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Chapter 21

The Best for Last: Bacteriophages

Bacteriophages attached to a bacterium.

21.1 Bacteriophage Research History

Bacteriophagesviruses that infect bacteria Discovered after recognition of bacterial hosts in the 1880s (the golden age of microbiology) Frederick W. Twort observed glassy transformation
Used a Chamberland filter to discover the filterable bacteriophages that lysed cultures of micrococci His work was not acknowledged for 5 years

Felix dHerelle (1873-1849)

Credited as the solediscoverer of bacteriophages 1917 report by dHerelle describes the lysis of dysentery-causing bacteria grown in liquid medium Tworts paper was not cited in dHerelles publication
SPL/Photo Researchers, Inc. .

Figure 21.01: Felix d'Herelle (1873-1949)

Felix dHerelles Research

2 directions
Determining the biological nature of bacteriophages Exploring the use of bacteriophages as therapy to treat bacterial infections in a preantibiotic era

dHerelle and Bacteriophage Therapy 1919 field trials to control an epidemic of chicken typhoid () caused by the bacterium Salmonella gallinarum
Inoculated chickens either orally or by injection with bacteriophages Flocks treated with bacteriophages suffered fewer deaths and shorter epidemics that did not reoccur

Positive results motivated dHerelle to conduct human trials in the 1920s

dHerelles Human Trials To prove that the bacteriophage preparations were safe: dHerelle injected himself Family members Coworkers No harmful effects observed

dHerelles Bacteriophage Experiments, cont.

1925 report by dHerelle regarding experiments at the League of Nations quarantine station in Alexandria, Egypt
Injected 4 patients suffering from laboratory confirmed bubonic () plague with bacteriophages into the bubos present in their lymph nodes

All 4 patients recovered rapidly After this success, dHerrelle traveled the world continuing bacteriophage therapy as a means of controlling cholera outbreaks

dHerelles Career Accepted an appointment as a professor of protobiology at Yale University in 1928 Played a role in establishing a bacteriophage institute in Tbilisi, Soviet Georgia in 1934
This institute exists today as the Georgia Eliava Institute of Bacteriophage, Microbiology and Virology

Bacteriophage Therapy Abandoned in the 1930s

Published reports were not consistent The therapy appeared to be hit or miss The main reason for the abandonment of bacteriophage therapy was the development of antibiotics Today the Western world has a renewed interest in bacteriophage therapy to combat antibiotic-resistant strains of pathogenic bacteria

The History of the Georgia Eliava Institute of Bacteriophage, Microbiology and Virology
Founded in 1923 by Professor Giorgyi Eliava Felix dHerelle visited Eliava at the Institute during 1934-1935 via invites by Josef Stalin
Stalin was interested in bacteriophage therapy for military use

Eliava was executed in 1937 dHerelle did not return to the Institute after his death

Activities of the Georgia Eliava Institute

Continued after Eliavas death Research focus centered around bacteriological and bacteriophage research The Institute change names in 1952 and was reorganized in 1988 The Institute manufactured bacteriophage sprays, salves , ointments and pills The Institute was damaged during the Georgian civil war. Thousands of bacteriophage samples were lost The Institute was revitalized by energetic entrepreneurs after a 1997 BBC Broadcast entitled The Virus That Cures

The American Phage Group

Formed in the 1940s Consisted of scientists from universities throughout the U.S. that were studying bacteria and bacteriophages
Max Delbruck Salvador Luria Alfred Hershey

The Phage Group spent summers doing research experiments at Cold Spring Harbor (Long Island, NY)

The American Phage Group, cont. Optimized experiments to study the biology of bacteriophages such as: One-step growth experiments Plaque assays Focused on a selected group of authorized bacteriophages such as the Type (T) bacteriophages

Bacteriophage Lysis and Plaque Assays

Figure 21.2a: Bacterial cells growing in log phase (left) and being lysed by bacteriophages (right).
David B. Fankhauser, PhD, University of Cincinnati Clermont College

Figure 21.2b: Bacteriophage plaque assays from one-step growth experiments.

21.2 Bacteriophage Ecology Scientific community estimates that aquatic communities contain 4-6 X 1030 bacteria and 1 X 1031 bacteriophages Bacteriophages recycle bacterial carbon in the marine environment Marine microbial ecology is a rapidly developing field

21.3 The Biology of Bacteriophages Composition and Structure Over 5,100 bacteriophages have been analyzed using the transmission electron microscope ICTV recognizes
1 order 13 families 31 genera

Bacteriophage Structure 4 basic shapes or symmetries

Binary (head and tail structure) Icosahedral (also called cubic) Helical (filamentous) Pleomorphic ()

The majority of bacteriophages contain a head and tail structure

Representatives of the 13 Bacteriophage Families Grouped by their types of nucleic acid genomes.

