The document summarizes research characterizing newly isolated bacteriophages (viruses that infect bacteria) that prey on Mycobacterium smegmatis bacteria. Twelve bacteriophages were found and nine were analyzed by electron microscopy, showing a tailed "siphoviridae" structure. The genome of one phage, Bipolar, was fully sequenced and found to be similar to the F1 subcluster. The document then analyzes whether two genes, Tape Measure Protein (TMP) and Lysin A, can accurately predict phage cluster relationships on their own. Results showed that TMP was highly accurate, while Lysin A was less so, supporting the hypothesis that Lysin A is more diverse

1. Methods
• Twelve mycobacteriophages were isolated from the College of Idaho campus and 9 of
these were characterized by electron microscopy. All are consistent with siphoviridae
morphotypes (bacteriophages with head and tail).
• The genome of mycobacteriophage Bipolar was sequenced. Comparison by whole
genome nucleotide alignment places Bipolar within the F1 mycobacteriophage subcluster.
• Annotation of the Bipolar genome indicates that the Bipolar genome is 58985bp, including
107 predicted genes. The genome contains a predicted programmed translational
frameshift at position 9096bp (in the putative tail assembly chaperone gene).
• The gene encoding Tape Measure Protein (TMP) was found to accurately predict the
subcluster assignment for Bipolar. More broadly, TMP was found to accurately predict the
subcluster assignment for 166 mycobacteriophages (97.5% accuracy) based on dot plot
analysis.
• The gene encoding Lysin A was found to accurately predict the subcluster assignment for
Bipolar. Lysin A was somewhat less accurate (93.4%) in predicting the subcluster
assignments for other mycobacteriophages.
• Our hypothesis was supported. These results are important for understanding
mycobacteriophage diversity and the genes involved in lysis of Mycobacterium
smegmatis.
Conclusions
Genomic Analysis of the Novel F1 Subcluster Mycobacteriophage Bipolar
Shandee J. Tachick, McKayla M. Stevens, Aliza M. Auces, Ljuvica R. Kolich, Hana L. Hoang, Pamela A. Dockstader, Ann P. Koga, and Richard L. Daniels
Biology Department, The College of Idaho, Caldwell, Idaho 83605.
Acknowledgements
We would like to thank the students in the Fall BIO201 (Molecules to Cells) course for mycobacteriophage isolation,
especially our TAs Macey Horch, Laura Holden, Megan Brock, Jessie Lambright and Juan Cervantes. We are very
grateful to the HHMI SEA Staff, the laboratory of Graham Hatfull (University of Pittsburgh) and DNA sequencing facility
at Virginia Commonwealth University. Karthik Chinnathambi and Rick Ubic provided technical assistance with electron
microscopy at the Boise State University Center for Materials Characterization. The project described was supported in
part by the INBRE Program, NIH Grant Nos. P20 RR016454 (National Center for Research Resources) and P20
GM103408 (National Institute of General Medical Sciences).
Tape Measure Protein
Figure 2. Bipolar genome and
single gene analysis.
A) Dot plot of 166
mycobacteriophage genomes. Colors
on axes represent the phage clusters
as depicted on the legend. Graph
generated by Genome Pair - Rapid
Dotter (GEPARD). B) Dot plot of
Tape Measure Protein (TMP) gene
from 166 mycobacteriophages. C)
Dot plot of Lysin A gene from 166
mycobacteriophages.
A
Hypothesis
Kalliden
CuteCollette
Alcor
MrMud
Gary1
Achille13
BipolarBactina
Oink
Figure 1. Electron
micrographs of 9
mycobacteriophages
isolated from soil samples
on the College of Idaho
campus. All isolates were
identified as siphoviridae
(tailed bacteriophages). Using
ImageJ (a freely available
image analysis program
provided by the National
Institutes of Health) we found
an average capsid diameter
of 73.3 nm and the average
tail length of 164.8 nm.
Lysin A
Bacteriophages are viruses that infect bacteria. As a group, bacteriophages are the single most
abundant biological entity in the biosphere, with a population estimated at 1031 particles1. As part
of the HHMI SEA-PHAGES program, the College of Idaho’s Fall 2013 BIO201 class isolated and
characterized 12 mycobacteriophages from Southwestern Idaho that infect Mycobacterium
smegmatis. Electron microscopy revealed that each of these mycobacteriophages displayed
morphological characteristics consistent with a siphoviridae morphotype (tailed bacteriophages).
One of these newly-isolated mycobacteriophages (Bipolar) was selected for sequencing and
further genomic analysis. We found that the Bipolar genome is 59.0kb in length and contains 107
predicted protein-coding genes, with a nucleotide sequence similar to the F1 mycobacteriophage
subcluster. This annotation included gene function predictions and the results are being reviewed
for submission to Genbank, a repository for biological sequence information hosted by the National
Institutes of Health’s National Center for Biotechnology Information (NCBI).
