The Discovery of Bacteriophage~1896-1920

"L'action bactericide des eaux de la Jumna et du Gange sur le vibrion du cholera” Annales de l'Institut Pasteur 10: 511-523 (1896)

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/mlFrom Agra

"L'action bactericide des eaux de la Jumna et du Gange sur le vibrion du cholera” Annales de l'Institut Pasteur 10: 511-523 (1896)

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 2.5 1.5 1 0.5 0 0 0

Filt.+boiled 5 4 6 10 6 10 36

Frederick Twort1877-1950

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

Félix d'Herelle1873-1949

•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

The Bacteriophage: It Role in Immunity (1921)

Félix d'Herelle1873-1949

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

The plaque assay

phage

Bacteria + top agar

•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)

A brief introduction to the phage world

• Bacteriophages are viruses of bacteria

Phage life cycle

temperate phage form lysogens

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: 1025 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

Virion morphologies

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

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

Kim Davis, in the redwood forest where she isolated LRRHood

(Little Red Riding Hood)

Phage LRRHood

Bio121L Course Overview:

1. Isolate your own phage(s)2. Purify phage3. Isolate phage DNA and characterize by

restriction enzyme digestions (and PCR if time permits)

4. Visualize phage by electron microscopy5. Term paper, ~6 pages on a topic relevant to

phage biology6. Special projects

Mycobacteriophage genomics

• First mycobacteriophage sequence published in

1993

• ~250 completely sequenced genomes

•Genomes range in size from 42-153kbp (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

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)

Cluster A genome organizations

Lysis

PackagingHead Tail Integration

ReplicationRepressor

Kim Davis, in the redwood forest where she isolated LRRHood

(Little Red Riding Hood)

• 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

LRRHood Genome Sequence

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

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?

John Paul Donohue

The UCSC Genome Browser

genes

conservation

homology with group C1 phages

John Paul Donohue

genes

conservation

homology with group C1 phages

insertions

The UCSC Genome Browser

LRRHood has acquired a transcriptional repressor via a novel sequence insertion

LRRHood has acquired a transcriptional repressor via a novel sequence insertion

= 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?

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?

• 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

Supressor tRNA?

Integration of phage Ogopogo in M. smeg. Disrupts tRNALys

Integration of phage Ogopogo in M. smeg. Disrupts tRNALys

• 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?