Mycobacteriophages

Hatfull, Graham F.

doi:10.1128/microbiolspec.gpp3-0026-2018

Cited by 76 publications

(86 citation statements)

References 133 publications

(223 reference statements)

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“…to Cluster AC) and ten singleton phages (each with no close relative) 3 ; all but one of these clusters (Cluster C, myoviridae) have siphoviral morphotypes. For the mycobacteriophage siphoviruses, the virion structure and assembly genes are syntenically organized and transcribed rightwards in the left parts of the genomes 4 . Genes in the right parts of the genomes have nonstructural roles, are small relative to the structural genes, abundant, and most are of unknown function 4 .…”

Section: Resultsmentioning

confidence: 99%

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Identification of mycobacteriophage toxic genes reveals new features of mycobacterial physiology and morphology

Hatfull

2020

Sci Rep

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Double-stranded DNA tailed bacteriophages typically code for 50-200 genes, of which 15-35 are involved in virion structure and assembly, DNA packaging, lysis, and DNA metabolism. However, vast numbers of other phage genes are small, are not required for lytic growth, and are of unknown function. The 1,885 sequenced mycobacteriophages encompass over 200,000 genes in 7,300 distinct protein 'phamilies', 77% of which are of unknown function. Gene toxicity provides potential insights into function, and here we screened 193 unrelated genes encoded by 13 different mycobacteriophages for their ability to impair the growth of Mycobacterium smegmatis. We identified 45 (23%) mycobacteriophage genes that are toxic when expressed. The impacts on M. smegmatis growth range from mild to severe, but many cause irreversible loss of viability. Expression of most of the severely toxic genes confers altered cellular morphologies, including filamentation, polar bulging, curving, and, surprisingly, loss of viability of one daughter cell at division, suggesting specific impairments of mycobacterial growth. Co-immunoprecipitation and mass spectrometry show that toxicity is frequently associated with interaction with host proteins and alteration or inactivation of their function. Mycobacteriophages thus present a massive reservoir of genes for identifying mycobacterial essential functions, identifying potential drug targets and for exploring mycobacteriophage physiology. The bacteriophage population is vast, dynamic, old, and highly diverse 1. Phage genome sizes vary considerably from 5 to 500 kbp, but the average size of referenced phage genome sequences in publicly available databases is ~ 50 kbp 2. The genome sequences of over 3,300 phages isolated on bacteria in the phylum Actinobacteria (i.e. 'actinobacteriophages') have been determined, with an average genome length of 61 kbp and an average of 100 predicted protein-coding genes per genome 3. With an average gene length of about 600 bp, phage genes are substantially smaller than the average bacterial gene, reflecting an abundance of relatively small genes coding for non-structural proteins 3,4. Phage genomes are characteristically mosaic as a consequence of horizontal genetic exchange 5,6 , although the rates of exchange vary for different types of phages 7. Mosaicism is likely driven largely by microbial dynamics involving bacterial resistance to infection and phage co-evolution 8. The 3,300 sequenced actinobacteriophages include over 320,000 predicted protein coding genes, of which ~ 75% have no predicted function. Many of such genes are likely to be non-essential for viral lytic replication 9 , although most of them are expressed during lytic growth 10. The general roles for these proteins may be in modulating the dynamic interactions with their host, counteracting restriction, CRISPR-Cas, and other viral defense systems 11-13 , or to actively promote defense against heterotypic viral attack 11,14-16. Elucidating the roles and functions of these genes is of importance in understan...

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Section: Resultsmentioning

confidence: 99%

“…NT, Tested; Bulbous pole, cell pole enlarged; Filament, cells become filamentous; Branched fil., cells form branched filaments; Curved, curved cells. 4 Mass spectrometry analysis of HA-tagged toxic proteins' co-immunoprecipitation (co-IP) targets. Details can be found in Table S2.…”

Section: Resultsmentioning

confidence: 99%

Identification of mycobacteriophage toxic genes reveals new features of mycobacterial physiology and morphology

