NCUR 2014: ISOLATION AND CHARACTERIZATION OF NOVEL BACILLUS BACTERIOPHAGES Virginia Commonwealth University Bioinformatics 252: Phage Discovery II Akhil Garg and Jacob Jaminet Instructor: Dr. Allison Johnson Saturday, April 5, 2014

What the ph is a phage? Bacteriophage = bacteria eater A virus that infects bacteria One of the most abundant “organisms” 1031 predicted phages Found everywhere Jacob Highly specific – mentioned tail and receptor oceanworld.tamu.edu www2.iitd.pan.wroc

Bacterial Infection by a Lytic Phage Akhil

Phage Infecting bacterial in the lab Bacterial lawn Virus plated for isolated plaques A “Zone of Death” Contains billions of infectious and identical phage particles Jacob

Host: Bacillus ACT “ACT” group B. anthracis B. cereus B. thuringiensis Akhil Define Host Range Genotypically related, phenotypically different Host range similar due to genomtypically similar Some B. cereus strains cause food poisoning Btk used as a pesticide and is sprayed on trees along east coast of USA to control gypsy moth. microbewiki.kenyon.edu

Biological relevance: Phage influence pathogenicity of B. anthracis Akhil Can tell B. anthracis to be vegetative (non pathogenic) or spore (pathogenic!) 2009 Schuch PLoSONE

Biological relevance: Phage therapy Phage therapeutics were in active development in early 1900s, but the ‘west’ moved away from them with discovery of antibiotics. Useful for Persistent infections (eg. Lung infections in cystic fibrosis patients) Antibiotic resistant bacteria (eg. MRSA) Food safety (eg. Listeria contamination in deli meat) Jacob There are two FDA approved phage products for food safety: http://www.intralytix.com/Intral_products.htm Looks like a product for E coli 0157:H7 is partially approved or close…

Host: B. cereus 14579 ATCC strain (continued) Bacteria donated from Dr. Chris Ehrhardt in VCU Dept. of Forensic Science Anthrax used as a bioterror weapon, phages might be used as a detection tool. Phages can influence whether the bacteria is in its vegetative state or its pathogenic spore state Akhil Why he is interested in isolating phages Studies B. anthracis surface carbohydrates and lipids to determine Source Media used Strain Lab it is from

Technique Dirt Sample Enrichment culture Phage Purification Lysate DNA Akhil Use what you wish here. You might want to take out “stick streak”- irrelevant for this presentation. Might combine Plaque Enrichment and Phage Titer Assay into “Phage Purification” and talk more generally about increasing purity and concentration of phage before collecting lysate and DNA.

VCU summer Pilot Summer 2013 6 soil samples 4 successful spot tests B. cereus T strain (FBI strain) B. cereus 14579 ~67% success rate Jacob TA’s made sure it was possible to isolate phages from soil

Unforseen difficulties Fall 2013 21 researchers 57 direct plating and enrichment plating attempts 4 successful enrichment spot tests ~7% success rate Lee et al, 20111 40 soil samples collected Only one sample presented two plaque types ~5% success rate Jacob More difficult than summer isolation 1Lee, W. J., Billington, J. A., Hudson, J. A., & Heinemann, J. A. (2010). Isolation and characterization of phages infecting Bacillus cereus. Applied Microbiology, 52, 456-464.

Plaque Enrichment Objective: Increase concentration of phage while decreasing concentration of bacteria Amplify our signal Bacteria transferred from “tip-dip” obscured our phage so filtration was necessary Jacob We set up 24 hour plaque enrichments in B. cereus from dipping a p 200

Plaque enrichment (continued) Incubate 24 hour @ 30° C Filter 100 10-1 10-2 10-3 10-4 3. Dilute 2. B. cereus 14579 culture aliquot Jacob 4. Infect and plate

Pictures taken by Teaching Assistants Plaque morphology B. cereus B. thuringiensis Kurstaki Princess Philip Akhil Nigalana Larbie Pictures taken by Teaching Assistants

Phage Phound! Myoviridae Head diameter: ~150nm Tail length: ~120nm Very little DNA 15 plate infection Jacob Contractile tail 30 degree celsius Imaged by Dr. Bertino via TEM at VCU Nanocharacterization Facility

NotTheCreek Bella SageFayge Vinny Nigalana By genome, these are ‘the same phage’ TEM of close homolog from phagesdb TEM unavailable Akhil Same morphology based on sequencing Contractile tails can be visualized in their extended or contracted state NotTheCreek and Bella “Uncomfortably” similar?- 8 nucleotides different Myoviridae morphology includes phages with contractile tail.  We believe TEM shows tails in both extended (after DNA injected) and contracted (before injection) conformations. Eyuki = myoviridae Terra = myoviridae Bastille = myoviridae BPS13 = myoviridae

Annotation of bacteriophage Nigalana genes Software predicts protein-coding genes Students curate predictions using bioinformatics Share by submitting annotation to GenBank Jacob

Genome comparison Phage Genome Length Terminal repeat %GC # predicted genes # tRNAs Nigalana 163041 2867 38.8 305 NotTheCreek 159336 2585 38.2 296 SageFayge 159547 2812 38.1 299 Vinny 162865 2358 36.4 7 Akhil

Dot plot comparison Nigalana NotTheCreek SageFayge Vinny Nigalana Jacob How it works What it shows Dotplot- carefully trace diagonal and walk across one row. Annotation of Nigalana- phamerator map SageFayge Vinny

Current Research projects Comparative genomics project to determine the relationship between host range and tail fibers Evolutionary comparison of gene families Sigma factors, endolysin, structural genes, etc. Jacob Tail proteins responsible for identifying receptor proteins ebi.ac.uk

semester Projects (continued) Determining a better RNA binding sequence for annotation tool Shine-Dalgarno Identify transcription regions promoters and terminators Annotate additional genomes Akhil RNA binding sequence = ribosome “Optimize” the SD in our annotation tool Exciting stuff!

Acknowledgements Dr. Allison Johnson Dr. Chris Ehrhardt Our Teaching Assistants Harry Caufield Michael Kiflezghi Sindhu Karnam Mariam Sankoh Our Classmates! Jacob + Akhil

Questions? ? Welcome to Alyssa Pagel CAPTURE AND DETECTION OF BACILLUS ANTHRACIS SPORES USING APTAMER BASED SURFACE ENHANCED RAMAN SPECTROSCOPY