The official website of the HHMI Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science program.

SEA-PHAGES Publications

Below is a list of publications that are based on data generated by participants in the SEA-PHAGES program and are authored by SEA-PHAGES students or faculty.

Genome Announcements. 2016.
Genome Sequence of Bacillus cereus Group Phage SalinJah
Ivan Erill, Steven M. Caruso, on behalf of the 2015 UMBC Phage Hunters
The double-stranded DNA (dsDNA) Myoviridae Bacillus cereus group bacteriophage SalinJah was isolated from soil collected in Gyeonggi-do, South Korea. SalinJah, a cluster C phage with a broad host range, suggests the need to create a new subcluster with SalinJah and Helga as founding members.
Virology. Vol 489, Pages 243-251, 2016.
Genomic characterization and comparison of seven Myoviridae bacteriophage infecting Bacillus thuringiensis
Sauder A.B., Quinn M.R., Brouillette A., Caruso S., Cresawn S., Erill I., Lewis L., Loesser-Casey K., Pate M., Scott C., Stockwell S., Temple L.
Bacillus thuringiensis Kurstaki, a bacterium that is a source of biopesticides and a safe simulant for pathogenic Bacillus species, was used to isolate seven unique bacteriophages. The phage genomes were sequenced and ranged in size from 158,100 to 163,019bp encoding 290-299 genes, and the GC content of ~38% was similar to that of the host bacterium. All phages had terminal repeats 2-3kb long. Three of the phages encoded tRNAs and three contained a self-splicing intron in the DNA polymerase gene. They were categorized as a single cluster (>60% nucleotide conservation) containing three subclusters (>80% nucleotide conservation), supported by genomic synteny and phylogenetic analysis. Considering the published genomes of phages that infect the genus Bacillus and noting the ability of many of the Bacillus cereus group phages to infect multiple species, a clustering system based on gene content is proposed.
Genome Announcements. Vol 4, Issue 1, 2016.
Paenibacillus larvae Phage Tripp Genome Has 378-Base-Pair Terminal Repeats
J. Abraham, A.-C. Bousquet, E. Bruff, N. Carson, A. Clark, A. Connell, Z. Davis, J. Dums, C. Everington, A. Groth, N. Hawes, N. McArthur, C. McKenney, A. Oufkir, B. Pearce, S. Rampal, H. Rozier, J. Schaff, T. Slehria, S. Carson, E. S. Miller
Paenibacillus larvae bacteriophage Tripp was isolated from an American foulbrood diseased honey bee hive in North Carolina, USA. The 54,439-bp genome is 48.3% G+C, encodes 92 proteins, no tRNAs, and has 378-bp direct terminal repeats. It is currently unique in Genbank.
Genome Announcements. Vol 3, Issue 3, 2015.
Genome Sequences of Six Paenibacillus larvae Siphoviridae Phages
Carson S, Bruff E, DeFoor W, Dums J, Groth A, Hatfield T, Iyer A, Joshi K, McAdams S, Miles D, Miller D, Oufkir A, Raynor B, Riley S, Roland S, Rozier H, Talley S, Miller ES.
Six sequenced and annotated genomes of Paenibacillus larvae phages isolated from the combs of American foulbrood-diseased beehives are 37 to 45 kbp and have approximately 42% G+C content and 60 to 74 protein-coding genes. Phage Lily is most divergent from Diva, Rani, Redbud, Shelly, and Sitara.
Genome Announcements. Vol 2015 Jun 18;3(3), Pages e00586-15, 2015.
Genome Sequences of Mycobacteriophages AlanGrant, Baee, Corofin, OrangeOswald, and Vincenzo, New Members of Cluster B.
Pope WH, Carbonara ME, Cioffi HM, Cruz T, Dang BQ, Doyle AN, Fan OH, Gallagher M, Gentile GM, German BA, Farrell ME, Gerwig M, Hunter KL, Lefever VE, Marfisi NA, McDonnell JE, Monga JK, Quiroz KG, Pong AC, Rimple PA, Situ M, Sohnen PC, Stockinger AN, Thompson PK, Torchio NM, Toner CL, Ulbrich MC, Vohra NI, Zakir A, Adkins NL, ...
