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

Bipolar Bactina

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

Melv

in (

A4)

Dha

nu

sh (

A4)

Bellu

sT

err

a (

A4)

ICle

are

d (

A4)

Flu

x (

A4)

Art

uro

(A

4)

Pukovnik

(A

2)

SkiP

ole

(A

1)

Kugel (A

1)

KS

SJE

B (

A1)

BP

Bie

bs3

1 (

A1)

Lese

di (

A1)

JC27 (A

1)

Rose

bush

(B

2)

Qyr

zula

(B

2)

Arb

iter (B

2)

DaV

inci

(A

6)Eric

B (A6)

Jeffa

bunn

y (A

6)

Gla

diat

or (A

6)

Ham

mer

(A6)

Blue7

(A6)

Zaka

(A6)

HIN

deR (A7)

Timsh

el (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)

Giles (Q

)

Suffolk (B

1)

Firecracker (O)

Corndog (O

)

Dylan (O

)

BAKA (J)

Optim

us (J)

Macn

Cheese

(K3)

Pixie

(K3)

Phaedru

s (B3)

PackM

an (A

9)

Alm

a (A

9)

Lin

Stu

(C1)

Ple

ione (C

1)

Nappy (C

1)

Dandelio

n (C

1)

Spud (C

1)

Arc

herS

7 (C

1)

Ava3 (C

1)

Bru

jita (

I1)

Isla

nd3

(I1

)

Dori (

Sin

gle

ton

)

Bu

tte

rs (

N)

Fis

hb

urn

e (

P)

Don

ova

n (

P)

Jebeks (

P)

LittleE

(J)

Phru

x (

E)

Rakim

(E

)

Phatb

acte

r (E

)M

urp

hy (

E)

Phaux (

E)

Bask

21 (

E)

DS

6A

(S

ingle

ton)

Trixi

e (A

2)

Ech

ild (A

2)

L5 (A

2)

Eag

leE

ye (A

2)

D29

(A2)

Adz

zy (A2)

Gad

jet (

B3)

Bon

go (M

)

Peg

Leg

(M)

Isaa

cEli (B

1)

Three

Oh3

D2

(B1)

Kinbote

Dra

ft (Q

)

UncleHowie

(B1)

Fang (B1)

TallGra

ssMM (B

1)

Thora (B1)

RockyHorror (

F1)

Fruitloop (F

1)

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)

Microwolf (A3)

Vix (A3)

Methuselah (A3)

ElTiger69 (A5)

Benedict (A

5)

SG

4 (F1)

Bernardo (B

3)

Ram

sey (F1)

Jabbawokkie (F

2)

TM

4 (K2)

Rey (M

)

Patie

nce

(Sin

gle

ton)

Rum

pelstiltskin

(L2)

UP

IE (L

1)

JoeD

irt (L1)

LeB

ron (L

1)

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

(K

1)

JA

WS

(K

1)

Barr

elR

oll

(K1)

Anaya (

K1)

Angelic

a (

K1)

Pix

ie (

K3)

Eagle

Eye (

A2)

Jabbaw

okkie

(F

2)

Alm

a (

A9)

Court

house (

J)

Lie

fie (

G)

Redi (

N)

Bongo (

M)

Fang (B

1)

Tw

iste

r (A

10)

D29 (A

2)

Ski

Pol

e (A

1)

Ros

ebus

h (B

2)Are

s (B

2)

Hed

gero

w (B2)

Arb

iter (

B2)

Qyr

zula

(B2)

Valid

us (K

1)

Akom

a (B

3)

Heath

cliff

(B3)

Bernard

o (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)

Charlie (N

)

Corndog (O

)

BPs (G

)

Hope (G

)

Avrafan (G

)

Angel (G

)

Phrux (E

)

Phatb

acte

r (E)

Phaux (E

)

Rakim

(E)

Bask2

1 (E

)

Murp

hy ( E

)

Toto

(E)

Bip

ola

r (F

1)

Shauna1 (

F1)

DeadP

(F

1)

SG

4 (

F1)

Fru

itlo

op (

F1)

GU

mbie

(F

1)

Ram

sey (

F1)

RockyH

orr

or

(F1)

DS

6A

(S

ing

leto

n)