Following the genomic annotation of Bipolar, we further investigated whether the clustering
relationships that result from whole genome similarity are also evident when single genes are
analyzed. A recent study demonstrated that mycobacteriophages could be accurately assigned
into their respective clusters and subclusters based on the nucleotide sequence of two genes:
Tape Measure Protein (TMP) and Major Capsid Protein2. Here we examine whether ubiquitous
non-structural genes, such as Lysin A, can also be used to predict phage cluster assignments.
While structural genes are generally found in a similar 5’ region of mycobacteriophage genomes
and are highly conserved, Lysin A exhibits a greater degree of positional freedom and does not
display the same high degree of similarity between mycobacteriophages3. We hypothesize that this
diversity makes Lysin A a less suitable gene for cluster assignment. Using dot plot analysis and
phylogenetic trees, we test this hypothesis by visualizing the relationships predicted by both whole
genome analysis and single gene analysis of Tape Measure Protein (TMP) and Lysin A from 166
mycobacteriophages.
REFERENCES
1. Hatfull et al., Comparative genomic analysis of sixty mycobacteriophage genomes: Genome clustering, gene acquisition and
gene size. J Mol Biol. 2010.
2. Smith et al., Phage cluster relationships identified through single gene analysis. BMC Genomics. 2013.
3. Payne and Hatfull, Mycobacteriophage Endolysins: Diverse and Modular Enzymes with Multiple Catalytic Activiites. PLOS
ONE. 2012.
Background & Introduction
Whole Genome
Singleton
A
B
C
D
E
F
G
H
I
J
K
L
M
O
P
Q
R
N
B
C
Melvin(A4)
Dhanush(A4)
BellusTerra(A4)
ICleared(A4)
Flux(A4)Arturo(A4)
Pukovnik(A2)
SkiPole(A1)
Kugel(A1)
KSSJEB(A1)
BPBiebs31(A1)
Lesedi(A1)JC27(A1)
Rosebush(B2)
Qyrzula(B2)
Arbiter(B2)
DaVinci(A6)
EricB
(A6)
Jeffabunny(A6)
G
ladiator(A6)
Ham
m
er(A6)
Blue7
(A6)
Zaka
(A6)
HINdeR
(A7)
Tim
shel (A7)
Thibault (J)
Courthouse (J)
Cucu (A5)
George (A5)
LittleCherry (A5)
Tiger (A5)
RidgeCB (A1)
Saintus (A8)
Astro (A8)
Winky (L2)
Faith1 (L2)
Crossroads (L2)
Muddy (Singleton)
Predator (H1)
BigNuz (P)
Konstantine (H1)
Nova (D1)
Troll4 (D1)
Butterscotch (D1)
Adjustor (D1)
Gumball (D1)
SirHarley (D1)
Avani (F2)
Jovo (A5)
Toto (E)Babsiella (I1)Hedgerow (B2)Ares (B2)
Send513 (R)
Papyrus (R)
Akoma (B3)
Phyler (B3)
Daisy (B3)
Heathcliff (B3)
Liefie (G)
Hope
(G)Angel (G
)
G
iles
(Q
)
Suffolk
(B1)
Firecracker(O
)
Corndog
(O)
Dylan
(O)
BAKA
(J)
Optimus(J)
MacnCheese(K3)
Pixie(K3)
Phaedrus(B3)
PackMan(A9)
Alma(A9)
LinStu(C1)
Pleione(C1)
Nappy(C1)
Dandelion(C1)
Spud(C1)ArcherS7(C1)
Ava3(C1)
Brujita(I1)
Island3(I1)
Dori(Singleton)
Butters(N)
Fishburne(P)Donovan(P)
Jebeks(P)
LittleE(J)
Phrux(E)
Rakim(E)
Phatbacter(E)
Murphy(E)
Phaux(E)
Bask21(E)
DS6A(Singleton)Trixie(A2)
Echild(A2)
L5(A2)
EagleEye(A2)
D29(A2)
Adzzy(A2)
Gadjet(B3)
Bongo
(M
)
PegLeg
(M
)
IsaacEli (B1)
ThreeO
h3D2
(B1)
Kinbote
Draft (Q)
UncleHowie
(B1)
Fang (B1)
TallGrassMM
(B1)
Thora (B1)
RockyHorror (F1)
Fruitloop (F1)
Shauna1 (F1)
DeadP (F1)
Gumbie (F1)Bipolar (F1)ChrisnMich (B4)JAMal (B4)Zemanar (B4)Nigel (B4)
Stinger (B4)
Cooper (B4)
Anaya (K1)
Angelica (K1)
Adephagia (K1)
JAWS (K1)
BarrelRoll (K1)
CrimD (K1)
Validus (K1)
Yoshi (F2)
Redi (N)
Charlie (N)
ZoeJ (K2)
Reprobate (B5)
Acadian (B5)
Twister (A10)
Rebeuca (A10)
Goose (A10)
Rockstar (A3)
HelDan (A3)
Jobu08 (A3)
JHC117 (A3)
M
icrowolf (A3)
Vix
(A3)
M
ethuselah
(A3)
ElTiger69
(A5)
Benedict(A5)
SG
4
(F1)
Bernardo
(B3)
Ramsey(F1)
Jabbawokkie(F2)
TM4(K2)
Rey(M)
Patience(Singleton)
Rumpelstiltskin(L2)
UPIE(L1)
JoeDirt(L1)LeBron(L1)
Single-gene analysis of Tape Measure Protein (TMP)
more accurately predicts mycobacteriophage cluster
relationships than Lysin A.
