Phage Lab @ BruceB, cluster G Et2Brutus, cluster

Phage Lab @ BruceB, cluster G Et2Brutus, cluster

Phage Lab @ BruceB, cluster G Et2Brutus, cluster A2 Cluster G phage: BruceB, discovered by Catherine Halpern Table 1. Genome characteristics of BruceB Phage Genome % GC # ORFs # tRNAs length BruceB Cluster G 41901 41837 66.6

66.6 63 62 0 0 Extremely conserved cluster Blastn 95-99% identity over genome length Some infect M. tuberculosis with decent efficiency Novel feature is MPME, pham 139 A1 Twister (A10) Theia (A5)

Wile (A4) Maverick (A4) Et2Brutus (A2) Turbido (A2) Trixie (A2) Pari (A1) Cluster A2 phage: Et2 Brutus, discovered by Jonathan Kindberg Pari (A1) Trixie (A2) A2

Turbido (A2) Et2Brutus (A2) A4 -Et2Brutus is a typical cluster A2 phage Maverick (A4) -Best Blastn homologue is D29 -Similar to two other VCU A2 phages Trixie Wile (A4) and Turbido A5 Theia (A5) A10 Twister (A10)

*No phamerator map yet because were struggling to get PCR to verify our 8bp 3-overhang Dotplot of Cluster A phages from VCU Table 2. Genome characteristics of Et2Brutus Phage Genome length % GC # ORFs Et2Brutus 52495 62.4 102 Cluster A2 52671 63.2 89 avg, range 77-98

# tRNAs 1 0-5 2009-2013: VCUs 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

Comparison of repressor proteins in VCU phages 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 Our collection of phages has four different repressor phams #3885 Charlie (N)

SkinnyPete (N) #54 Pari (A1) Wile (A4) Trixie (A2) Theia (A5) Turbido (A2) Twister (A10) Et2Brutus (A2) Batiatus (F1) Maverick (A4) Ovechkin (F1) #1377 #1425 BruceB (G) ShiLan (F1) Ovechkin (F1)

Batiatus (F1) -Phams are mostly organized by cluster -Phams 3885, 54 and 1377 are published by Hatfull lab -Were trying to figure out if 1425 is really a repressor or an antirepressor Pham 54 repressor protein groupings reflect clusters.sort of Functional portion is the HTH domain. Wed 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 Helix Superinfection immunity in wet lab: Wile (A4) /Maverick (A4)/Twister (A10) Trixie/Turbido (A2)

Need to test: Et2Brutus vs. Turbido & Trixie Batiatus/Pari/Ovechkin Twister (A10) Wile (A4) Maverick (A4) Theia (A5) Trixie (A2) Et2Brutus (A2) Turbido (A2) Batiatus (F1) Pari (A1) Ovechkin (F1) Turn Helix

Repressor binding site prediction Bind repressor to regulate transcription Maintenance of lysogeny 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 VCU cluster A RBS Consensus Sequence 250 Nucleotide Recurrence 200 150

100 50 0 1 2 3 4 5 6

7 8 9 10 11 12 13 14 Repressor binding site conservation within subcluster A2 Trixie

Turbido Et2Brutus Remember? . Pari/Ovechkin/Batiatus have identical HTH motifs in their repressor proteins. MEME didnt find any motifs in Ovechkin or Batiatus that look like Pari. Pari, A1 phage consensus Ovechkin, F1 phage one example, 5X 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 dont group into any of the threebut 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 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. ShiLan Saal BruceB BPs Ovechkin Halo Are DD-carboxypeptidase genes in phages related to bacterial beta-lactamases? Batiatus Ovechkin Ardmore

Functions associated with this pham: DD-carboxypeptidase beta-lactamase ShiLan We compared the second gene of a double-pham region in the minor tail proteins. Are DD-carboxypeptidase genes in phages related to bacterial beta-lactamases? Bacterial beta-lactamases Phage Carboxy-peptidases Phage and bacterial genes separate on a tree Phages proteins lack two functional domains SXXK M. tuberculosis Streptomyces

Bacillus Rhodococcus M. smegmatis Wally Nelitza Shilan Saal Ovechkin Ardmore Batiatus Redno2 Pari SDN KTG Thanks!! HHMI SEA-PHAGES

& Hatfull Lab

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