Complete Genome Sequences of 138 Mycobacteriophages

Abstract

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.T he bacteriophage population is large (9), dynamic (8), and old (3). The genetic diversity of the population is sufficiently great that phages of different bacterial hosts typically share little recognizable sequence similarity (2). Comparing the genomes of phages that infect a single common bacterial host-and are therefore likely to be in genetic contact with one another-provides insights into both viral diversity and the mechanisms giving rise to new viruses (4). An emerging central theme is that phage genomes are pervasively mosaic, with segments being readily exchanged between genomes by horizontal exchange and thus present in numerous genomic contexts with different genetic neighbors (2). Eighty-three complete mycobacteriophage genome sequences have been previously reported (5, 6) and are grouped into 22 clusters or subclusters except for 5 singletons that have no close relatives (5, 6).The isolation, purification, and genomic analysis of new bacteriophages offer an effective platform for providing authentic research experiences to novice scientists (1). Of the 138 phages whose genomes are reported here, 80 were isolated by participants in the Howard Hughes Medical Institute Science Education Alliance's Phage Hunters Advancing Genomics and Evolutionary Science Program (SEA-PHAGES), 10 were isolated in a mycobacterial genetics course at the University of KwaZulu-Natal, and 47 were isolated by teachers, undergraduates, and high school students in the Phage Hunters Integrating Research and Education Program (PHIRE) at the University of Pittsburgh. Each of the new phages was plaque purified, named, sequenced, and annotated by participating students and faculty. In addition, the previously isolated phage DS6A-the only one of the 138 phages that does not infect M. smegmatis (7)-was sequenced and annotated.Genomic DNA isolated from each phage was shotgun sequenced using either Sanger sequencing, 454 pyrosequencing (6), or Illumina sequencing. The qualities of the sequence assemblies were evaluated, and targeted Sanger sequencing was used to resolve weak areas and to determine genome ends. All studentannotated genomes were reviewed and revised as necessary at the University of Pittsburgh prior to GenBank submission.Of the 138 new genomes, 19 do not belong within an existing cluster and form five new clusters (J, L, M, N, and O), incorporating 3 prior singletons to form new clusters J, L, and O. Six new singletons were identified. Representatives of all existing clusters ...

The First Structure of a Mycobacteriophage, the Mycobacterium abscessus subsp. bolletii Phage Araucaria

“…Cutinase that degrades cutin, a polyester protecting leaves, is also present in mycobacteria, for which it might be involved in cell wall wax degradation (31,32). Finally, the ORF30 protein shares 38% sequence similarity with the holin (gp31) of the mycobacteriophage Fruitloop (cluster F1 [9,10]), a small protein forming membrane pores through which the folded endolysin reaches its substrate. Two transmembrane (iv) Virion assembly cassette.…”

“…Using the Pfam database, an integrase domain was detected toward the C terminus of this ORF protein, between amino acid positions 67 and 241 (Pfam: PF0058). Temperate mycobacteriophages typically encode either a serine or tyrosine integrase that mediates prophage integration (8)(9)(10)(11)13). In the Araucaria prophage genome, the ORF2 protein is perfectly identified as a tyrosine integrase and is located 22 kb distant from the cluster of structural proteins (Fig.…”

“…All mycobacteriophages characterized to date are doublestranded DNA (dsDNA)-tailed phages of the Caudovirales order and belong to the Siphoviridae or Myoviridae (9,10). We report here the electron microscopic (EM) reconstruction of the M. abscessus subsp.…”

“…Besides the interest in phages infecting a bacterial pathogen, studies of mycobacteriophages may reveal specific features of mycobacterium-phage interactions due to the peculiar cell wall features of these hosts (12). Despite the wealth of mycobacteriophages isolated and sequenced (8)(9)(10)(11)13), their infection and host recognition mechanisms are still poorly understood.…”

“…This large set of complete genomes reveals a considerable diversity (8)(9)(10)(11). Conversely, the large genetic repertoire of the predicted mycobacteriophage protein-coding genes possesses a low number of detectable homologues in the public databases (9,10).…”