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The World Health Organization (WHO) estimates that 40% of tuberculosis (TB) cases are not diagnosed and treated correctly. Even though there are several diagnostic tests available in the market, rapid, easy, inexpensive detection, and drug susceptibility testing (DST) of Mycobacterium tuberculosis is still of critical importance specially in low and middle-income countries with high incidence of the disease. In this work, we have developed a microscopy-based methodology using the reporter mycobacteriophage mCherrybombϕ for detection of Mycobacterium spp. and phenotypic determination of rifampicin resistance within just days from sputum sample collection. Fluoromycobacteriophage methodology is compatible with regularly used protocols in clinical laboratories for TB diagnosis and paraformaldehyde fixation after infection reduces biohazard risks with sample analysis by fluorescence microscopy. We have also set up conditions for discrimination between M. tuberculosis complex (MTBC) and non-tuberculous mycobacteria (NTM) strains by addition of p-nitrobenzoic acid (PNB) during the assay. Using clinical isolates of pre-XDR and XDR-TB strains from this study, we tested mCherrybombΦ for extended DST and we compared the antibiotic resistance profile with those predicted by whole genome sequencing. Our results emphasize the utility of a phenotypic test for M. tuberculosis extended DST. The many attributes of mCherrybombΦ suggests this could be a useful component of clinical microbiological laboratories for TB diagnosis and since only viable cells are detected this could be a useful tool for monitoring patient response to treatment.

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. Electronic supplementary material The online version of this article (10.1186/s12866-018-1159-y) contains supplementary material, which is available to authorized users.

Roger W. Hendrix was at the forefront of bacteriophage biology for nearly 50 years and was central to our understanding of both viral capsid assembly and phage genomic diversity and evolution. Roger's warm and gentle demeanor belied a razor-sharp mind and warmed him to numerous highly productive collaborations that amplified his scientific impact. Roger was always completely open with scientific ideas while at the same time quietly agitating with a stream of new ways of thinking about problems and nudging our communities to search for innovative solutions: a gentle but highly effective provocateur.

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.

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.

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.

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.

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.

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.

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.

Four subcluster L2 mycobacteriophages, Finemlucis, Miley16, Wilder, and Zakai, that infect Mycobacterium smegmatis mc2155 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.

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.

Mycobacteriophages Chancellor, Mitti, and Wintermute infect Mycobacterium smegmatis mc2155 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.

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.

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 .

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.

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.

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.

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.

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.

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.

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.

Mycobacteriophage CrystalP is a newly isolated phage infecting Mycobacterium smegmatis strain mc 2 155. CrystalP has a 76,483-bp genome and is predicted to contain 143 protein-coding and 2 tRNA genes, including repressor and integrase genes consistent with a temperate lifestyle. CrystalP is related to the mycobacteriophages Toto and Kostya and to other Cluster E phages.

Bacteriophages play key roles in microbial evolution1,2, marine nutrient cycling3, and human disease4. Phages are genetically diverse and their genome architectures are characteristically mosaic, driven by horizontal gene transfer (HGT) with other phages and host genomes5. As a consequence, phage evolution is complex and their genomes are composed of genes with distinct and varied evolutionary histories6,7. However, there are conflicting perspectives on the roles of mosaicism, and the extent to which it generates a spectrum of genome diversity8 or genetically discrete populations9,10. Here, we show that bacteriophages evolve within two general evolutionary modes that differ in the extent of HGT by an order of magnitude. Temperate phages distribute into high and low gene flux modes, whereas lytic phages share only the lower gene flux mode. The evolutionary modes are also a function of the bacterial host, and different proportions of temperate and lytic phages are distributed in either mode depending on the host phylum. Groups of genetically-related phages fall into either the high or low gene flux modes, suggesting there are genetic as well as ecological drivers of HGT rates. Consequently, genome mosaicism varies depending on the host, lifestyle, and genetic constitution of phages.

Bacteriophage discovery and genomics provides a powerful and effective platform for integrating missions in research and education. Implementation of the Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program facilitates a broad impact by including a diverse array of schools, faculty, and students. The program generates new insights into the diversity and evolution of the bacteriophage population and presents a model for introducing first-year undergraduate students to discovery-based research experiences.