AlanGrant, Baee, Corofin, OrangeOswald, and Vincenzo are newly isolated phages of Mycobacterium smegmatis mc(2)155 discovered in Pittsburgh, Pennsylvania, USA. All five phages share nucleotide similarity with cluster B mycobacteriophages but span considerable diversity with Corofin and OrangeOswald in subcluster B3, AlanGrant and Vincenzo in subcluster B4, and Baee in subcluster B5.
Genome Announcements. Vol 3, Issue 5, 2015.
Genome Sequences of Two Bacillus cereus Group Bacteriophages, Eyuki and AvesoBmore
Ivan Erill, Steven M. Caruso, on behalf of the 2013 UMBC Phage Hunters
The genomes of two double-stranded DNA (dsDNA) bacteriophages isolated on Bacillus thuringiensis show similarity to previously sequenced phages and provide evidence of the mosaicism of phage genomes.
Genome Announcements. Vol 3, Issue 5, 2015.
Complete Genome Sequence of Bacillus cereus Group Phage TsarBomba
Ivan Erill, Steven M. Caruso, on behalf of the 2014 UMBC Phage Hunters
The Bacillus cereus group bacteriophage TsarBomba, a double-stranded DNA Myoviridae, was isolated from soil collected in Saratov, Russia. TsarBomba was found to be similar to Bacillus phages BCP78 and BCU4, and to have a wide host range among Bacillus cereus group species.
eLIFE. Vol 4, 2015.
Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity
Pope WH, Bowman CA, Russell DA, Jacobs-Sera D, Asai DJ, Cresawn SG, Jacobs WR, Hendrix RW, Lawrence JG, Hatfull GF; Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science; Phage Hunters Integrating Research and Education; Mycobacterial Genetics Course
The bacteriophage population is large, dynamic, ancient, and genetically diverse. Limited genomic information shows that phage genomes are mosaic, and the genetic architecture of phage populations remains ill-defined. To understand the population structure of phages infecting a single host strain, we isolated, sequenced, and compared 627 phages of Mycobacterium smegmatis. Their genetic diversity is considerable, and there are 28 distinct genomic types (clusters) with related nucleotide sequences. However, amino acid sequence comparisons show pervasive genomic mosaicism, and quantification of inter-cluster and intra-cluster relatedness reveals a continuum of genetic diversity, albeit with uneven representation of different phages. Furthermore, rarefaction analysis shows that the mycobacteriophage population is not closed, and there is a constant influx of genes from other sources. Phage isolation and analysis was performed by a large consortium of academic institutions, illustrating the substantial benefits of a disseminated, structured program involving large numbers of freshman undergraduates in scientific discovery.
PLoS One. 2015.
Comparative genomics of Cluster O mycobacteriophages
Cresawn SG, Pope WH, Jacobs-Sera D, Bowman CA, Russell DA, Dedrick RM, Adair T, Anders KR, Ball S, Bollivar D, Breitenberger C, Burnett SH, Butela K, Byrnes D, Carzo S, Cornely KA, Cross T, Daniels RL, Dunbar D, Findley AM, Gissendanner CR, Golebiewska UP, Hartzog GA, Hatherill JR, Hughes LE, Jalloh CS, De Los Santos C, Ekanem K, Khambule SL, ...
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.
BMC Genomics. 2014.
