1. The Discovery of Bacteriophage~
D’Herelle was a French-Canadian microbiologist, worked for the French producing vaccines and antibiotics during WWI
Twort worked for British military, made initial observtions of phage infections, but did not pursue

2. Water Source [bacteria] Jumna river, near Agra 105/ml
The bacteriocidal action of the waters of the Jumna and the Ganges on Vibrio Cholera.
Ernest Hanbury Hankin, 1896.
Water Source [bacteria]
Jumna river, near Agra 105/ml
Jumna, 5 km downstream <102/ml
From Agra
"L'action bactericide des eaux de la Jumna et du Gange sur le vibrion du cholera” Annales de l'Institut Pasteur 10: (1896)

3. The bacteriocidal action of the waters of the Jumna and the Ganges on Vibrio Cholera.
Ernest Hanbury Hankin, 1896.
Experiment: Mix filtered river water with cultures of Vibrio cholerae and count bacteria
# Vibrio Cholerae (1000s) after time:
Water 0 1hr 2hr 3hr 4hr 25hr 49hr
Filtered
Filt.+boiled
"L'action bactericide des eaux de la Jumna et du Gange sur le vibrion du cholera” Annales de l'Institut Pasteur 10: (1896)

4. Observed glassy, clear areas in colonies of Stapphylococcus
An Investigation into the Nature of the Ultramicroscopic Viruses. Lancet, ii, 1241 (1915)
Observed glassy, clear areas in colonies of Stapphylococcus
Could not be cultured independently
Could be serially transferred and propagated on new bacterial colonies, but not on dead bacteria
Passed through a porcelin filter
Destroyed by heat
Twort worked for British military, made initial observtions of phage infections, but did not pursue
Frederick Twort

5. The Bacteriophage: It Role in Immunity (1921)
Bacterial growth media innoculated with stool from patient with dysentery
Culture became cloudy overnignt, indicating growth of bacteria
Culture was filtered and filtrate added to pure cultures of Shiga bacteria (the causative agent of the dysentery)
One day, the Shiga culture showed no growth. At the same time, the patient had shown marked improvement.
Sterile filtrate of cleared culture could kill other cultures of Shiga
D’Herelle was a French-Canadian microbiologist, worked for the French producing vaccines and antibiotics during WWI
In 1917, made the following observations……..
Félix d'Herelle

6. The Bacteriophage: It Role in Immunity (1921)
Observed plaques on solid media, developed plaque assay, concept of titering
Deduced that active agent was a virus!
Showed that bacteriophage could be used to treat infections
D’Herelle was a French-Canadian microbiologist, worked for the French producing vaccines and antibiotics during WWI
Twort worked for British military, made initial observtions of phage infections, but did not pursue
Félix d'Herelle

7. The plaque assay Bacteria + top agar phage
Serial dilutions of phage gave rise to decreased numbers of plaques
a discrete entity (i.e. the virus) is responsible for the plaques
inferred that 1 plaque arose from a single virus. Thus, plaque forming units=viruses
1 plaque is composed of many pfu
the viruses can replicate!
Final acceptance of the existence of bacteriophage came with electron micrographs (Helmut Ruska, 1939)

8. A brief introduction to the phage world
Bacteriophages are viruses of bacteria 8

9. Phage life cycle

10. temperate phage form lysogens

11. A brief introduction to the phage world
Bacteriophages are viruses of bacteria
Bacteriophages represent the majority of life forms: Estimated number 1031
The phage population is extremely dynamic: infections/sec
Phages are specific to particular bacterial hosts
Phages are genetically highly diverse, but relatively few have been characterized
Phages are the largest reservoir of unexplored genetic information 11

12. Virion morphologies

13. Why mycobacteriophages?
Mycobacteriophages are viruses that infect mycobacterial hosts
Phages can facilitate understanding of host physiology
Phages have therapeutic potential
Some mycobacteria cause important human diseases
M. tuberculosis kills more people than any other single infectious agent
We study phages that infect M. smegmatis, a useful surrogate bacterium that is not harmful to humans.
~10% of phage that infect M. smegmatis also infect M. tuberculosis

14. Mycobacteriophage Isolation
M. smegmatis is a fast-growing (~3hr doubling time), non-pathogen
Phage solated as plaque formers on lawns of M. smegmatis mc2155
Isolated from environmental samples such as soil, compost etc.
M. smegmatis may not be ‘preferred’ host
All phages isolated are dsDNA tailed phages
Have complete sequences for ~250 genomes
Safety-
- must not be immunosuppressed, talk to me if you have concerns
No food in lab
Wash hands, clean spills

15. Phage LRRHood
Kim Davis, in the redwood forest where she isolated LRRHood
(Little Red Riding Hood)
transition: Now I would like to tell you about several unique features of the LRRHood genome…….
6. LRRHood Genome Sequence
Double stranded circular genome of 154,349 bp.
 Member of group C1, most similar to Cali, which was isolated in nearby Santa Clara California by one of Graham Hatfull’s phage hunters.
 Glimmer and Genemark were used to identify 221 potential protein coding genes
 Used trnascan and Aragorn ( to identify
 32 predicted trna genes
 1 predicted tmrna.
Of these predicted structural RNAs, the tmrna and 3 of the trnas were only found by Aragorn

16. Bio121L Course Overview:
Isolate your own phage(s)
Purify phage
Isolate phage DNA and characterize by restriction enzyme digestions (and PCR if time permits)
Visualize phage by electron microscopy
Term paper, ~6 pages on a topic relevant to phage biology
Special projects

