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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.

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.

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.

Amela and Verse are two Streptomyces phages isolated by enrichment on Streptomyces venezuelae (ATCC 10712) from two different soil samples. Amela has a genome length of 49,452, with 75 genes. Verse has a genome length of 49,483, with 75 genes. Both belong to the BD3 subcluster of Actinobacteriophage.

The impact of orotate accumulation in the medically important bacterium Pseudomonas aeruginosa was studied by deleting pyrE, the gene encoding orotate phosphoribosyltransferase and responsible for converting orotate into orotate monophosphate within the de novo pyrimidine synthesis pathway. The pyrE mutant accumulated orotate and exhibited decreased production of hemolysin, casein protease, and elastase. Feeding orotate at a concentration of 51.25 μM to the wild type, PAO1, likewise decreased production of these factors except for hemolysin, which was not affected. A significant increase in the pigments pyocyanin and pyoverdin was also observed. Pyocyanin increase in the pyrE mutant was heightened when the mutant was supplemented with orotate. Although pyoverdin production in the wild-type PAO1 was unaffected by orotate supplementation, a decrease in the mutant’s production was observed when supplemented with orotate. These results indicate a significant reduction in virulence factor production in the pyrE mutant and reduction in some virulence factors in the wild type when supplemented with orotate.

Mycobacteriophages – viruses of mycobacterial hosts – are genetically diverse but morphologically are all classified in the Caudovirales with double-stranded DNA and tails. We describe here a group of five closely related mycobacteriophages – Corndog, Catdawg, Dylan, Firecracker, and YungJamal – designated as Cluster O with long flexible tails but with unusual prolate capsids. Proteomic analysis of phage Corndog particles, Catdawg particles, and Corndog-infected cells confirms expression of half of the predicted gene products and indicates a non-canonical mechanism for translation of the Corndog tape measure protein. Bioinformatic analysis identifies 8–9 strongly predicted SigA promoters and all five Cluster O genomes contain more than 30 copies of a 17 bp repeat sequence with dyad symmetry located throughout the genomes. Comparison of the Cluster O phages provides insights into phage genome evolution including the processes of gene flux by horizontal genetic exchange.

Genomic analysis of a large set of phages infecting the common host Mycobacterium smegmatis mc 2 155 shows that they span considerable genetic diversity. There are more than 20 distinct types that lack nucleotide similarity with each other, and there is considerable diversity within most of the groups. Three newly isolated temperate mycobacteriophages, Bongo, PegLeg, and Rey, constitute a new group (cluster M), with the closely related phages Bongo and PegLeg forming subcluster M1 and the more distantly related Rey forming subcluster M2. The cluster M mycobacteriophages have siphoviral morphologies with unusually long tails, are homoimmune, and have larger than average genomes (80.2 to 83.7 kbp). They exhibit a variety of features not previously described in other mycobacteriophages, including noncanonical genome architectures and several unusual sets of conserved repeated sequences suggesting novel regulatory systems for both transcription and translation. In addition to containing transfer-messenger RNA and RtcB-like RNA ligase genes, their genomes encode 21 to 24 tRNA genes encompassing complete or nearly complete sets of isotypes. We predict that these tRNAs are used in late lytic growth, likely compensating for the degradation or inadequacy of host tRNAs. They may represent a complete set of tRNAs necessary for late lytic growth, especially when taken together with the apparent lack of codons in the same late genes that correspond to tRNAs that the genomes of the phages do not obviously encode. IMPORTANCE The bacteriophage population is vast, dynamic, and old and plays a central role in bacterial pathogenicity. We know surprisingly little about the genetic diversity of the phage population, although metagenomic and phage genome sequencing indicates that it is great. Probing the depth of genetic diversity of phages of a common host, Mycobacterium smegmatis , provides a higher resolution of the phage population and how it has evolved. Three new phages constituting a new cluster M further expand the diversity of the mycobacteriophages and introduce novel features. As such, they provide insights into phage genome architecture, virion structure, and gene regulation at the transcriptional and translational levels.

Recent recommendations for educational research encourage empirically tested, theory-based, completely transparent, and broadly applicable studies. In light of these recommendations, we call for a research standard and community of practice in the evaluation of technology use in the undergraduate life science classroom. We outline appropriate research methodology, review and critique the past research on technology usage and, lastly, suggest a new and improved focus for research on emerging technologies.