Avan

i (F

2)

Yosh

i (F

2)

Gile

s (

Q)

Kin

bote

(Q

)H

H92 (

Q)

JH

C117 (

A3)

Vix

(A

3)

Jobu08 (

A3)

Mic

row

olf (

A3)

PegLeg (

M)

TM

4 (

K2)

Ech

ild (

A2)

Crim

D (

K1)

Macn

Cheese

(K

3)

JAM

al (

B4)

Zem

anar (B

4)

Arc

herS

7 (C

1)

Dan

delio

n (C

1)

Spu

d (C

1)

Nap

py (C

1)

Ava

3 (C

1)

Ple

ione

(C1)

Nigel

(B4)

Stinge

r (B4)

Acadia

n (B5)

Dylan (O

)

Firecr

acker (

O)

ZoeJ (K2)

Blue7 (A6)

Hammer (A6)

Gladiator (A6)

DaVinci (A6)

EricB (A6)L5 (A2)LinStu (C

1)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 (H

1)

Papyrus (R

)

Send513 (R

)

Patience (S

ingleton)

Faith1 (L2)

Win

ky (L2)

Cro

ssroads (L

2)

Rum

pelstiltskin

(L2)

Muddy (S

ingle

ton)

UP

IE (L

1)

JoeD

irt (L1)

LeB

ron (L

1)

C

B Phatb

acte

r (E

)

Pha

ux (

E)

Murp

hy (

E)

Bask21 (

E)

Phru

x (

E)

Rakim

(E

)

Toto

(E

)B

igN

uz (

P)

Donovan (

P)

Fis

hburn

e (

P)

Jebeks

(P

)

Rebeuca

(A

10)

Tw

iste

r (A

10)

Goose

(A

10)

Mic

row

olf

(A3)

Jobu08 (A

3)

JHC

117

(A3)

Vix

(A3)

Met

huse

lah

(A3)

ElT

iger

69 (A5)

Ben

edict (

A5)

LeBro

n (L

1)

UPIE

(L1)

JoeD

irt (L

1)

Rum

pelstilts

kin (L

2)

Faith1 (L

2)

Winky

(L2)

Cross

roads (

L2)

Arturo

(A4)

Flux (A4)

ICleared (A

4)

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 (H1)

Lesedi (A1)

BPBiebs31 (A

1)

SkiP

ole (A1)

JC27 (A

1)

KSSJE

B (A

1)

Kugel (A

1)

Rid

geC

B (A

1)

Bru

jita (I1

)

Island3 (I1

)

Babsie

lla (I1

)

TM

4 (K

2)

ZoeJ (K

2)

DS

6A

(Sin

gle

ton)

TallG

RassM

M (B

1)

Fire

cra

cker (O

)

Corn

dog (O

)

Dyla

n (O

)

Kin

bote

Dra

ft (Q)

Gile

s (Q

)

HH

92

(Q)

Nova

(D

1)

Tro

ll4 (

D1

)

Adju

tor

(D1)

Butt

ers

cotc

h (

D1)

SirH

arl

ey (

D1)

Gum

ball

(D1)

Patience (

Sin

gle

ton)

PackM

an (

A9)

Alm

a (

A9)

Rocksta

r (A

3)

HelD

an (

A3)

Sain

tus (

A8)

Ast

ro (A

8)

Lin

Stu

(C

1)

Spud (C

1)

Litt

leE

(J)

Courthouse

(J)

Thib

ault

(J)

BAKA (J)

Opt

imus

(J)

Sen

d513

(R

)

PAPYRUS (R

)

Mud

dy (S

ingl

eton

)Rey

(M)

Bonga

(M)

PegLeg (M)

Phlyer (

B3)

Phaedrus

(B3)

Heathcli

ff (B

3)

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)

Phelemich (B5)

Acadian (B5)

Kayacho (B4)

Ares (B2)

Rosebush (B

2)

Hedgerow

(B2)

Qyrzula (B

2)

Arbiter (B

2)

Dori (S

ingleton)

Ram

sey (F1)

RockyH

orro

r (F1)

DeadP

(F1)

SG

4 (F

1)

Shauna1 (F

1)

Fru

itloop (F

1)

Bip

ola

r (F1)

GU

mbie

(F1)