Genomic comparison of 93 Bacillus phages reveals 12 clusters, 14 singletons and remarkable diversity
Grose JH, Jensen GL, Burnett SH, Breakwell DP
The Bacillus genus of Firmicutes bacteria is ubiquitous in nature and includes one of the best characterized model organisms, B. subtilis, as well as medically significant human pathogens, the most notorious being B. anthracis and B. cereus. As the most abundant living entities on the planet, bacteriophages are known to heavily influence the ecology and evolution of their hosts, including providing virulence factors. Thus, the identification and analysis of Bacillus phages is critical to understanding the evolution of Bacillus species, including pathogenic strains. RESULTS: Whole genome nucleotide and proteome comparison of the 93 extant Bacillus phages revealed 12 distinct clusters, 28 subclusters and 14 singleton phages. Host analysis of these clusters supports host boundaries at the subcluster level and suggests phages as vectors for genetic transfer within the Bacillus cereus group, with B. anthracis as a distant member of the group. Analysis of the proteins conserved among these phages reveals enormous diversity and the uncharacterized nature of these phages, with a total of 4,922 protein families (phams) of which only 951 (19%) had a predicted function. In addition, 3,058 (62%) of phams were orphams (phams containing a gene product from a single phage). The most populated phams were those encoding proteins involved in DNA metabolism, virion structure and assembly, cell lysis, or host function. These included several genes that may contribute to the pathogenicity of Bacillus strains. CONCLUSIONS: This analysis provides a basis for understanding and characterizing Bacillus phages and other related phages as well as their contributions to the evolution and pathogenicity of Bacillus cereus group bacteria. The presence of sparsely populated clusters, the high ratio of singletons to clusters, and the large number of uncharacterized, conserved proteins confirms the need for more Bacillus phage isolation in order to understand the full extent of their diversity as well as their impact on host evolution.
Journal of Virology. Vol Oct 88, Issue 20, Pages 11846-60, 2014.
The genomes, proteomes, and structures of three novel phages that infect the Bacillus cereus group and carry putative virulence factors
Grose JH, Belnap DM, Jensen JD, Mathis AD, Prince JT, Merrill BD, Burnett SH, Breakwell DP
This article reports the results of studying three novel bacteriophages, JL, Shanette, and Basilisk, which infect the pathogen Bacillus cereus and carry genes that may contribute to its pathogenesis. We analyzed host range and superinfection ability, mapped their genomes, and characterized phage structure by mass spectrometry and transmission electron microscopy (TEM). The JL and Shanette genomes were 96% similar and contained 217 open reading frames (ORFs) and 220 ORFs, respectively, while Basilisk has an unrelated genome containing 138 ORFs. Mass spectrometry revealed 23 phage particle proteins for JL and 15 for Basilisk, while only 11 and 4, respectively, were predicted to be present by sequence analysis. Structural protein homology to well-characterized phages suggested that JL and Shanette were members of the family Myoviridae, which was confirmed by TEM. The third phage, Basilisk, was similar only to uncharacterized phages and is an unrelated siphovirus. Cryogenic electron microscopy of this novel phage revealed a T=9 icosahedral capsid structure with the major capsid protein (MCP) likely having the same fold as bacteriophage HK97 MCP despite the lack of sequence similarity. Several putative virulence factors were encoded by these phage genomes, including TerC and TerD involved in tellurium resistance. Host range analysis of all three phages supports genetic transfer of such factors within the B. cereus group, including B. cereus, B. anthracis, and B. thuringiensis. This study provides a basis for understanding these three phages and other related phages as well as their contributions to the pathogenicity of B. cereus group bacteria. Importance: The Bacillus cereus group of bacteria contains several human and plant pathogens, including B. cereus, B. anthracis, and B. thuringiensis. Phages are intimately linked to the evolution of their bacterial hosts and often provide virulence factors, making the study of B. cereus phages important to understanding the evolution of pathogenic strains. Herein we provide the results of detailed study of three novel B. cereus phages, two highly related myoviruses (JL and Shanette) and an unrelated siphovirus (Basilisk). The detailed characterization of host range and superinfection, together with results of genomic, proteomic, and structural analyses, reveal several putative virulence factors as well as the ability of these phages to infect different pathogenic species.
Journal of Basic Microbiology. Vol Oct 54, Issue 10, Pages 1140-5, 2014.
A web-based restriction endonuclease tool for mycobacteriophage cluster prediction
Gissendanner CR, Wiedemeier AM, Wiedemeier PD, Minton RL, Bhuiyan S, Harmson JS, Findley AM
A recent explosion in the amount of genomic data has revealed a large genetic diversity in the bacteriophages that infect Mycobacterium smegmatis. In an effort to assess the novelty of newly described mycobacteriophage isolates and provide a preliminary determination of their probable cluster assignment prior to full genome sequencing, we have developed a systematic approach that relies on restriction endonuclease analysis. We demonstrate that a web-based tool, the Phage Enzyme Tool (or PET), is capable of rapidly facilitating this analysis and exhibits reliability in the putative placement of mycobacteriophages into specific clusters of previously sequenced phages. We propose that this tool represents a useful analytical step in the initial study of phage genomes and that this tool will increase the efficiency of phage genome characterization and enhance the educational activities involving mycobacteriophage discovery.
mBio. Vol Feb 4, 5, Issue 1, Pages e01051-13, 2014.