Hatfull

2020

Sci Rep

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“…Genes are grouped into phams based on amino acid sequence similarity, and phages sharing at least 35% pham similarity are clustered together (Hatfull, 2018). Therefore, a SplitsTree analysis of pham conservation between Giantsbane and other AU phages was used to further examine intra-cluster similarities, which showed four distinct groups: the first containing Loretta and Ingrid; the second containing Makai, Giantsbane, and Shepard; the third containing Caterpillar and MediumFry, and a fourth containing all of the remaining AU1 phages (Figure 6b).…”

Section: Splitstree Results Consistent With Nucleotide Sequence Analysismentioning

confidence: 99%

“…Phages are typically sorted into clusters and subclusters to show evolutionary relatedness and facilitate phage characterization. While phages are primarily clustered based on pham similarity, nucleotide similarity is an important factor as well (Hatfull, 2018). Batch ANI analysis with all AU phages helped examine intra-cluster similarities (Figure 6a).…”

Section: Giantsbane Shares Highest Nucleotide Similarity With Au2 Phamentioning

confidence: 99%

Characterization of novel Actinobacteriophage Giantsbane reveals unexpected cluster AU relationships

Liu¹,

Chen²,

Dang³

et al. 2020

Preprint

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Bacteriophages that infect Arthrobacter , a genus of bacteria which play key ecological roles in soil, warrant further study. A novel Actinobacteriophage, Giantsbane, was isolated on Arthrobacter globiformis and purified. Particle stability was tested at various temperatures and salinity concentrations by observing titer differences; unpaired Student's t-tests revealed these differences to be insignificant. Transmission electron microscopy and Illumina whole-genome sequencing revealed that Giantsbane's morphology and genome length, respectively, were characteristic of Siphoviridae phages. 94 putative open reading frames were determined using Glimmer, GeneMark, and manual review; none were associated with lysogeny. Giantsbane was placed into phage cluster AU, and SplitsTree and batch ANI analyses revealed similarities with other AU phages. The annotated genome was further analyzed using Phamerator and MEME-Suite, which identified repeated motifs present in several other phages. These findings help further our understanding of the physiological and genomic aspects of phage biology.

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“…The discovery of novel mycobacteriophages is typically conducted using Mycobacterium smegmatis, a nonpathogenic strain that serves a model host for evaluating a mycobacteriophage's therapeutic potential. Over 150,000 genes have been identified in the collection of over 1,800 mycobacteriophages which have been sequenced (2). This incredible degree of genetic diversity warrants mycobacteriophages to be organized into clusters, and further subclusters, based on genomic similarity (3).…”

Section: Introductionmentioning

confidence: 99%

Nine Novel Cluster E Mycobacteriophages Isolated from Soil

Jm¹,

Freise²,

Jm³

2020

Preprint

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Novel mycobacteriophages FireRed, MISSy, MPhalcon, Murica, Sassay, Terminus, Willez, YassJohnny, and Youngblood were isolated from soil samples on the host Mycobacterium smegmatis. Transmission electron microscopy revealed phage structures suggestive of the Siphoviridae family of phages. Phage genomes were sequenced and annotated, revealing that they belong to actinobacteriophage Cluster E. Here, we describe the features of these phage genomes and discuss their similarity to previously discovered cluster E phages. Mycobacteriophages were isolated by undergraduates in the Research Immersion in Virology course-based undergraduate research experience in the Microbiology, Immunology, and Molecular Genetics Department at UCLA. This project was funded by the Dean of Life Sciences Division at UCLA, with additional support for sequencing from the HHMI Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program. We thank Krisanavane Reddi for preparation of materials, lysate archiving, and management of the instructional laboratories; Hong Zhou of the UCLA Electron Imaging Center for NanoMachines for electron microscopy support; Rebecca A. Garlena and Daniel A. Russell at the Pittsburgh Bacteriophage Institute for phage sequencing and assistance with genome assembly;and Debbie Jacobs-Sera, Welkin Pope, Graham Hatfull, and the SEA-PHAGES community for programmatic support.

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Mycobacteriophages

Cited by 76 publications

References 133 publications

Identification of mycobacteriophage toxic genes reveals new features of mycobacterial physiology and morphology

Identification of mycobacteriophage toxic genes reveals new features of mycobacterial physiology and morphology

Characterization of novel Actinobacteriophage Giantsbane reveals unexpected cluster AU relationships

Nine Novel Cluster E Mycobacteriophages Isolated from Soil

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