1. Comparison of Cluster S Phages
Monique L. Mills, Judd Bragg,  Asri Bruce, Ari Dehn, Morgan Hefner, Dylan Katon, Dustin McHugh, Payton Petersen, Franck Zeba,
Gerard P. Zegers, Shallee T. Page.
University of Maine at Machias, Machias, ME 04655
Methods: Marvin, MosMoris and Gattaca’s genomic DNA were sequenced at the Joint Genome Institute (Sanger sequencing), and the Pittsburgh Bacteriophage Institute (Ion Torrent and Illumina sequencing), respectively. Sequences were stored in the database at Phagesdb.org. The genome annotation workbench, DNA Master, was used to auto-annotate the genes of Marvin, MosMoris and Gattaca using the algorithm programs Glimmer and GeneMark. These auto-annotated genes were then manually verified and corrected as necessary. BLAST was used to find regions of similarity between Marvin, MosMoris and Gattaca’s gene sequences and those of other phages or organisms as a substantiation step in the annotation process. The Frame feature of DNA Master was used to verify start and stop codons for individual genes based on Shine-Dalgarno scores and also the open reading frame (ORF) length. The ORF reveals coding potential for a gene sequence and a longer ORF is typically more indicative of the proper start sequence for the gene. Phamerator was used to map comparative genomics. HHpred was used to detect protein homology and structure using pairwise comparison of profile hidden Markov models (HMMs). Gepard was used to calculate the dot plot. Aragorn was used to search for tRNAs.
Abstract:
Mycobacterium phages MosMoris, Gattaca, and Marvin are the only members of the S cluster. Marvin was the first S cluster phage discovered in 2009 and remained a singleton until the discovery of a similar phage: MosMoris. The cluster makes up of only 0.2% of currently known phages and currently has no subclusters. The average genome of an S cluster phage is 65,193 bp long, has a 64.3% GC content, 110 genes, and a 3’ sticky overhang of 11 base pairs containing a sequence of GCGCGCAGCGC. Marvin, MosMoris and Gattaca have two individual reverse strand genes, 22 and 30 among its forward genes, then a cluster of reverse strand genes near the end of the genome. A syntenic group of tail protein genes is found at an unusual location: spanning genes (Figure 4).
3.0 kb
1.0 kb
Figure 4: Phamerator output for minor tail protein area
(Top to bottom: Mycobacteriophages MosMoris, Gattaca, and Marvin.)
0.5 kb
Results: Annotations of the three S cluster phages revealed that Marvin, MosMoris, and Gattaca are 99% identical. This identity is shown by similar traits shared among the cluster. The three genomes have an average of 110 ORFs. All of these genes are forward transcribed except for a block of ORFs near gp100 and two single reverse genes earlier in the genomes: DNA methylase and a hypothetical protein (figure 5). Many of these genes in the downstream half of the genome cannot be assigned a function, however the following were identified amongst all three phages: terminase, capsid maturation protease, major capsid protein, major tail subunit, minor tail proteins tail assembly, chaperone, and tape measure protein. The location of the minor tail proteins were of particular interest because all three phages were located at some distance from the other structural proteins (Figure 4). This displacement in Marvin, MosMoris and Gattaca groups them evolutionarily suggesting that they share a common ancestor, but the functional significance is yet to be determined. The rest of the genes in the S cluster phages are syntenic and tightly packed, except for a region in which the direction of transcription changes. There is an inserted region not seen in Marvin adjacent to and presumably mediated by an HNH endonuclease. In addition, Marvin has a putative insertion with low coding-potential near 9500 bp that is not seen in the other two members of the cluster.
The individual phages in the S cluster were also compared pairwise using a Gepard map (Figure 3). Marvin and Gattaca are indistinguishable on the Gepard plot, while MosMoris has only a few differences from the two throughout the genome. To observe other differences in the three phages a restriction digest were performed (Figure 2). The digest of all three phages are identical meaning the enzyme cut the DNA in each of the phages at the same length, reflecting the sequence similarity of all three S cluster phages – Marvin, MosMoris, and Gattaca. Comparison of the diversity of phages by Splitstree determined that the phages with closest sequence similarity to the S phages are Cluster V and singleton Sparky (Pope, 2015).
Figure 2:
Agarose gel electrophoresis restriction digest of MosMoris. Lane 1-MW standard 2-log ladder, Lane 2-BamHI enzyme, Lane 3-ClaI, Lane 4-EcoRI, Lane 5-HaeIII, Lane 6-HindIII.
Figure 1:
Images top to bottom
1.Electron microscope image for MosMoris.
2.Electron microscope image for Gattaca.
3. Electron microscope image for Marvin.
Scale: Bar= 0.1μm
Figure 3:
This graph to shows the similarity between Marvin/Gattaca and MosMoris and was done using the program Gepard. The appearance of a fairly solid line diagonally from the top left to the bottom right corner with few gaps indicates a high degree of similarity between the genomes.
Background: Mycobacteriophages are viruses that infect and kill bacteria by injecting their DNA, which codes for various parts of the phage, into the bacteria. Structural components of phages include a capsid head, a spiral tail, and tail fibers. The phage parts are assembled inside the bacterial host cell and new phages are made until the bacterial cell bursts and the new phages are released to infect more cells. If the phage is ‘lysogenic’, the viral DNA is incorporated into the bacterial DNA where it can remain inactive, but can be replicated and conserved by bacterial duplication until an activation signal is received.
The mycobacteriophages Marvin, MosMoris, and Gattaca were isolated from non-enriched soil samples. The environmental samples were collected from a garden in Radnor, PA, chicken manure in Milbridge, ME and a compost pile in Milbridge, ME, respectively, and then filtered at 0.22mm in order to isolate a phage. Through direct plating, the phages were grown in a culture of Mycobacterium smegmatis mc2155. The phages were put through many rounds of purification and then grown to create a high titer assay. These isolated phages were examined under the electron microscope (Figure 1). DNA from each phage was extracted in order to prepare it for sequencing, and the DNA was cut and subjected to agarose electrophoresis (Figure 2). The DNA was sequenced and mapped out for analysis and annotation during the bioinformatics course. In this course, students studied the genomic properties of the phage and helped to identify its genomic traits.
Figure 5: MosMoris gene map from DNA Master
Acknowledgements: Kim Bastille, and Janelle Grendler, TAs; Sam Cheeney, soil samples; UMM IT-computer support; Sherrie Sprangers, grants administration; and Howard Hughes Medical Institute and U. Maine Machias for support and funding. Maine INBRE: Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM
Reference: Pope et al Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity. eLife. 2015; 4: e06416.