The Life-Changing Magic of SallySpecial: A Viral Tale of a True Minimalist�Caleigh Charlebois, Kodey Silknitter, William Petterson, Isaac Johnson, Morgan Cavagnaro, Benjamin King, and Sally Dixon Molloy�The Honors College and Molecular and Biomedical Sciences, University of Maine, sally.dixon@maine.edu
SallySpecial codon usage is similar to that of
G. terrae and M. smegmatis
Acknowledgements
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Submission No.
Research reported in this project was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103423.
Figure 3. Partial Phamerator genome maps for Emperor and SallySpecial aligned at the location of gp14 in Sallyspecial.
GP14, to be or not to be?
Figure 4. Start positions of the open reading frame for gp14 in SallySpecial (A) and in the equivalent open reading frame in Emperor (C). The open reading frames in both Sally Special (B) and Emperor (D) have high coding potential.
SallySpecial’s genome represents the minimal genes necessary for lytic and lysogenic infection
Figure 5. A genome map showing the genes in the SallySpecial genome. Each box represents a gene. Genes above the ruler are transcribed forward, while genes beneath the ruler are transcribed in the reverse orientation. Text above or below the genes indicates the function if known. The blue bars below the genome indicate the 13 genes belonging to phams which also have members in clusters other than DM.
Figure 1. (A) Electronmicrograph of the SallySpecial virion with a bar for scale. (B)(C) Pie charts showing the distribution of sequenced Gordonia phage and Mycobacteriophage into clusters. Each “slice” represents a cluster. The group of “singletons,” phage not sorted into clusters, is represented as a cluster in each, but it should be noted that each singleton is sufficiently distinct from other phage as to form its own cluster. Colors are assigned in the same order starting from cluster A in both diagrams.
Gordonia phage are incredibly diverse
Figure 10. Phyre2 predicted structure of SallySpecial repressor protein, gp19. The structure of gp19, shown in green, is superposed over the lambda repressor-like binding domain, shown in grey (2).
The AttP integration site of SallySpecial is located in the repressor protein, GP19.
References
SallySpecial forms a new cluster, DM, with phage Emperor
Bacteriophage (phage), viruses that infect bacteria, are highly diverse and abundant. Knowledge of virus-host interactions gives insight into the biology of both entities and has applications in a variety of fields including medicine and environmental studies. Phage are sorted into clusters based on genome structure and sequence similarity. Using wet lab techniques and bioinformatics programs, we characterized the novel cluster DM phage SallySpecial. Cluster DM contains only one other member, Emperor. SallySpecial is a temperate phage, one that can insert its DNA into a bacterial genome, forming a lysogen. The SallySpecial genome is short for a tailed phage, with 15,895 nucleotides encoding 23 putative genes. SallySpecial is unusual in that it has a GC content of 70%. To learn more about the biology of SallySpecial, gene functions and regulatory sequences will be predicted with a focus on sequences involved in lysogen formation.
Figure 8. Model of predicted integration mechanism of SallySpecial into the host genome. attP of the phage genome is located in the 3’ end of the repressor gene and recombines with the homologous attB sequence in the host bacterium Gordonia terrae 3612 genome. After integration, the repressor gene is truncated, with results in a more stable and active repressor protein (1).
Figure 9. A sequence alignment showing G. terrae double-stranded DNA on top (grey) and Lysine tRNA beneath (blue). Green and blue bars mark the bounds of the attB sequence.
Figure 2. A Phamerator map showing the genomes of phage Emperor and SallySpecial. Bars of color link regions where the sequence is very similar between the genomes. In other words, the bars indicate areas of high sequence identity. To be in the same cluster, two phage must have at least 50% sequence identity across their genomes.
Figure 11. A partial Phamerator genome map showing the aligned immunity cassettes of SallySpecial and four phage known to have integration-dependent immunity (1).
GP20 is a Cro-like protein
Figure 12. Predicted protein folding structure of gp20 based on alignment with mycobacteriophage Pukovnik Xis (2). The diagram is rainbow colored from red at the N terminus to blue at the C terminus.
Figure 13. HHPred protein alignment of gp20 with Cro/C1-type HTH DNA-binding domain (3). The protein sequence of gp20 is shown above that of the HTH domain, with symbols in the center indicating the level of match at each amino acid.
A.
B.
C.
Abstract
Emperor
SallySpecial
B
C
A
B
C
D
Figure 7. Comparisons of codon usage in SallySpecial to that of Emperor �(A), M. chelonae (B), M. smegmatis (C), and G. terrae (D). SallySpecial’s codon usage differs from that of Emperor and M. chelonae and is more similar to that of its isolation host, G. terrae, and M. smegmatis.
A
Cro-like Gene
attP
Integrase
Repressor
Phage Genome
attB
tRNA Gene
Bacterial Genome
attL
attR
repressor
Integrase
Cro-like gene
Integration
attP
attB
B
C
D
Figure 6. Histogram chart showing phage genome length distribution. Genome length categories are displayed from left to right on the x axis, while the heights of the bars represent how many sequenced Gordonia phage are in each category. SallySpecial’s position in the chart is indicated with an arrow.