1. 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

2. 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

3. Bacterial Infection by a Lytic Phage
Akhil

4. 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

5. 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

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

7. 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:
Looks like a product for E coli 0157:H7 is partially approved or close…

8. 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

9. 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.

10. 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

11. 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,

12. 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

13. Plaque enrichment (continued)
Incubate 24 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

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

15. 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

16. 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

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

18. 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

19. 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

20. 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

21. 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!

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

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