcluster L3 phages reveal strong nucleotide similarity and gene conservation, with several large insertions/deletions near their right genome ends., (Copyright © 2017 Chung et al.)

Bacteriophages AlleyCat, Edugator, and Guillsminger were isolated on Mycobacterium smegmatis mc 2 155 from enriched soil samples. All are members of mycobacteriophage subcluster K5, with genomes of 62,112 to 63,344 bp. Each genome contains 92 to 99 predicted protein-coding genes and one tRNA. Guillsminger is the first mycobacteriophage to carry an IS 1380 family transposon., (Copyright © 2017 King et al.)

Mycobacteriophages Chancellor, Mitti, and Wintermute infect Mycobacterium smegmatis mc 2 155 and are closely related to phages Cheetobro and Fionnbharth in subcluster K4. Genome sizes range from 57,697 bp to 58,046 bp. Phages are predicted to be temperate and to infect the pathogen Mycobacterium tuberculosis ., (Copyright © 2017 Edgington et al.)

We report here the genome sequences of three newly isolated phages that infect Mycobacterium smegmatis mc 2 155. Phages Findley, Hurricane, and TBond007 were discovered in geographically distinct locations and are related to cluster K mycobacteriophages, with Findley being similar to subcluster K2 phages and Hurricane and TBond007 being similar to subcluster K3 phages., (Copyright © 2017 Saha et al.)

Caterpillar, Nightmare, and Teacup are cluster AU siphoviral phages isolated from enriched soil on Arthrobacter sp. strain ATCC 21022. These genomes are 58 kbp long with an average G+C content of 50%. Sequence analysis predicts 86 to 92 protein-coding genes, including a large number of small proteins with predicted transmembrane domains., (Copyright © 2017 Adair et al.)

We report here the genome sequences of six newly isolated bacteriophages infecting Arthrobacter sp. ATCC 21022. All six have myoviral morphologies and have double-stranded DNA genomes with circularly permuted ends. The six phages are closely related with average nucleotide identities of 73.4 to 93.0% across genomes lengths of 49,797 to 51,347 bp., (Copyright © 2017 Adair et al.)

Seven mycobacteriophages from distinct geographical locations were isolated, using Mycobacterium smegmatis mc 2 155 as the host, and then purified and sequenced. All of the genomes are related to cluster A mycobacteriophages, BobSwaget and Lokk in subcluster A2; Fred313, KADY, Stagni, and StepMih in subcluster A3; and MyraDee in subcluster A18, the first phage to be assigned to that subcluster., (Copyright © 2017 Butela et al.)

The global bacteriophage population is large, dynamic, old, and highly diverse genetically. Many phages are tailed and contain double-stranded DNA, but these remain poorly characterized genomically. A collection of over 1,000 phages infecting Mycobacterium smegmatis reveals the diversity of phages of a common bacterial host, but their relationships to phages of phylogenetically proximal hosts are not known. Comparative sequence analysis of 79 phages isolated on Gordonia shows these also to be diverse and that the phages can be grouped into 14 clusters of related genomes, with an additional 14 phages that are "singletons" with no closely related genomes. One group of six phages is closely related to Cluster A mycobacteriophages, but the other Gordonia phages are distant relatives and share only 10% of their genes with the mycobacteriophages. The Gordonia phage genomes vary in genome length (17.1 to 103.4 kb), percentage of GC content (47 to 68.8%), and genome architecture and contain a variety of features not seen in other phage genomes. Like the mycobacteriophages, the highly mosaic Gordonia phages demonstrate a spectrum of genetic relationships. We show this is a general property of bacteriophages and suggest that any barriers to genetic exchange are soft and readily violable. IMPORTANCE Despite the numerical dominance of bacteriophages in the biosphere, there is a dearth of complete genomic sequences. Current genomic information reveals that phages are highly diverse genomically and have mosaic architectures formed by extensive horizontal genetic exchange. Comparative analysis of 79 phages of Gordonia shows them to not only be highly diverse, but to present a spectrum of relatedness. Most are distantly related to phages of the phylogenetically proximal host Mycobacterium smegmatis , although one group of Gordonia phages is more closely related to mycobacteriophages than to the other Gordonia phages. Phage genome sequence space remains largely unexplored, but further isolation and genomic comparison of phages targeted at related groups of hosts promise to reveal pathways of bacteriophage evolution., (Copyright © 2017 Pope et al.)

We describe the genome sequences of three closely related mycobacteriophages, Kerberos, Pomar16, and StarStuff, isolated at similar times but from geographically distinct regions. All three genomes are similar to those of other subcluster A2 phages, such as L5 and D29, are temperate, and have siphoviral virion morphologies., (Copyright © 2017 Jacobs-Sera et al.)

The vast bacteriophage population harbors an immense reservoir of genetic information. Almost 2000 phage genomes have been sequenced from phages infecting hosts in the phylum Actinobacteria, and analysis of these genomes reveals substantial diversity, pervasive mosaicism, and novel mechanisms for phage replication and lysogeny. Here, we describe the isolation and genomic characterization of 46 phages from environmental samples at various geographic locations in the U.S. infecting a single Arthrobacter sp. strain. These phages include representatives of all three virion morphologies, and Jasmine is the first sequenced podovirus of an actinobacterial host. The phages also span considerable sequence diversity, and can be grouped into 10 clusters according to their nucleotide diversity, and two singletons each with no close relatives. However, the clusters/singletons appear to be genomically well separated from each other, and relatively few genes are shared between clusters. Genome size varies from among the smallest of siphoviral phages (15,319 bp) to over 70 kbp, and G+C contents range from 45-68%, compared to 63.4% for the host genome. Although temperate phages are common among other actinobacterial hosts, these Arthrobacter phages are primarily lytic, and only the singleton Galaxy is likely temperate.