Figure 21-3
Adapted from H. -W. Ackermann, Arch. Virol. 146 (2001): 843-857.

Electron Micrographs of Tailed Bacteriophages

Figure 21-4

Enterobacter virus P2

Bacillus licheniformis virus

Bacteriophage 3/K26

Courtesy of Maria Schnos and used with permission of Ross Inman, Department of Molecular Virology, Bock Laboratories, University of Wisconsin-Madisonx

Photos courtesy of ICTVdB Picture Gallery. Used with permission from Hans-W. Ackermann, Department of Medical Biology, Laval University.

Bacteriophage Structure and Genomes

3 families of bacteriophages are enveloped 96% of all bacteriophages contain dsDNA genomes The remaining 4% contain ssDNA, ssRNA or dsRNA genomes Genomes may code for as few as 3-5 genes to as many as 100 The genomes of bacteriophages contain unusual or modified bases that protect them from degradation by host nucleases during phage infection Most famous group of bacteriophages T-Type

Only Two Forms of DNA Genomes in Prokaryotes

Genome Linear d.s. Circular s.s. 25~120 (x106 daltons) 1.7~2.7 (x106 daltons) Virion Complex morphology Simple icosahedral or helical capsids



21.4 Overview of Bacteriophage Infection

Bacteriophages Possess Alternative Lifestyles: Lytic vs Temperate Phages Bacteriophages adsorb to the surface receptor molecules of bacteria during the first step of infection Receptors may be: Pili Proteins Oligosaccharides Lipopolysaccharides (LPS)

Adsorption and Penetration

T4 bacteriophages anchors its tail fibers in the LPS layer of its bacterial host This adsorption step causes a conformational change, resulting in contraction of the tail sheath and penetration of the cell membrane and the bacteriophage The bacteriophage DNA genome is subsequently injected into the host cell through the tail tube

Bacteriophage T4 Adsorption and Penetration

Figure 21.5 Bacteriophage T4 penetrating through the E. coli K-12 Omp C outer membrane protein receptor.

Other Bacteriophages
May bind to other receptors Bacteriophages that do not have tails penetrate the host by producing polysaccharide-degrading enzymes that digest components of the bacterial envelope or cell wall

Resistance to Bacteriophage Infection

Bacteria become resistant to phage infection when their host cell receptors are altered by mutation There is interest in engineering new receptorrecognition elements into the tail fibers of wellcharacterized bacteriophages so they can infect genetically distant hosts

Genome Penetration
Not an injection process Bacteriophages may package lysozyme in the base of its tail and uses the enzyme to degrade a portion of the peptidoglycan of the bacterial cell wall The DNA is drawn into the cell by a process that is not well understood for most bacteriophages After the DNA enters the cell, it circularizes rapidly by sticky ends or termini or the linear ends are modified and protected from bacterial nucleases

Transcription and Translation are Coupled

Bacterial host RNA polymerase recognizes the viral DNA promoters and begins transcription of early genes Translation of the early genes is coupled with transcription Late genes are transcribed and translated

Temporal Gene Expression

Class I: 0-6 min, transcribed by E. coli RNA polymerase. Class II: 6-12 min, involving DNA synthesis and nuclease. Class III: 8-20 min, for viral structural proteins.

Transcribed by T7 RNA polymerase

Assembly and Release Process Lytic Infection

After all bacteriophage parts are produced, new bacteriophages are assembled A copy of the genomic DNA is reeled into a preassembled icosahedral head A few molecules of lysozyme are packaged into the tail plate Phage lysin, endolysins, muramidases or virolysins hydrolyze bonds in the murein or peptidoglycan of the cell wall, allowing the viruses to escape or be released Holin is used to create pores in the inner membrane of the host, allowing the lysin or other enzymes to facilitate bacteriophage release

Lytic Infection

Figure 21.06: E. coli bacteriophage T4 lytic infection cycle.