A
Figure 3. Phylogenetic
trees generated from
166 A) Lysin A nucleotide
sequences using
Maximum Likelihood (ML)
analysis. B) Tape
Measure Protein (TMP)
nucleotide sequences. C)
whole mycobacteriophage
genomes. Phylogenetic
trees created using
Molecular Evolutionary
Genetics Analysis
software (MEGA6).
Adephagia(K1)
JAWS(K1)
BarrelRoll(K1)
Anaya(K1)
Angelica(K1)
Pixie(K3)
EagleEye(A2)
Jabbawokkie(F2)
Alma(A9)
Courthouse(J)
Liefie(G)
Redi(N)
Bongo(M)Fang(B1)
Twister(A10)D29(A2)
SkiPole(A1)
Rosebush(B2)
Ares(B2)
Hedgerow
(B2)
Arbiter(B2)
Q
yrzula
(B2)
Validus
(K1)
Akom
a
(B3)
Heathcliff (B3)
Bernardo
(B3)
Gadjet (B3)
Phlyer (B3)
Daisy (B3)
Cuco (A5)
George (A5)
Tiger (A5)
Phaedrus (B3)
Brujita (I1)
Island3 (I1)
HINdeR (A7)
Timshel (A7)
Suffolk(B1)
ThreeOh3D2 (B1)
IsaacEli (B1)
Thora (B1)
TallGrassMM (B1)
UncleHowie (B1)
Astro (A8)
Saintus (A8)
ElTiger69 (A5)
Phelemich (B5)
Rockstar (A3)
HelDan (A3)PackMan (A9)Pukovnik (A2)BellusTerra (A4)
TiroTheta9 (A4)Flux (A4)Melvin (A4)
ICleared (A4)
Arturo (A4)
Methuselah (A3)
Trixie (A2)
Goose (A10)
Rebeuca (A10)
Reprobate (B5)
Kayacho
(B4)
Rey (M
)
Butters
(N)
C
harlie
(N)
C
orndog
(O
)
BPs
(G
)
Hope
(G
)
Avrafan
(G)
Angel(G)
Phrux(E)
Phatbacter(E)
Phaux(E)
Rakim
(E)
Bask21(E)
Murphy(E)
Toto(E)
Bipolar(F1)Shauna1(F1)
DeadP(F1)SG4(F1)
Fruitloop(F1)
GUmbie(F1)
Ramsey(F1)
RockyHorror(F1)
DS6A(Singleton)
Avani(F2)
Yoshi(F2)
Giles(Q)
Kinbote(Q)HH92(Q)
JHC117(A3)
Vix(A3)
Jobu08(A3)
Microwolf(A3)
PegLeg(M)
TM4(K2)
Echild(A2)
CrimD(K1)
MacnCheese(K3)
JAMal(B4)
Zemanar(B4)
ArcherS7(C1)
Dandelion(C1)
Spud(C1)
Nappy(C1)
Ava3
(C1)
Pleione
(C1)
N
igel(B4)
Stinger (B4)
Acadian
(B5)
Dylan
(O)
Firecracker (O)
ZoeJ (K2)
Blue7 (A6)
Hammer (A6)
Gladiator (A6)
DaVinci (A6)
EricB (A6)L5 (A2)LinStu (C1)Kugel (A1)Lesedi (A1)Dhanush (A4)JC27 (A1)RidgeCB (A1)Donovan (P)
Jebeks (P)
Fishburne (P)
BigNuz (P)
LittleE (J)
BAKA (J)
Optimus (J)
Thibault (J)
ChrisnMich (B4)
Dori (Singleton)
LittleCherry (A5)
Jovo (A5)
Jeffabunny (A6)
Zaka (A6)
BPBiebs31 (A1)
KSSJEB (A1)
Benedict (A5)
Adzzy (A2)
Babsiella (I1)
Cooper (B4)
Predator (H1)
Gumball (D1)
Nova (D1)
SirHarley (D1)
Troll4
(D1)
Adjutor (D1)
Butterscotch
(D
1)
Konstantine
(H1)