A broadly implementable research course in phage discovery and genomics for first-year undergraduate students
Jordan TC, Burnett SH, Carson S, Caruso SM, Clase K, DeJong RJ, Dennehy JJ, Denver DR, Dunbar D, Elgin SC, Findley AM, Gissendanner CR, Golebiewska UP, Guild N, Hartzog GA, Grillo WH, Hollowell GP, Hughes LE, Johnson A, King RA, Lewis LO, Li W, Rosenzweig F, Rubin MR, Saha MS, Sandoz J, Shaffer CD, Taylor B, Temple L, Vazquez E, ...
Engaging large numbers of undergraduates in authentic scientific discovery is desirable but difficult to achieve. We have developed a general model in which faculty and teaching assistants from diverse academic institutions are trained to teach a research course for first-year undergraduate students focused on bacteriophage discovery and genomics. The course is situated within a broader scientific context aimed at understanding viral diversity, such that faculty and students are collaborators with established researchers in the field. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) course has been widely implemented and has been taken by over 4,800 students at 73 institutions. We show here that this alliance-sourced model not only substantially advances the field of phage genomics but also stimulates students' interest in science, positively influences academic achievement, and enhances persistence in science, technology, engineering, and mathematics (STEM) disciplines. Broad application of this model by integrating other research areas with large numbers of early-career undergraduate students has the potential to be transformative in science education and research training. IMPORTANCE: Engagement of undergraduate students in scientific research at early stages in their careers presents an opportunity to excite students about science, technology, engineering, and mathematics (STEM) disciplines and promote continued interests in these areas. Many excellent course-based undergraduate research experiences have been developed, but scaling these to a broader impact with larger numbers of students is challenging. The Howard Hughes Medical Institute (HHMI) Science Education Alliance Phage Hunting Advancing Genomics and Evolutionary Science (SEA-PHAGES) program takes advantage of the huge size and diversity of the bacteriophage population to engage students in discovery of new viruses, genome annotation, and comparative genomics, with strong impacts on bacteriophage research, increased persistence in STEM fields, and student self-identification with learning gains, motivation, attitude, and career aspirations.
Journal of Virology. Vol Mar 88, Issue 5, Pages 2461-80, 2014.
Cluster M mycobacteriophages Bongo, PegLeg, and Rey with unusually large repetoires of tRNA isotypes
Pope WH, Anders KR, Baird M, Bowman CA, Boyle MM, Broussard GW, Chow T, Clase KL, Cooper S, Cornely KA, DeJong RJ, Delesalle VA, Deng L, Dunbar D, Edgington NP, Ferreira CM, Weston Hafer K, Hartzog GA, Hatherill JR, Hughes LE, Ipapo K, Krukonis GP, Meier CG, Monti DL, Olm MR, Page ST, Peebles CL, Rinehart CA, Rubin MR, Russell ...
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.
Genome Announcements. Vol 1, Issue 6, 2013.
Genome sequences of five b1 subcluster mycobacteriophages
Breakwell DP1, Barrus EZ, Benedict AB, Brighton AK, Fisher JN, Gardner AV, Kartchner BJ, Ladle KC, Lunt BL, Merrill BD, Morrell JD, Burnett SH, Grose JH.
Mycobacteriophages infect members of the Mycobacterium genus in the phylum Actinobacteria and exhibit remarkable diversity. Genome analysis groups the thousands of known mycobacteriophages into clusters, of which the B1 subcluster is currently the third most populous. We report the complete genome sequences of five additional members of the B1 subcluster.
Genome Announcements. Vol 1, Issue 6, 2013.
