The Isolation and Characterization of Novel Mycobacteriophages J-Gladiator, Uncle Howie, and Angelica

Transcription

1 The Isolation and Characterization of Novel Mycobacteriophages J-Gladiator, Uncle Howie, and Angelica Suchita Rastogi, Martin Y. Fan, Ho Yee Joyce Fung, Michael B. Schultz, Joseph Marcus, Samantha Alford, Alexander Anderson, William Barshop, Lisa Deng, Vincent Huang, Peter M. Hynes, Patrick Ng, Hannah S. Rabinowitz, Corwin N. Rhyan, Erika Fieldman Sims, Emilie G. Weisser, Bo Zhang, and Thomas Shull Funded by the Howard Hughes Medical Institute

2 Discovery and Isolation J-Gladiator: Heman Park, St. Louis, at N W Uncle Howie: Heman Park, St. Louis, at N W Angelica: Koenig House, Washington University, at N W Purified on M. smegmatis

3 Physical Characteristics of J-Gladiator Plaques for J Gladiator Halo structure These may indicate that J Gladiator is lysogenic EM Photograph of J Gladiator Tail: 93 nm long Capsid: 61 nm in diameter Short tail for phage

4 Physical Characteristics of Angelica Plaques for Angelica Cloudy plaques with halo May indicate that Angelica is lysogenic EM Photograph of Angelica Tail: 210 nm long Capsid: 70 nm in diameter Capsids appear filled with stain

5 Physical Characteristics of Uncle Howie Plaques for Uncle Howie Small plaques with halo EM Photograph of Uncle Howie Tail: 250 nm long Capsid: 57 nm in diameter Visible base plate at end of tail

6 J-Gladiator Belongs to Cluster A2 J Gladiator genome Similar global architectures Mycobacteriophages in the A2 family

7 Angelica: Non-clustered or Cluster G? Global architectures for Angelica and its closest relatives. s Angelica BPs Halo TM4 Percent Identity Between Angelica and Other Mycobacteriophages Angelica has G{ a high degree of similarity to both Cluster G and TM4. Cluster Nonclustered

8 Uncle Howie Belongs to Cluster B1 Similar global architectures: Uncle Howie PG1 Uncle Howie is most similar to phage PG1

9 Sequence of J-Gladiator Genome Size: 49.7 kbp GC%: 61.44% Terminase Major Head Subunit Portal Tape Measure DNA Polymerase ThyX Endonuclease Anti repressor RecB nuclease trnas Genome Map of J Gladiator

10 Distribution of Gene Functions for J Gladiator Genome contains 90 ORF s 3 trna s Novel no significant BLAST hits* Structural capsid and tail subunits Enzymatic proteins involved in gene regulation, enzymes Unknown no known function Structural, 8, 9% Novel, 19, 21% Enzymatic, 14, 16% Unknown, 49, 54% * significant hits have E values lower than 1E 5

11 Genes of Interest in J Gladiator Thymidylate Synthase DNA Polymerase Replication Speed Vertical Gene Transfer

12 Replication Speed DNA Polymerase (gp 44) J-Glad gp44 is more similar to eukaryotic polymerase than to that of M. Smeg: Consistent with current classification of DNA polymerases 1. Polymerase function (?) Replication speed 1 Jonathan Filée, Patrick Forterre, Tang Sen-Lin and Jacqueline Laurent. Evolution of DNA Polymerase Families: Evidences for Multiple Gene Exchange Between Cellular and Viral Proteins. Journal of Molecular Evolution (2002) 54: 763

13 Replication Speed Thymidylate Synthase (gp 48) Conserved RHRX 7-8 SXR sequence corroborates ThyX activity 2. ThyX could regulate the speed of viral replication 3. 2 Parthasarathy Sampathkumar, et. al. Structure of the Mycobacterium tuberculosis Flavin Dependent Thymidylate Synthase (MtbThyX) at 2.0 A Resolution. Journal of Molecular Biology (2005) 352: Se bastien Graziani, et. al. Functional Analysis of FAD-dependent Thymidylate Synthase ThyX from Paramecium bursaria Chlorella Virus-1. Journal of Biological Chemistry (2004) 279:

14 Vertical Gene Transfer DNA Polymerase (gp 44) High conservation among phages Phamily Circle of DNA polymerase Low host-phage gene transfer Vertical gene transfer

15 Vertical Gene Transfer Thymidylate Synthase (gp 48) Each phage s ThyX is homologous to a unique pool of bacteria. Variable placement of ThyX in phages of different clusters.

16 Acknowledgements Special thanks to Dr. Elgin, Dr. Hafer, Dr. Shaffer, Dr. Berg, Ryan, Kelsey, Anthony, Matt, the Sarah Elgin lab, the Graham Hatfull lab, and the Washington University sequencing center.