17. Mycobacteriophage genomics
First mycobacteriophage sequence published in 1993
~250 completely sequenced genomes
Genomes range in size from kbp (kbp= 1000 basepairs)
Average genome size: ~72kbp
Average number of genes: 88/genome
High genetic diversity
High gene density
Genomes are genetically mosaic (meaning we can’t construct a simple “tree of life” for these viruses)
Most genes of unknown function

18. Complete bacteriophage genome sequence
What do we want to know about this sequence?
where are the genes?
what do they do?
how is this virus related to other viruses?
How do we study DNA sequences?
Bioinformatics- computational tools for analysis of biological sequence information (DNA/RNA/Protein)

20. Cluster A genome organizations
Lysis
Replication
Head
Tail
Integration
Repressor
Packaging

21. LRRHood Genome Sequence
Double stranded circular genome of 154,349 bp.
Member of group C1, highly similar to Cali.
Identified 221 potential protein coding genes
Encodes a transcription repressor protein that is not found in any other group C phage
Also found:
32 tRNA genes
1 tmRNA
1 tRNA
Kim Davis, in the redwood forest where she isolated LRRHood
(Little Red Riding Hood)
transition: Now I would like to tell you about several unique features of the LRRHood genome…….
6. LRRHood Genome Sequence
Double stranded circular genome of 154,349 bp.
 Member of group C1, most similar to Cali, which was isolated in nearby Santa Clara California by one of Graham Hatfull’s phage hunters.
 Glimmer and Genemark were used to identify 221 potential protein coding genes
 Used trnascan and Aragorn ( to identify
 32 predicted trna genes
 1 predicted tmrna.
Of these predicted structural RNAs, the tmrna and 3 of the trnas were only found by Aragorn

22. temperate phage form lysogens
Formation of stable lysogens depends on transcriptional repressor proteins that bind to and turn off transcription of almost all of the phage genes

23. temperate phage form lysogens
Formation of stable lysogens depends on transcriptional repressor proteins that bind to and turn off transcription of almost all of the phage genes
LRRHood does not form lysogens
The LRRHood genome does not have DNA binding sites for the repressor
Why does LRRHood have a repressor?

24. The UCSC Genome Browser
genes
conservation
homology with group C1 phages
. The UCSC Genome Browser
            To complement the bioinformatic tools provided to us by the HHMI, and taking advantage of local expertise in bioinformatics, we developed the UCSC Phage Genome Browser.
this figure shows 1st 30 kb of LRRHood
A) explain tracks
John Paul Donohue 24

25. The UCSC Genome Browser
genes
conservation
homology with group C1 phages
C) and insertions
point to the large insertion, and say that you will spend a few moments talking about this one insertion in particular
insertions
John Paul Donohue 25

26. LRRHood has acquired a transcriptional repressor via a novel sequence insertion
A)  phamerator generated this, shows 4 genes in the insert relative to cali 26

27. LRRHood has acquired a transcriptional repressor via a novel sequence insertion
B)  insertion is flanked by 29 bp direct repeat sequence that only occurs once in Cali and other grp C1 phages. suggests that insertion may have occurred by creation of staggered nicks followed by cut and paste of novel sequence
= 29 bp sequence flanking insertion in LRRHood and found at apparent insertion site in group C1 phages (not to scale).
Does this repeat explain the mechanism of the insertion? 27

28. LRRHood has acquired a transcriptional repressor via a novel sequence insertion
LRRHood gp44:
similar to transcriptional repressor protein found in grp A1, A2 phages and the group F1 phage fruitloop
169/170 amino acids are identical to Bxb1 gp69
gp69 DNA binding site is not present in LRRHood genome
does repressor provide a growth advantage to LRRHood in a mixed infection?
C) No sequenced phage has this entire sequence, suggesting we do not know where thus sequence was originally derived from.  However, I would like to tell you about one particularly interesting gene in this insertion
LRRHood gp44:
 member of Pham 54.  The other members of this pham are found in grp A1 and A2 phages and they encode a protein similar to transcriptional repressor protein.  The one A1/A2 phage that lacks this sequence is D29, which is highly lytic
 In pg44, 169/170 amino acids are identical to phage Bxb1 gp69, the single difference between these two proteins is far from the proteins putative DNA-binding domain.  Thus the proteins are likely to bind highly similar or identical sequences
 The binding sites recognized by gp69 have been determined, and a similar sequence motif is not present in LRRHood genome
This suggesets that LRRHood may not be use gp44 to regulate expression of its own genes.  Does it provide immunity to superinfection? 28

29. Supressor tRNA? One tRNA with a CUA anti-codon
Thus, it appears to recognize UAG stop codons
Homology to other bacterial tRNAs suggest it may charged with tryptophan
36 LRRHood genes end in UAG, and in several cases, readthrough of a UAG stop codon would join two annotated ORFs
7. Suppressor tRNA
One tRNA with a CUA anti-codon
 Thus, it appears to be a UAG suppressor tRNA
 Homology to other phage tRNAs suggest it is charged with tryptophan
36 LRRHood genes end in UAG, and in several cases, readthrough of a UAG stop codon would join two annotated ORFs
Thus, it may serve to regulate expression of one or more fusion proteins. 29

30. Integration of phage Ogopogo in M. smeg. Disrupts tRNALys

31. Integration of phage Ogopogo in M. smeg. Disrupts tRNALys

32. Do the insertions of Trouble or Ogopogo in lysogens preserve the function of groEL and tRNALys? If they do, do the altered genes have exactly the same function or altered functions?