Papyrus
(R)
Send513
(R)
Patience(Singleton)
Faith1(L2)
W
inky(L2)Crossroads(L2)
Rumpelstiltskin(L2)
Muddy(Singleton)
UPIE(L1)
JoeDirt(L1)LeBron(L1)
C
B
Phatbacter(E)
Phaux(E)Murphy(E)
Bask21(E)
Phrux(E)
Rakim(E)
Toto(E)
BigNuz(P)Donovan(P)
Fishburne(P)
Jebeks(P)
Rebeuca(A10)Twister(A10)Goose(A10)
Microwolf(A3)
Jobu08(A3)
JHC117(A3)Vix(A3)
Methuselah
(A3)
ElTiger69
(A5)
Benedict(A5)
LeBron
(L1)
U
PIE
(L1)
JoeDirt (L1)
Rum
pelstiltskin
(L2)
Faith1
(L2)
W
inky (L2)
Crossroads (L2)
Arturo (A4)
Flux (A4)
ICleared (A4)
Adzzy (A2)
Pukovnik (A2)
EagleEye (A2)
Tiger (A5)
Jovo (A5)
Cuco (A5)
George (A5)
LittleCherry (A5)
ArcherS7 (C1)
Ava3 (C1)
Pleione (C1)
Nappy (C1)
Dandelion(C1)
TiroTheta9 (A4)
Dhanush (A4)
BellusTerra (A4)
Melvin (A4)
HINdeR (A7)Timshel (A7)Trixie (A2)Echild (A2)L5 (A2)D29 (A2)DaVinci (A6)EricB (A6)Zaka (A6)
Jeffabunny (A6)
Gladiator (A6)
Blue7 (A6)
Hammer (A6)
Yoshi (F2)
Avani (F2)
Jabbawokkie
(F2)
Predator (H1)
Konstantine
(H
1)
Lesedi(A1)
BPBiebs31
(A1)
SkiPole
(A1)
JC27
(A1)
KSSJEB
(A1)
Kugel(A1)
RidgeCB(A1)
Brujita(I1)
Island3(I1)
Babsiella(I1)
TM4(K2)
ZoeJ(K2)
DS6A(Singleton)
TallGRassMM(B1)
Firecracker(O)
Corndog(O)
Dylan(O)
KinboteDraft(Q)
Giles(Q)
HH92(Q)
Nova(D1)
Troll4(D1)
Adjutor(D1)
Butterscotch(D1)SirHarley(D1)Gumball(D1)
Patience(Singleton)
PackMan(A9)Alma(A9)
Rockstar(A3)
HelDan(A3)
Saintus(A8)
Astro(A8)
LinStu(C1)
Spud(C1)
LittleE(J)
Courthouse(J)
Thibault(J)
BAKA
(J)
Optimus(J)
Send513
(R)
PAPYRUS
(R)
M
uddy
(Singleton)
R
ey
(M
)
Bonga
(M
)
PegLeg
(M
)
Phlyer (B3)
Phaedrus (B3)
Heathcliff (B3)
Akoma (B3)
Bernardo (B3)
Gadjet (B3)
Daisy (B3)
Charlie (N)Redi (N)Butters (N)Validus (K1)CrimD (K1)BPs (G)
Avrafan (G)Hope (G)
Angel (G)
Liefie (G)
MacnCheese (K3)
Pixie (K3)
Anaya (K1)
Adephagia (K1)
Angelica (K1)
JAWS (K1)
BarrelRoll (K1)
Fang (B1)
ThreeOh3D2 (B1)
Thora (B1)
IsaacEli (B1)
UncleHowie (B1)
Suffolk (B1)
Cooper (B4)
Stinger (B4)
Nigel (B4)
JAMal (B4)
ChrisnMich (B4)
Zemanar (B4)
Reprobate (B5)
Phelem
ich
(B5)
Acadian
(B5)
Kayacho
(B4)
Ares
(B2)
R
osebush
(B2)
Hedgerow
(B2)
Q
yrzula
(B2)
Arbiter(B2)
Dori(Singleton)
Ramsey(F1)
RockyHorror(F1)
DeadP(F1)
SG4(F1)
Shauna1(F1)Fruitloop(F1)
Bipolar(F1)GUmbie(F1)