T7 bacteriophage DNA

Lysogenic (Temperate) Infections

These bacteriophages infect their hosts but do not kill them. Instead their genome becomes integrated into a specific region of the host chromosome The integrated bacteriophage genome is called a prophage The viral DNA replicates every time the cell copies its chromosomal DNA during cell division

Lysogenic (Temperate) Infections, cont

Some temperate phages encode transposase which allows the bacteriophage to insert randomly into the chromosome Other bacteriophages integrate into site specific locations within the chromosome All bacteriophage gene expression is repressed by a repressor protein

Infection Cycle of a Temperate Bacteriophage

Figure 21.07: Infection cycle of a temperate bacteriophage.

Derepression or Induction If the repressor loses function (becomes inactivated), viral DNA is excised from the bacterial chromosome and the excised DNA acts like a lytic virus Spontaneous derepression or induction happens about 1 in every 10,000 cell divisions Temperate bacteriophages can carry host genes from one bacterial cell to another in a process called transduction

Icosahedral head (55 nm in diameter) Tail 15x135 nm long Linear double-stranded DNA About 50 kb with 12 unpaired bases at both ends called cohesive ends. Biological active form is circular rather than linear. Lytic and lysogenic life cycle. Rolling circle replication mechanism.

Lytic cycle and lysogenic cycle

Regulatory elements that control lysogenic and lytic development

Repression of the lytic genes in a lysogenic cells CI: OR1 > OR2 > OR3


1. The CI gene of phage encodes a repressor. 2. CI: 27 kD and form dimmer. 3. Binds to OL (for transcription in a left direction) 4. Binds to OR (for transcription in a right direction)

Autogenous regulation of phage repressor synthesis

1. CIOL1,OR1 (1>2>3) 2. OR1PR, OL1PL, OR3 PRM 3. PR:cro 4. PL :N 5. CIbind OL1,L2,R1, R2, 6. RNA polymerase bind PRPL (cro, N)

Autogenous regulation of phage repressor synthesis

7. CI Bind OR2PRM , CI 8. PRM: Repressor Maintenance 9. Repressor,bind OR1,R2 10. Repressor,bind OR1,R2,R3 11. Repressor bind OR3 Block PRM (self regulation) 12. OR2, CI repressor 13. U.V.activate protease CI repressor

Lytic Cycle (45 mins)

1. Immediately early genes:

Cro: control of repressor and other things N: antiterminator, prevent termination at TL1 and TR1.
2. Delayed-early genes:

O and P proteins: for circular DNA replication. Q: 2nd antiterminator, prevent termination at TR4.
3. Late genes:

Block early genes, rolling-circle replication.

Genes and recognition sites involved in the lytic regulatory cascade

Express delayed early genes

Express late genes

1. Cro, N, Q: regulatory proteins required for lytic development. 2. ORPR , OLPL: leftward and rightward transcription. 3. OR1, OR2, OR3: three repressor binding sites. 4. nutL, nutR: N protein binding site prevent transcription termination at tL1 and tR1.

Functions of cro at early and late stages of lytic cycle

CI: OR1 > OR2 > OR3 Cro: OR3 > OR2 > OR1

N protein functions as an antiterminator at tR1 and tL1 N: antiterminator

N, RNA polymerasetR1

1. N, RNA polymerase readthrough stop site (tR1) 2. nut: N utilization 3. Nus: N utilization substance 4. RNA polymeraseN, NusB/S10, NusA,pass tR1,

lysogenic or lytic cycle?

1. [cro] [CI] OR, OL 2. CIICIII 3. PRE: promoter for repressor establishment,CI cro antisense mRNA

lysogenic or lytic cycle?


Poor growth condition CIIICII lysogeny Good growth condition CIII (less active)CII lytic

Regulatory elements that control lysogenic and lytic development

Phage rolling circle replication

21.5 Bacteriophages Create Pathogenic Bacteria in Nature

Lysogenic conversiona prophage may carry genes that alter the phenotype of a lysogenic bacterium.
e.g. Corynebacterium diphtheriae ( ) produces a toxin responsible for diphtheria only if it carries the temperate bacteriophage called phage. The tox gene is located near one end of the phage genome
1883 Klebs diphtheriae1884Loeffer

Other Examples of Lysogenic Conversion

If Streptococcus pyogenes ( contains a temperate bacteriophage T2, the bacterium changes to a pyrogenic or erythrogenic exotoxin producing bacterium This exotoxin causes the rash of scarlet fever ()
Figure 21.08: This child has the Scarlet Fever rash caused by a lysogenic Streptococcus pyogenes bacterium.


