1. ● 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 Source
Vorrps5 1716 1147 DNA Methylase BLAST
Vorrps6 2047 1508 DNA Methylase Phamerator
Vorrps10 3349 2729 Endo VII BLAST
Vorrps20 7753 10116 DNA Primase/Polymerase BLAST
Vorrps27 12011 12364 HNH Endonuclease BLAST
Vorrps30 13116 14630 Terminase BLAST
Vorrps31 14689 15918 Portal Phamerator
Vorrps32 15915 16499 O-Methyltransferase Phamerator
Vorrps34 17358 18050 Glycosyltransferase BLAST
Vorrps35 18047 18667 Glycosyltransferase BLAST
Vorrps38 19688 20920 Major Capsid BLAST
Vorrps46 23445 24260 Major Tail Subunit Phamerator
Vorrps51 25336 25890 Tail Assembly Chaperone Phamerator
Vorrps52 25943 26287 Tail Assembly Chaperone Phamerator
Vorrps53 26287 26886 HNH Phamerator
Vorrps54 27047 31717 Tape Measure Protein BLAST
Vorrps55 31749 33473 Minor Tail Protein BLAST
Vorrps56 33517 35253 Minor Tail Subunit Phamerator
Vorrps57 35293 36141 Minor Tail Subunit BLAST
Vorrps58 36171 38648 Minor Tail Subunit Phamerator
Vorrps59 38641 40131 D-ala-D-ala-carboxypeptidase BLAST
Vorrps60 40143 40544 Minor Tail Subunit Phamerator
Vorrps61 40548 40949 Minor Tail Subunit Phamerator
Vorrps62 40959 41183 Minor Tail Subunit Phamerator
Vorrps63 41196 41384 Minor Tail Subunit Phamerator
Vorrps64 41384 42070 Minor Tail Subunit Phamerator
Vorrps66 42275 43459 Lysin A Phamerator
Vorrps67 43461 44501 Lysin B BLAST
Vorrps68 44514 44810 Holin BLAST
Vorrps73 46894 46487 HTH DNA Binding Protein BLAST
Vorrps80 51141 50098 DNA Poly III Beta Subunit BLAST
Vorrps87 54475 53621 Ku BLAST
Vorrps89 56724 55216 ParB-like Domain BLAST
Vorrps97 61485 59707 AAA ATPase BLAST
Collection
Date: September 1st, 2014
Temperature: 27.7° C
Coordinates: 38°38'54.8"N 90°18'44.0"W
Depth: 12 cm
Notes: Slightly moist, artificially fertilized
soil near planted flowers, boulder,
foot traffic area.
Isolation and Purification
Titer: 3.32 x 109
pfu/mL
4 rounds of purification
10 plate phage infection
Morphology: Small,
circular, clear plaques
Diameter: 1mm
Infection: Lytic
Isolation and Genomic Analysis of Cluster O Mycobacteriophage Vorrps
by 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%