The genome sequences of eight Streptomyces phages are presented, four of which were isolated for this study. Phages R4, TG1, ϕHau3, and SV1 were isolated previously and have been exploited as tools for understanding and genetically manipulating Streptomyces spp. We also extracted five apparently intact prophages from recent Streptomyces spp. genome projects and, together with six phage genomes in the database, we analyzed all 19 Streptomyces phage genomes with a view to understanding their relationships to each other and to other actinophages, particularly the mycobacteriophages. Fifteen of the Streptomyces phages group into four clusters of related genomes. Although the R4-like phages do not share nucleotide sequence similarity with other phages, they clearly have common ancestry with cluster A mycobacteriophages, sharing many protein homologues, common gene syntenies, and similar repressor-stoperator regulatory systems. The R4-like phage ϕHau3 and the prophage StrepC.1 (from Streptomyces sp. strain C) appear to have hijacked a unique adaptation of the streptomycetes, i.e., use of the rare UUA codon, to control translation of the essential phage protein, the terminase. The Streptomyces venezuelae generalized transducing phage SV1 was used to predict the presence of other generalized transducing phages for different Streptomyces species.

Bacteriophages represent a majority of all life forms, and the vast, dynamic population with early origins is reflected in their enormous genetic diversity. A large number of bacteriophage genomes have been sequenced. They are replete with novel genes without known relatives. We know little about their functions, which genes are required for lytic growth, and how they are expressed. Furthermore, the diversity is such that even genes with required functions - such as virion proteins and repressors - cannot always be recognized. Here we describe a functional genomic dissection of mycobacteriophage Giles, in which the virion proteins are identified, genes required for lytic growth are determined, the repressor is identified, and the transcription patterns determined. We find that although all of the predicted phage genes are expressed either in lysogeny or in lytic growth, 45% of the predicted genes are non-essential for lytic growth. We also describe genes required for DNA replication, show that recombination is required for lytic growth, and that Giles encodes a novel repressor. RNAseq analysis reveals abundant expression of a small non-coding RNA in a lysogen and in late lytic growth, although it is non-essential for lytic growth and does not alter lysogeny.

We report here the genome sequences of 38 newly isolated bacteriophages using Gordonia terrae 3612 (ATCC 25594) and Gordonia neofelifaecis NRRL59395 as bacterial hosts. All of the phages are double-stranded DNA (dsDNA) tail phages with siphoviral morphologies, with genome sizes ranging from 17,118 bp to 93,843 bp and spanning considerable nucleotide sequence diversity.

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.

Genetic switches are critical components of developmental circuits. Because temperate bacteriophages are vastly abundant and greatly diverse, they are rich resources for understanding the mechanisms and evolution of switches and the molecular control of genetic circuitry. Here, we describe a new class of small, compact, and simple switches that use site-specific recombination as the key decision point. The phage attachment site attP is located within the phage repressor gene such that chromosomal integration results in removal of a C-terminal tag that destabilizes the virally encoded form of the repressor. Integration thus not only confers prophage stability but also is a requirement for lysogenic establishment. The variety of these self-contained integration-dependent immunity systems in different genomic contexts suggests that these represent ancestral states in switch evolution from which more-complex switches have evolved. They also provide a powerful toolkit for building synthetic biological circuits.

Mycobacteriophage DS6A is unique among the more than 8,000 isolated mycobacteriophages due to its ability to form plaques exclusively on mycobacteria belonging to the Mycobacterium tuberculosis complex (MTBC). Speculation surrounding this specificity has led to unsupported assertions in published studies and patents that nontuberculous mycobacteria (NTM) are wholly resistant to DS6A infection. In this study, we identified two independent nonessential regions in the DS6A genome and replaced them with an mVenus-expressing plasmid to generate fluorescent reporter phages Φ 2 GFP12 and Φ 2 GFP13. We show that even though DS6A is able to form plaques only on MTBC bacteria, infection of various NTM results in mVenus expression in transduced cells. The efficiency of DS6A in delivering DNA varied between NTM species. Additionally, we saw a striking difference in the efficiency of DNA delivery between the closely related members of the Mycobacterium abscessus complex, M. abscessus and Mycobacterium massiliense . We also demonstrated that TM4 and DS6A, two phages that do not form plaques on M. massiliense , differ in their ability to deliver DNA, suggesting that there is a phage-specific restriction between mycobacterial species. Phylogenetic analysis reveals that the DS6A genome has a characteristically mosaic structure but provided few insights into the basis for the specificity for MTBC hosts. This study demonstrates that the inability of the MTBC-specific phage DS6A to form plaques on NTM is more complex than previously thought. Moreover, the DS6A-derived fluorophages provide important new tools for the study of mycobacterial biology. IMPORTANCE The coevolution of bacteria and their infecting phages involves a constant arms race for bacteria to prevent phage infection and phage to overcome these preventions. Although a diverse array of phage defense systems is well characterized in bacteria, very few phage restriction systems are known in mycobacteria. The DS6A mycobacteriophage is unique in the mycobacterial world in that it forms plaques only on members of the Mycobacterium tuberculosis complex. However, the novel DS6A reporter phages developed in this work demonstrate that DS6A can infect nontuberculous mycobacteria at various efficiencies. By comparing the abilities of DS6A and another phage, TM4, to infect and form plaques on various mycobacterial species, we can begin to discern new phage restriction systems employed within the genus.