() ()



21.6 Control of Bacteriophages in Industrial Fermentations

Bacteria are used in a variety of food fermentation processes:

Yogurt Cheese Sauerkraut Soy sauce

Pharmaceutical and biotechnology industries use bacteria on a large scale to produce products such as:
Alcohols Vitamins Amino acids Enzymes Hormones Biopolymers Antibiotics Other therapeutics

Bacteriophages are a Threat to Fermentation and Pharmaceutical Industries

Attacks by bacteriophages can results in considerable economic losses About 1-10% of dairy product fermentation batches are lost to bacteriophage infection
Often happens because the raw starting materials are contaminated with undetectable numbers of bacteriophages

Laboratory staff are trained to expect and respond to bacteriophage outbreaks

Bacteriophages are ubiquitous Bacteriophage seasons are January to March and October to November

Bacteriophages are ubiquitous If a bacteriophage were the size of a cockroach- these cockroaches would cover the surface of the earth in a layer that is 31,071 miles or 50,000 km deep!
- Bacteriophage researcher Roger Hendrix

Microbe Fermentation Vessel Used to Make Enzymes for Medical and Industrial Use

Courtesy of Shawn Walshaw-Wertz, Cherokee Pharmaceuticals LLC

Figure 21-9

21.7 Biofilms and Bacteriophages

Bacteria form complex communities called biofilms on solid wet surfaces in natures such as
River rocks Walls of limestone caves Pipes Industrial equipment Hulls of ships Teeth Lining of the colon Sutures Lungs of Cystic Fibrosis Patients Medical devices such as catheters (), heart valves Surrounding tissues of the heart Orthopedic devices Facial implants

Biofilms, cont.
Biofilms may consist of a single bacterial species to hundreds or thousands of bacterial species The bacteria produce extracellular polysaccharide polymers that surround and encase the microbes, facilitating their adhesion to surfaces Biofilms can cause environmental problems

Biofilms Can Cause Chronic Bacterial Infections in Humans

Biofilms are a challenge to medical care Bacteria commonly isolated from medical devices:
Enterococcus faecalis S. aureus S. epidermidis Streptococcus viridans E. coli Klebsiella pneumoniae Proteus mirabilis Pseudomonas aeruginosa

Biofilms Can Cause Chronic Bacterial Infections in Humans, cont.

These bacteria originate from the skin of the patient or the healthcare workers, entry ports of catheters etc. Preventative strategies to treat biofilms on medical devices:
Application of silver-impregnated catheters Coating devices with antibiotics Disinfection at the surface Adding inline filters into the ports of catheters

Bacteriophages studies to pretreat catheter surfaces to control biofilms are promising

EM of Staphylococcus aureus Bacteria Found on a Patients Catheter

Figure 21.10: EM of Staphylococcus aureus bacteria found on the surface of a patient's catheter. Magnified 2363X.

21.8 FDA-Approved Listeria-Specific Bacteriophage Preparation on Ready-to-Eat Meats Listeriosis () is a foodborne illness caused by the bacterium Listeria monocytogenes Listeriosis occurs in pregnant women (can cause a miscarriage, stillbirth or premature delivery), newborns and people with weakened immune systems 2006FDA approved LISTEX P100 which is a cocktail of 6 bacteriophages in the form of an application spray

LISTEX P100 will be used in clean rooms of cheese, meat, and poultry processing plants to inhibit the growth of Listeria on products such as lunch meat and hot dogs LISTEX P100 will not be declared as an ingredient on the label of a treated product

Virus File 21-1


Bacteriophage Therapy Makes a Comeback

3 Georgia woodsman suffered from burns caused by Soviet-era makeshift heaters packed with radiation (Strontium 90) Their burns became infected with Staphylococcus aureus Antibiotics failed to prevent the bacteria from invading deeply into the wounds

Bacteriophage Therapy Makes a Comeback, cont. The men were rushed to a hospital in Tbilisi where doctors placed bacteriophage patches on the wounds The infections cleared in a few weeks The men were stable enough to undergo skin grafting abroad

Figure 21.VF01: These wounds were treated with the bacteriophage powder PhagoBioDerm.

Bacteriophage Therapy Hurdles Being Addressed Contamination of bacteriophage stocks with bacterial toxins and debris that causes side effects in patients Narrow host range of bacteriophages The elimination of bacteriophages by the hosts immune system (antibodies) Bacterial host resistance to phage infection Lytic bacteriophages are ideal for therapy
50% of all bacteriophages are temperate