Phage Lab @

BruceB, cluster G Et2Brutus, cluster A2

Cluster G phage: BruceB, discovered by Catherine Halpern

Extremely conserved clusterBlastn 95-99% identity over genome lengthSome infect M. tuberculosis with decent efficiencyNovel feature is MPME, pham 139

Table 1. Genome characteristics of BruceBPhage Genome

length% GC # ORFs # tRNAs

BruceB 41901 66.6 63 0Cluster G 41837 66.6 62 0

Table 2. Genome characteristics of Et2BrutusPhage Genome length % GC # ORFs # tRNAsEt2Brutus 52495 62.4 102 1Cluster A2 52671 63.2 89 avg, range

77-980-5

-Et2Brutus is a typical cluster A2 phage-Best Blastn homologue is D29-Similar to two other VCU A2 phages Trixie and Turbido*No phamerator map yet because we’re struggling to get PCR to verify our 8bp 3’-overhang

Dotplot of Cluster A phages from VCU

Cluster A2 phage: Et2 Brutus, discovered by Jonathan Kindberg

A1

A2

A4

A5

A10

Pari (A1)

Trixie (A2)

Turbido (A2)

Et2Brutus (A2)

Maverick (A4)

Wile (A4)

Theia (A5)

Twister (A10)

Pari

(A1)

Trix

ie (A

2)

Turb

ido

(A2)

Et2B

rutu

s (A

2)

Mav

eric

k (A

4)

Wile

(A4)

Thei

a (A

5)

Twist

er (A

10)

2009-2013: VCU’s 19 sequenced phages

Comparative genomics projects• Start with an interesting figure from a paper• Develop a research question• Collect literature supporting the question

Develop a project using • VCU phage sequences• Simple, free bioinformatics tools

Topics to present today:1. Repressor proteins- Hasan Alkhairo & Dominique Grim2. Repressor binding sites- Saman Raftari3. Capsid protein- Winston Sanders4. Recombination- Alexandra Ii & Catherine Halpern5. DD-carboxypeptidases- Shantel Brown & Tori Diesel

Repressor protein•Binds phage promoters•Bind repressor to regulate transcription•Maintenance of lysogeny•Prevents infection by second phage with similar repressor binding site

Lysogen lawnLysed by lytic phage

Immune to infection by its own virus

Comparison of repressor proteins in VCU phages

Pari (A1) Wile (A4)Trixie (A2) Theia (A5) Turbido (A2) Twister (A10)Et2Brutus (A2) Batiatus (F1)Maverick (A4) Ovechkin (F1)

Charlie (N)SkinnyPete (N)

ShiLan (F1)Ovechkin (F1)Batiatus (F1)

BruceB (G)

Our collection of phages has four different repressor phams

-Phams are mostly organized by cluster-Phams 3885, 54 and 1377 are published by Hatfull lab-We’re trying to figure out if 1425 is really a repressor or an antirepressor

#3885 #54 #1377 #1425

Twister (A10)Wile (A4)

Maverick (A4)Theia (A5)Trixie (A2)

Et2Brutus (A2)Turbido (A2)Batiatus (F1)

Pari (A1)Ovechkin (F1)

Helix Turn Helix

Pham 54 repressor protein groupings reflect clusters….sort of

Functional portion is the HTH domain. We’d predict phages with pham 54 HTH domains could- display superinfection immunity in the wet lab- have similar/identical prediction of repressor

binding sites….data in progress

Superinfection immunity in wet lab:

Wile (A4) /Maverick (A4)/Twister (A10)Trixie/Turbido (A2)

Need to test:Et2Brutus vs. Turbido & TrixieBatiatus/Pari/Ovechkin

Repressor binding site prediction

Methods:

Motif detection using MEME suite

Compiled motifs for each phage in Excel

Ran through python program to count frequency at each position

Results: Graphed in Excel and generated consensus logos using Weblogo

Bind repressor to regulate transcription

Maintenance of lysogeny

1 2 3 4 5 6 7 8 9 10 11 12 13 14

A 2 11 15 37 27 102 20 17 7 2 156 168 9 15

C 5 9 13 61 36 24 10 2 0 133 34 26 9 22

T 8 86 94 1 17 20 138 15 152 9 2 1 0 108

G 154 65 50 62 91 30 3 136 10 1 2 1 151 24

25

75

125

175

225

VCU cluster A RBS Consensus Sequence

Nuc

leoti

de R

ecur

renc

e

Trixie

Turbido

Et2Brutus

Repressor binding site conservation within subcluster A2

Pari, A1 phageconsensus

Ovechkin, F1 phageone example, 5X

Remember? …. Pari/Ovechkin/Batiatus have identical HTH motifs in their repressor proteins.MEME didn’t find any motifs in Ovechkin or Batiatus that look like Pari.

Can we explore structural relationships using capsid protein sequences?

Structure-based phylogeny was used to group capsid proteins of dsDNA viruses into 3 groups (2005 Bamford CoSB)

Capsid proteins from 18/19 VCU phages matched HK97 group

The capsid of Frederick, a B4 phage, looked more interesting!

Can we explore structural relationships using capsid protein sequences?

Frederick is fairly closely related to TPA2 capsid

They don’t group into any of the three…but not sureour tree is valid (sequences are not related)

Ideas for what to do next?

Explorations of recombination in mycobacteriophages

Possible example of sequence-directed recombination19bp sequence repeat in Rizal may have allowed elimination of gp35

Propose intramolecular recombination since pham 666 is conserved in cluster C phages

ShiLanSaal

BruceBBPs

OvechkinHalo

Explorations of recombination in mycobacteriophages

MPMEs described in cluster G and F phages (Sampson 2009 Microbiology)We found pham 139 in BruceB , Saal, Shilan and OvechkinMPME1 vs MPME2 sequences are identical within their groups.

Batiatus

Ovechkin

Ardmore

ShiLan

Are DD-carboxypeptidase genes in phages related to bacterial beta-lactamases?

We compared the second gene of a double-pham region in the minor tail proteins.

Functions associated with this pham:DD-carboxypeptidasebeta-lactamase

Bacterial beta-lactamases

PhageCarboxy-peptidases

M. tuberculosisStreptomyces

BacillusRhodococcus

M. smegmatisWally

NelitzaShilan

SaalOvechkinArdmoreBatiatusRedno2

Pari

SXXK SDN KTG

Are DD-carboxypeptidase genes in phages related to bacterial beta-lactamases?

Phage and bacterial genes separate on a treePhages proteins lack two functional domains

Thanks!!

HHMI SEA-PHAGES&Hatfull Lab