Five newly isolated mycobacteriophages -Angelica, CrimD, Adephagia, Anaya, and Pixie - have similar genomic architectures to mycobacteriophage TM4, a previously characterized phage that is widely used in mycobacterial genetics. The nucleotide sequence similarities warrant grouping these into Cluster K, with subdivision into three subclusters: K1, K2, and K3. Although the overall genome architectures of these phages are similar, TM4 appears to have lost at least two segments of its genome, a central region containing the integration apparatus, and a segment at the right end. This suggests that TM4 is a recent derivative of a temperate parent, resolving a long-standing conundrum about its biology, in that it was reportedly recovered from a lysogenic strain of Mycobacterium avium, but it is not capable of forming lysogens in any mycobacterial host. Like TM4, all of the Cluster K phages infect both fast- and slow-growing mycobacteria, and all of them - with the exception of TM4 - form stable lysogens in both Mycobacterium smegmatis and Mycobacterium tuberculosis; immunity assays show that all five of these phages share the same immune specificity. TM4 infects these lysogens suggesting that it was either derived from a heteroimmune temperate parent or that it has acquired a virulent phenotype. We have also characterized a widely-used conditionally replicating derivative of TM4 and identified mutations conferring the temperature-sensitive phenotype. All of the Cluster K phages contain a series of well conserved 13 bp repeats associated with the translation initiation sites of a subset of the genes; approximately one half of these contain an additional sequence feature composed of imperfectly conserved 17 bp inverted repeats separated by a variable spacer. The K1 phages integrate into the host tmRNA and the Cluster K phages represent potential new tools for the genetics of M. tuberculosis and related species. © 2011 Pope et al. Fil: Pope, Welkin H.. University of Pittsburgh; Estados Unidos Fil: Ferreira, Christina M.. University of Pittsburgh; Estados Unidos Fil: Jacobs Sera, Deborah. University of Pittsburgh; Estados Unidos Fil: Benjamin, Robert C.. University of North Texas; Estados Unidos Fil: Davis, Ariangela J.. Calvin College; Estados Unidos Fil: DeJong, Randall J.. Calvin College; Estados Unidos Fil: Elgin, Sarah C. R.. Washington University in St. Louis; Estados Unidos Fil: Guilfoile, Forrest R.. University of Pittsburgh; Estados Unidos Fil: Forsyth, Mark H.. The College Of William And Mary; Estados Unidos Fil: Harris, Alexander D.. Calvin College; Estados Unidos Fil: Harvey, Samuel E.. The College Of William And Mary; Estados Unidos Fil: Hughes, Lee E.. University of North Texas; Estados Unidos Fil: Hynes, Peter M.. Washington University in St. Louis; Estados Unidos Fil: Jackson, Arrykka S.. The College Of William And Mary; Estados Unidos Fil: Jalal, Marilyn D.. University of North Texas; Estados Unidos Fil: MacMurray, Elizabeth A.. The College Of William And Mary; Estados Unidos Fil: Manley, Coreen M.. University of North Texas; Estados Unidos Fil: McDonough, Molly J.. The College Of William And Mary; Estados Unidos Fil: Mosier, Jordan L.. University of North Texas; Estados Unidos Fil: Osterbann, Larissa J.. Calvin College; Estados Unidos Fil: Rabinowitz, Hannah S.. Washington University in St. Louis; Estados Unidos Fil: Rhyan, Corwin N.. Washington University in St. Louis; Estados Unidos Fil: Russell, Daniel A.. University of Pittsburgh; Estados Unidos Fil: Saha, Margaret S.. The College Of William And Mary; Estados Unidos Fil: Shaffer, Christopher D.. Washington University in St. Louis; Estados Unidos Fil: Simon, Stephanie E.. University of North Texas; Estados Unidos Fil: Sims, Erika F.. Washington University in St. Louis; Estados Unidos Fil: Tovar, Isabel G.. University of North Texas; Estados Unidos Fil: Weisser, Emilie G.. Washington University in St. Louis; Estados Unidos Fil: Wertz, John T.. Calvin College; Estados Unidos Fil: Weston-Hafer, Kathleen A.. Washington University in St. Louis; Estados Unidos Fil: Williamson, Kurt E.. The College Of William And Mary; Estados Unidos Fil: Zhang, Bo. Washington University in St. Louis; Estados Unidos Fil: Cresawn, Steven G.. James Madison University; Estados Unidos Fil: Jain, Paras. Albert Einstein College Of Medicine Of Yeshiva University; Estados Unidos Fil: Piuri, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. University of Pittsburgh; Estados Unidos Fil: Jacobs, William R.. Albert Einstein College Of Medicine Of Yeshiva University; Estados Unidos Fil: Hendrix, Roger W.. University of Pittsburgh; Estados Unidos Fil: Hatfull, Graham F.. University of Pittsburgh; Estados Unidos

Using 5-fluoropyrimidine analogues, high-performance liquid chromatography (HPLC), and the feeding of pyrimidine compounds to pyrimidine auxotrophs, the pathways for salvage of exogenous pyrimidine nucleosides and bases in Streptomyces were established. Selection for resistance to the analogues resulted in the isolation of strains of S. griseus lacking the following enzyme activities: uracil phosphoribosyltransferase (upp) and cytidine deaminase (cdd). The conversion of substrates in the pathway was followed using reverse-phase HPLC. The strains deficient in salvage enzymes were also verified by this method. In addition, feeding of exogenous pyrimidines to strains lacking the biosynthetic pathway confirmed the salvage pathway. Data from the analogue, HPLC, and feeding experiments showed that Streptomyces recycles the pyrimidine base uracil, as well as the nucleosides uridine and cytidine. Cytosine is not recycled due to a lack of cytosine deaminase.