Complete genome sequences of 63 mycobacteriophages
Hatfull GF; Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) Program; KwaZulu-Natal Research Institute for Tuberculosis and HIV (K-RITH) Mycobacterial Genetics Course; University of California—Los Angeles Research Immersion Laboratory in Virology; Phage Hunters Integrating Research and Education (PHIRE) Program
Mycobacteriophages are viruses that infect mycobacterial hosts. The current collection of sequenced mycobacteriophages-all isolated on a single host strain, Mycobacterium smegmatis mc(2)155, reveals substantial genetic diversity. The complete genome sequences of 63 newly isolated mycobacteriophages expand the resolution of our understanding of phage diversity.
Virology. Vol Sept 444, Issue 1-2, Pages 374-83, 2013.
Genomic characterization of six novel Bacillus pumilus bacteriophages
Lorenz L, Lins B, Barrett J, Montgomery A, Trapani S, Schindler A, Christie GE, Cresawn SG, Temple L
Twenty-eight bacteriophages infecting the local host Bacillus pumilus BL-8 were isolated, purified, and characterized. Nine genomes were sequenced, of which six were annotated and are the first of this host submitted to the public record. The 28 phages were divided into two groups by sequence and morphological similarity, yielding 27 cluster BpA phages and 1 cluster BpB phage, which is a BL-8 prophage. Most of the BpA phages have a host range restricted to distantly related strains, B. pumilus and B. simplex, reflecting the complexities of Bacillus taxonomy. Despite isolation over wide geographic and temporal space, the six cluster BpA phages share most of their 23 functionally annotated protein features and show a high degree of sequence similarity, which is unique among phages of the Bacillus genera. This is the first report of B. pumilus phages since 1981.
BMC Genomics. 2013.
Phage cluster relationships identified through single gene analysis
Smith KC, Castro-Nallar E, Fisher JN, Breakwell DP, Grose JH, Burnett SH.
Phylogenetic comparison of bacteriophages requires whole genome approaches such as dotplot analysis, genome pairwise maps, and gene content analysis. Currently mycobacteriophages, a highly studied phage group, are categorized into related clusters based on the comparative analysis of whole genome sequences. With the recent explosion of phage isolation, a simple method for phage cluster prediction would facilitate analysis of crude or complex samples without whole genome isolation and sequencing. The hypothesis of this study was that mycobacteriophage-cluster prediction is possible using comparison of a single, ubiquitous, semi-conserved gene. Tape Measure Protein (TMP) was selected to test the hypothesis because it is typically the longest gene in mycobacteriophage genomes and because regions within the TMP gene are conserved. RESULTS: A single gene, TMP, identified the known Mycobacteriophage clusters and subclusters using a Gepard dotplot comparison or a phylogenetic tree constructed from global alignment and maximum likelihood comparisons. Gepard analysis of 247 mycobacteriophage TMP sequences appropriately recovered 98.8% of the subcluster assignments that were made by whole-genome comparison. Subcluster-specific primers within TMP allow for PCR determination of the mycobacteriophage subcluster from DNA samples. Using the single-gene comparison approach for siphovirus coliphages, phage groupings by TMP comparison reflected relationships observed in a whole genome dotplot comparison and confirm the potential utility of this approach to another widely studied group of phages. CONCLUSIONS: TMP sequence comparison and PCR results support the hypothesis that a single gene can be used for distinguishing phage cluster and subcluster assignments. TMP single-gene analysis can quickly and accurately aid in mycobacteriophage classification.
PLoS One. Vol 8, Issue 7, Pages e69273, 2013.
Cluster J mycobacteriophages:intron splicing in capsid and tail genes
Pope WH, Jacobs-Sera D, Best AA, Broussard GW, Connerly PL, Dedrick RM, Kremer TA, Offner S, Ogiefo AH, Pizzorno MC, Rockenbach K, Russell DA, Stowe EL, Stukey J, Thibault SA, Conway JF, Hendrix RW, Hatfull GF.
