● Tail assembly chaperones are upstream of the tape measure gene● construct the major tail subunit around the tail core● First half is coded from bp 25336 to 25839 in +1 frame● shifts forward skipping a base (+1 translational)● 7.8 bps/nm, typical of Cluster O phage

Cluster O Phage Repeats

Electron Microscopy● Unusual prolate head shape (175 nm long and 40 nm wide) ● Straight tail (275 nm long and 7 nm wide). ● Identified Vorrps as Cluster O phage

Gene Function Summary● 126 identified genes

● Around 27% of these were assigned putative

functions ● Two categories;

enzymes and structural products

Gene Name Start Position Stop Position Function Function SourceVorrps5 1716 1147 DNA Methylase BLASTVorrps6 2047 1508 DNA Methylase PhameratorVorrps10 3349 2729 Endo VII BLASTVorrps20 7753 10116 DNA Primase/Polymerase BLASTVorrps27 12011 12364 HNH Endonuclease BLASTVorrps30 13116 14630 Terminase BLASTVorrps31 14689 15918 Portal PhameratorVorrps32 15915 16499 O-Methyltransferase PhameratorVorrps34 17358 18050 Glycosyltransferase BLASTVorrps35 18047 18667 Glycosyltransferase BLASTVorrps38 19688 20920 Major Capsid BLASTVorrps46 23445 24260 Major Tail Subunit PhameratorVorrps51 25336 25890 Tail Assembly Chaperone PhameratorVorrps52 25943 26287 Tail Assembly Chaperone PhameratorVorrps53 26287 26886 HNH PhameratorVorrps54 27047 31717 Tape Measure Protein BLASTVorrps55 31749 33473 Minor Tail Protein BLASTVorrps56 33517 35253 Minor Tail Subunit PhameratorVorrps57 35293 36141 Minor Tail Subunit BLASTVorrps58 36171 38648 Minor Tail Subunit PhameratorVorrps59 38641 40131 D-ala-D-ala-carboxypeptidase BLASTVorrps60 40143 40544 Minor Tail Subunit PhameratorVorrps61 40548 40949 Minor Tail Subunit PhameratorVorrps62 40959 41183 Minor Tail Subunit PhameratorVorrps63 41196 41384 Minor Tail Subunit PhameratorVorrps64 41384 42070 Minor Tail Subunit PhameratorVorrps66 42275 43459 Lysin A PhameratorVorrps67 43461 44501 Lysin B BLASTVorrps68 44514 44810 Holin BLASTVorrps73 46894 46487 HTH DNA Binding Protein BLASTVorrps80 51141 50098 DNA Poly III Beta Subunit BLASTVorrps87 54475 53621 Ku BLASTVorrps89 56724 55216 ParB-like Domain BLASTVorrps97 61485 59707 AAA ATPase BLAST

CollectionDate: September 1st, 2014Temperature: 27.7° CCoordinates: 38°38'54.8"N 90°18'44.0"WDepth: 12 cmNotes: Slightly moist, artificially fertilized soil near planted flowers, boulder, foot traffic area.

Isolation and PurificationTiter: 3.32 x 109 pfu/mL4 rounds of purification

10 plate phage infectionMorphology: Small,

circular, clear plaquesDiameter: 1mmInfection: Lytic

Isolation and Genomic Analysis of Cluster O Mycobacteriophage Vorrpsby Madeleine Mullon, Rahul Ramaswamy, Peeti Sithiyopasakul, and Leslie Sterling

We would like to thank Washington University in St. Louis and HHMI SEA-PHAGES for their support of the Phage Hunters program, the Donald Danforth Plant Science Center for the electron microscopy pictures, and the WashU Genome Institute for sequencing our phages. We would like to especially thank our TAs Rohan Khazanchi, Nathan Kopp, and Ruchik Patel and our professors Dr. Hafer, Dr. Shaffer, and Dr. Elgin for giving us direction and support when we needed it most.

Mycobacteriophage Vorrps was isolated in 2014 from soil on the Washington University campus. The phage was isolated and purified by repeatedly infecting host bacteria Mycobacterium smegmatis. Vorrps produces small, circular, clear plaques 1 mm in diameter, indicating that it is a lytic phage. Electron microscopy reveals that Vorrps has an unusual prolate head and a long tail characteristic of cluster O mycobacteriophage. Vorrps is very similar to the five fully analyzed cluster O phages; Corndog, Catdawg, YungJamal, Firecracker and Dylan, as well as cluster O phage Mori, another phage isolated and analyzed by Washington University students this year. Vorrps and Mori are the first cluster O phage to be isolated at Washington University. Vorrps has a genome of 71,721 base pairs long. 126 genes have been annotated, 68 of which run in the negative direction, the remaining 58 run in the positive direction. A majority of the suggested genes have no known function, much like other cluster O phage. Currently each team member is investigating one of the following interesting features of the Vorrps genome: gene overlaps, tape measure gene chaperone frameshift, cluster O phage common repeat sequences, and the functionality of the prolate head.

Abstract

Results

Results

Objectives● To isolate a novel mycobacteriophage from the environment and

characterize it using both wet bench and bioinformatic analyses.● To carefully determine where each potential gene starts and stops.● To ascertain the possible functions of Vorrps gene protein products.● To annotate Vorrps so that information about its genome can be

accessed by other researchers in order to further understanding of mycobacteriophage in relation to others in the GenBank database.

Putative Gene Functions

Overlap between Genes 6 and 7

Frameshift and Tape Measure Ratio

Conclusions

● Cluster O phage heads: long length/short width

● Conserved on BLASTx across O phage

● Similarity to HK97 phage isometric capsid protein

Cluster O phage contain a conserved 17 bp palindromic repeat sequence 5’-TGTTCGGNNNCCGAACA-3’.Vorrps contains 36 of these repeats.

● Vorrps is a member of mycobacteriophage cluster O● Contains 126 genes, 34 with an annotated function● Interesting genome features characteristic of other cluster O phage

Cluster O Unusual Prolate Head Shape

Acknowledgements

● 209 bp overlap between Vorrps.6 & Vorrps.7● Both identified as DNA methylase ● Both genes highly conserved across the Cluster● Next most similar Cluster is F and compromised

of one gene

73%

12%

15%