Phage BruceB, cluster G Et2Brutus, cluster A2

Cluster G phage: BruceB, discovered by Catherine Halpern Extremely conserved cluster Blastn 95-99% identity over genome length Some infect M. tuberculosis with decent efficiency Novel feature is MPME, pham 139 Table 1. Genome characteristics of BruceB Phage Genome length % GC# ORFs# tRNAs BruceB Cluster G

Table 2. Genome characteristics of Et2Brutus PhageGenome length% GC# ORFs# tRNAs Et2Brutus Cluster A avg, range 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) Trixie (A2) Turbido (A2) Et2Brutus (A2) Maverick (A4) Wile (A4) Theia (A5) Twister (A10)

: 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 Grim 2.Repressor binding sites- Saman Raftari 3.Capsid protein- Winston Sanders 4.Recombination- Alexandra Ii & Catherine Halpern 5.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 lawn Lysed 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 & Trixie Batiatus/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

Trixie Turbido Et2Brutus Repressor binding site conservation within subcluster A2

Pari, A1 phage consensus Ovechkin, F1 phage one 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 sure our tree is valid (sequences are not related) Ideas for what to do next?

Explorations of recombination in mycobacteriophages Possible example of sequence-directed recombination 19bp sequence repeat in Rizal may have allowed elimination of gp35 Propose intramolecular recombination since pham 666 is conserved in cluster C phages

ShiLan Saal BruceB BPs Ovechkin Halo 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 Ovechkin MPME1 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-carboxypeptidase beta-lactamase

Bacterial beta-lactamases Phage Carboxy-peptidases M. tuberculosis Streptomyces Bacillus Rhodococcus M. smegmatis Wally Nelitza Shilan Saal Ovechkin Ardmore Batiatus Redno2 Pari SXXK SDN KTG Are DD-carboxypeptidase genes in phages related to bacterial beta-lactamases? Phage and bacterial genes separate on a tree Phages proteins lack two functional domains

Thanks!! HHMI SEA-PHAGES & Hatfull Lab