Lactic acid bacteria (LAB) have many applications in food and industrial fermentations. Prophage induction and generation of new virulent phages is a risk for the dairy industry. We identified three complete prophages (PLE1, PLE2, and PLE3) in the genome of the well-studied probiotic strain Lactobacillus casei BL23. All of them have mosaic architectures with homologous sequences to Streptococcus, Lactococcus, Lactobacillus, and Listeria phages or strains. Using a combination of quantitative real-time PCR, genomics, and proteomics, we showed that PLE2 and PLE3 can be induced—but with different kinetics—in the presence of mitomycin C, although PLE1 remains as a prophage. A structural analysis of the distal tail (Dit) and tail associated lysin (Tal) baseplate proteins of these prophages and other L. casei/paracasei phages and prophages provides evidence that carbohydrate-binding modules (CBM) located within these “evolved” proteins may replace receptor binding proteins (RBPs) present in other well-studied LAB phages. The detailed study of prophage induction in this prototype strain in combination with characterization of the proteins involved in host recognition will facilitate the design of new strategies for avoiding phage propagation in the dairy industry. Fil: Dieterle, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Fina Martin, Joaquina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Durán, Rosario. Instituto Pasteur de Montevideo; Uruguay Fil: Nemirovsky, Sergio Ivan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Sanchez Rivas, Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina Fil: Bowman, Charles. University of Pittsburgh; Estados Unidos. The Scripps Research Institute; Estados Unidos Fil: Russell, Daniel. University of Pittsburgh; Estados Unidos Fil: Hatfull, Graham F.. University of Pittsburgh; Estados Unidos Fil: Cambillau, Christian. Centre National de la Recherche Scientifique; Francia. Marseille Universite; Francia Fil: Piuri, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina

We describe the genomes of two mycobacteriophages, MosMoris and Gattaca, newly isolated on Mycobacterium smegmatis . The two phages are very similar to each other, differing in 61 single nucleotide polymorphisms and six small insertion/deletions. Both have extensive nucleotide sequence similarity to mycobacteriophage Marvin and together form cluster S.

Here, we report the complete genome sequence of Gordonia terrae 3612, also known by the strain designations ATCC 25594, NRRL B-16283, and NBRC 100016. The genome sequence reveals it to be free of prophage and clustered regularly interspaced short palindromic repeats (CRISPRs), and it is an effective host for the isolation and characterization of Gordonia bacteriophages.

Hotorobo, Woes, and Monty are newly isolated bacteriophages of Gordonia terrae 3612. The three phages are related, and their genomes are similarly sized (76,972 bp, 73,752 bp, and 75,680 bp for Hotorobo, Woes, and Monty, respectively) and organized. They have extremely long tails and among the longest tape measure protein genes described to date.

Gordonia phages BaxterFox, Kita, Nymphadora, and Yeezy are newly characterized phages of Gordonia terrae , isolated from soil samples in Pittsburgh, Pennsylvania. These phages have genome lengths between 50,346 and 53,717 bp, and encode on average 84 predicted proteins. All have G+C content of 66.6%.

Emalyn is a newly isolated bacteriophage of Gordonia terrae 3612 and has a double-stranded DNA genome 43,982 bp long with 67 predicted protein-encoding genes, 32 of which we can assign putative functions. Emalyn has a prolate capsid and has extensive nucleotide similarity with several previously sequenced phages.