Bacteriophages isolated on Mycobacterium smegmatis mc(2)155 represent many distinct genomes sharing little or no DNA sequence similarity. The genomes are architecturally mosaic and are replete with genes of unknown function. A new group of genomes sharing substantial nucleotide sequences constitute Cluster J. The six mycobacteriophages forming Cluster J are morphologically members of the Siphoviridae, but have unusually long genomes ranging from 106.3 to 117 kbp. Reconstruction of the capsid by cryo-electron microscopy of mycobacteriophage BAKA reveals an icosahedral structure with a triangulation number of 13. All six phages are temperate and homoimmune, and prophage establishment involves integration into a tRNA-Leu gene not previously identified as a mycobacterial attB site for phage integration. The Cluster J genomes provide two examples of intron splicing within the virion structural genes, one in a major capsid subunit gene, and one in a tail gene. These genomes also contain numerous free-standing HNH homing endonuclease, and comparative analysis reveals how these could contribute to genome mosaicism. The unusual Cluster J genomes provide new insights into phage genome architecture, gene function, capsid structure, gene mobility, intron splicing, and evolution.
Journal of Virology. Vol 86, Issue 4, Pages 2382-4, 2012.
Complete genome sequences of 138 mycobacteriophages
Hatfull GF; Science Education Alliance Phage Hunters Advancing Genomics and Evolutionary Science Program; KwaZulu-Natal Research Institute for Tuberculosis and HIV Mycobacterial Genetics Course Students; Phage Hunters Integrating Research and Education Program
Bacteriophages are the most numerous biological entities in the biosphere, and although their genetic diversity is high, it remains ill defined. Mycobacteriophages-the viruses of mycobacterial hosts-provide insights into this diversity as well as tools for manipulating Mycobacterium tuberculosis. We report here the complete genome sequences of 138 new mycobacteriophages, which-together with the 83 mycobacteriophages previously reported-represent the largest collection of phages known to infect a single common host, Mycobacterium smegmatis mc(2) 155.
Virology. Vol 434, Issue 2, Pages 187-201, 2012.
On the nature of mycobacteriophage diversity and host preference
Jacobs-Sera D, Marinelli LJ, Bowman C, Broussard GW, Guerrero Bustamante C, Boyle MM, Petrova ZO, Dedrick RM, Pope WH; Science Education Alliance Phage Hunters Advancing Genomics And Evolutionary Science Sea-Phages Program, Modlin RL, Hendrix RW, Hatfull GF.
The complete genome sequences of over 220 mycobacteriophages reveal them to be highly diverse, with numerous types sharing little or no nucleotide sequence identity with each other. We have determined the preferences of these phages for Mycobacterium tuberculosis and for other strains of Mycobacterium smegmatis, and find there is a correlation between genome type (cluster, subcluster, singleton) and host range. For many of the phages, expansion of host range occurs at relatively high frequencies, and we describe several examples in which host constraints occur at early stages of infection (adsorption or DNA injection), and phages have the ability to expand their host range through mutations in tail genes. We present a model in which phage diversity is a function of both the ability of phages to rapidly adapt to new hosts and the richness of the diversity of the bacterial population from which those phages are isolated.
Journal of Virology. 2012.
Mycobacteriophage Marvin: a new singleton phage with an unusual genome organization.
Mageeney C, Pope WH, Harrison M, Moran D, Cross T, Jacobs-Sera D, Hendrix RW, Dunbar D, Hatfull GF.
Mycobacteriophages represent a genetically diverse group of viruses that infect mycobacterial hosts. Although more than 80 genomes have been sequenced, these still poorly represent the likely diversity of the broader population of phages that can infect the host, Mycobacterium smegmatis mc(2)155. We describe here a newly discovered phage, Marvin, which is a singleton phage, having no previously identified close relatives. The 65,100-bp genome contains 107 predicted protein-coding genes arranged in a noncanonical genomic architecture in which a subset of the minor tail protein genes are displaced about 20 kbp from their typical location, situated among nonstructural genes anticipated to be expressed early in lytic growth. Marvin is not temperate, and stable lysogens cannot be recovered from infections, although the presence of a putative xis gene suggests that Marvin could be a relatively recent derivative of a temperate parent. The Marvin genome is replete with novel genes not present in other mycobacteriophage genomes, and although most are of unknown function, the presence of amidoligase and glutamine amidotransferase genes suggests intriguing possibilities for the interactions of Marvin with its mycobacterial hosts.
CBE Life Sciences Education. Vol 10, Issue 3, Pages 279-286, 2011.
Classroom-based science research at the introductory level: changes in career choices and attitude
Harrison M, Dunbar D, Ratmansky L, Boyd K, Lopatto D
Our study, focused on classroom-based research at the introductory level and using the Phage Genomics course as the model, shows evidence that first-year students doing research learn the process of science as well as how scientists practice science. A preliminary but notable outcome of our work, which is based on a small sample, is the change in student interest in considering different career choices such as graduate education and science in general. This is particularly notable, as previous research has described research internships as clarifying or confirming rather than changing undergraduates' decisions to pursue graduate education. We hypothesize that our results differ from previous studies of the impact of engaging in research because the students in our study are still in the early stages of their undergraduate careers. Our work builds upon the classroom-based research movement and should be viewed as encouraging to the Vision and Change in Undergraduate Biology Education movement advocated by the American Association for the Advancement of Science, the National Science Foundation, and other undergraduate education stakeholders.
BMC Bioinformatics. 2011.
Phamerator: a bioinformatic tool for comparative bacteriophage genomics.
Cresawn SG, Bogel M, Day N, Jacobs-Sera D, Hendrix RW, Hatfu
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.
PLoS One. Vol 6, Issue 10, 2011.
Cluster K mycobacteriophages: insights into the evolutionary origins of mycobacteriophage TM4
Pope WH, Ferreira CM, Jacobs-Sera D, Benjamin RC, Davis AJ, DeJong RJ, Elgin SC, Guilfoile FR, Forsyth MH, Harris AD, Harvey SE, Hughes LE, Hynes PM, Jackson AS, Jalal MD, MacMurray EA, Manley CM, McDonough MJ, Mosier JL, Osterbann LJ, Rabinowitz HS, Rhyan CN, Russell DA, Saha MS, Shaffer CD, Simon SE, Sims EF, Tovar IG, Weisser EG, Wertz JT, ...
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.
PLoSOne. Vol 6, Issue 1, Pages e16329, 2011.
Expanding the diversity of mycobacteriophages: Insights into genome architecture and evolution
Pope WH, Jacobs-Sera D, Russell DA, Peebles CL, Al-Atrache Z, Alcoser TA, Alexander LM, Alfano MB, Alford ST, Amy NE, Anderson MD, Anderson AG, Ang AA, Ares M Jr, Barber AJ, Barker LP, Barrett JM, Barshop WD, Bauerle CM, Bayles IM, Belfield KL, Best AA, Borjon A Jr, Bowman CA, Boyer CA, Bradley KW, Bradley VA, Broadway LN, Budwal K, ...
Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.
Biochemistry and Molecular Biology Education. Vol 38, Issue 1, Pages 23-8, 2010.
Genomics and bioinformatics in undergraduate curricula: Contexts for hybrid laboratory/lecture courses for entering and advanced science students
Temple L, Cresawn SG, Monroe JD.
Emerging interest in genomics in the scientific community prompted biologists at James Madison University to create two courses at different levels to modernize the biology curriculum. The courses are hybrids of classroom and laboratory experiences. An upper level class uses raw sequence of a genome (plasmid or virus) as the subject on which to base the experience of genomic analysis. Students also learn bioinformatics and software programs needed to support a project linking structure and function in proteins and showing evolutionary relatedness of similar genes. An optional entry-level course taken in addition to the required first-year curriculum and sponsored in part by the Howard Hughes Medical Institute, engages first year students in a primary research project. In the first semester, they isolate and characterize novel bacteriophages that infect soil bacteria. In the second semester, these young scientists annotate the genes on one or more of the unique viruses they discovered. These courses are demanding but exciting for both faculty and students and should be accessible to any interested faculty member.
CBE Life Sciences Education. Vol Winter 8, Issue 4, Pages 278-82, 2009.
Non-STEM undergraduates become enthusiastic phage-hunters
Caruso SM, Sandoz J, Kelsey J.
To increase science literacy and appreciation among nonscience majors, we offered a course in which 20 non-STEM (science, technology, engineering, math) undergraduates participated in a unique, two-semester research experience. Each student isolated and characterized his or her own bacteriophage from soil samples. One bacteriophage was selected for sequencing and together, the class annotated the genome of the newly sequenced bacteriophage. The class produced a group poster and gave PowerPoint presentations, and one student presented the joint work at a science symposium

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