Gordonia phages Bowser and Schwabeltier are newly isolated phages infecting Gordonia terrae 3612. Bowser and Schwabeltier have similar siphoviral morphologies and their genomes are related to each other, but not to other phages. Their lysis cassettes are atypically situated among virion tail genes, and Bowser encodes two tyrosine integrases.

BetterKatz is a bacteriophage isolated from a soil sample collected in Pittsburgh, Pennsylvania using the host Gordonia terrae 3612. BetterKatz's genome is 50,636 bp long and contains 75 predicted protein-coding genes, 35 of which have been assigned putative functions. BetterKatz is not closely related to other sequenced Gordonia phages.

Bacteriophages Phinally and Vivi2 were isolated from soil from Pittsburgh, Pennsylvania, USA, using host Gordonia terrae 3612. The Phinally and Vivi2 genomes are 59,265 bp and 59,337 bp, respectively, and share sequence similarity with each other and with GTE6. Fewer than 25% of the 87 to 89 putative genes have predictable functions.

Mycobacteriophages—viruses of mycobacteria—provide insights into viral diversity and evolution as well as numerous tools for genetic dissection of Mycobacterium tuberculosis . Here we report the complete genome sequences of 61 mycobacteriophages newly isolated from environmental samples using Mycobacterium smegmatis mc 2 155 that expand our understanding of phage diversity.

We describe three newly isolated phages—Obliviate, UmaThurman, and Guacamole—that infect Gordonia terrae 3612. The three genomes are related to one another but are not closely related to other previously sequenced phages or prophages. The three phages are predicted to use integration-dependent immunity systems as described in several mycobacteriophages.

Gordonia bacteriophage Yvonnetastic was isolated from soil in Pittsburgh, PA, using Gordonia terrae 3612 as a host. Yvonnetastic has siphoviral morphology and a genome of 98,136 bp, with 198 predicted protein-coding genes and five tRNA genes. Yvonnetastic does not share substantial sequence similarity with other sequenced bacteriophage genomes.

Attis and SoilAssassin are two closely related bacteriophages isolated on Gordonia terrae 3612 from separate soil samples in Pittsburgh, PA. The Attis and SoilAssassin genomes are 47,881 bp and 47,880 bp, respectively, and have 74 predicted protein-coding genes, including toxin-antitoxin systems, but no tRNAs.

Lucky10 is a newly isolated phage of Gordonia terrae 3612 that was recovered from a soil sample in Pittsburgh, PA. Lucky10 has siphoviral morphology and a double-stranded DNA (dsDNA) genome of 42,979 bp, with 70 predicted protein-coding genes. Lucky10 shows little similarity to previously reported Gordonia phages.

Five bacteriophages that infect the Rhodobacter capsulatus strain YW1 were isolated from stream water near Bloomington, Illinois, USA. Two distinct genome types are represented in the newly isolated bacteriophages. These genomes are different from other bacteriophage genomes previously described.

Bacteriophages Katyusha and Benczkowski14 are newly isolated phages that infect Gordonia terrae 3612. Both have siphoviral morphologies with isometric heads and long tails (500 nm). The genomes are 75,380 bp long and closely related, and the tape measure genes (9 kbp) are among the largest to be identified.

More than 180 individual phages infecting hosts in the phylum Actinobacteria have been sequenced and grouped into Cluster A because of their similar overall nucleotide sequences and genome architectures. These Cluster A phages are either temperate or derivatives of temperate parents, and most have an integration cassette near the center of the genome containing an integrase gene and attP. However, about 20% of the phages lack an integration cassette, which is replaced by a 1.4 kbp segment with predicted partitioning functions, including plasmid-like parA and parB genes. Phage RedRock forms stable lysogens in Mycobacterium smegmatis in which the prophage replicates at 2.4 copies/chromosome and the partitioning system confers prophage maintenance. The parAB genes are expressed upon RedRock infection of M. smegmatis, but are down-regulated once lysogeny is established by binding of RedRock ParB to parS-L, one of two centromere-like sites flanking the parAB genes. The RedRock parS-L and parS-R sites are composed of eight directly repeated copies of an 8 bp motif that is recognized by ParB. The actinobacteriophage parABS cassettes span considerable sequence diversity and specificity, providing a suite of tools for use in mycobacterial genetics.