Bacteriology at UW-Madison

The Microbial World

Lectures in Microbiology by Kenneth Todar PhD    University of Wisconsin-Madison    Department of Bacteriology

Bacteriophage

Bacteriophage T4

Introduction

Viruses are smaller and less complex than bacteria. As science became aware of the role of the viruses in human disease, the techniques of bacteriology were modified to accomodate the viruses and the discipline of virology grew up within bacteriology. Because of this, we will begin this unit on viruses with bacteriophages, the viruses that infect bacterial cells. Animal viruses will be dealt with separately. But the lessons learned from the replication events of the bacteriophages will be directly applied to understanding the replication of viruses such as Herpes and HIV.

Viruses are the cause of many diseases in humans ranging from AIDS and cancer to the common cold.  Microbiologists have developed vaccines for many viral diseases, but haven not been as successful in discovery of treatments for the diseases. It is the opposite in bacteriology, at least since the discovery of antibiotics. We have generally been able to treat bacterial disease, but besides the toxoid vaccines, vaccination against bacterial diseases has been hit-and-miss.

Biological Identity of Viruses

Viruses consist of nucleic acid (DNA or RNA) surrounded by a protein coat called a capsid. The capsid is made up of individual structural subunits called capsomeres. Some viruses have additional structural features, such as the envelope of animal viruses or the tail of bacteriophages.
 
 
 

Figure 1. The most common viral morphologies. Left to Right. A naked icosahedral virus (e.g. poliovirus), an enveloped icosahedral virus (e.g. herpes virus), a naked helical virus (e.g. tobacco mosaic virus) and an enveloped helical virus (e.g. influenza virus). Individual capsomeres are arranged to form a capsid which encloses the nucleic acid (DNA or RNA) of the virus. Many animal viruses also contain an envelope, which is partly derived from the host cell membtrane but which always contains unique viral proteins drawn here as "spikes".
 

Viruses are considered obligate intracellular parasites because they require a host cell in order to replicate. The host cell may be any form of eucaryote or procaryote.

Viruses are noncellular entities that are not considered alive by most microbiologists. They are very different from cells. Viruses lack membranes and cannot produce energy; they lack enzymes for metabolic functions; and they lack ribosomes for protein synthesis.
The general features of viruses are outlined in the table below.

Table 1. General Features of Viruses

2. characteristic shapes - spherical (complex), helical, rod or polyhedral, sometimes with tails or envelopes. Most common polyhedron is the icosahedron which as 20 triangular faces.

3. obligate intracellular parasites Viruses do not contain within their coats the machinery for replication.  For this they depend upon a host cell and this accounts for their existence as obligate intracellular parasites. Each virus can only infect certain species of cells. This refers to the virus host range.

4. no built-in metabolic machinery Viruses have no metabolic enzymes and cannot generate their own energy.

5. no ribosomes Viruses cannot synthesize their own proteins. For this they utilize host cell ribosomes during replication. Features 4 and 5 acccount for the obligate intracellular parasitism of viruses.

6. only one type of nucleic acid Viruses contain either DNA or RNA (never both) as their genetic material. The nucleic acid can be single-stranded or double stranded. 

7. do not grow in size Unlike cells, viruses do not grow in size and mass leading to a division process. Rather viruses grow by separate synthesis and assembly of their components resulting in production of a "crop" of mature viruses.

Viruses are classified on the basis of host range (see below), morphology (size, shape), type of nucleic acid (DNA,  RNA, single-stranded, double-stranded, linear, circular, segmented, etc.) and occurrence of auxilliary structures such as tails or envelopes.

Host range refers to the the type of cell in which the virus is able to replicate. In its broadest sense host range arranges viruses into four groups:  bacterial viruses (bacteriophage), animal viruses, insect viruses (bacculoviruses) and plant viruses. However, viruses in archaea, protista, yeast, molds and fungi have also been described. In a narrower sense, host range may be defined by specific species that are infected by the virus. Thus, each bacterial virus only infects certain species of bacteria; each animal virus only infects certain species of animals; and so on. In a more limited sense, when a virus infects a multicellular organism, it usually infects only a certain type of cell in the organism. Hence, the rhinoviruses which cause the common cold only infect cells of the upper respiratory tract, and the human immunodeficiency virus (HIV) only infects primarily a specific type of cell (CD4+ cells) of the human immune system.
 
 

Figure 2. Comparative size and shape of various groups of viruses representing diversity of form and host range. A. Smallpox virus  B. Orf virus  C. Rhabdovirus D. Paramyxovirus  E. Bacteriophage T2  F. Flexuous-tailed bacteriophage  G. Herpes virus  H. Adenovirus  I. Influenza virus  J. Filamentous flexuous virus K. Tobacco mosaic virus  L.  Polyoma/papilloma virus  M. Alflafa mosaic virus  N. poliovirus  O. Bacteriophage phiX174. Viruses have fundamentally three morphologies: 1. polygonal, the most common polygon being the icosahedron (E, F, G,  H, L, N);  2. helical, wherein the capsomeres assemble as a helix enclosing the nucleic acid) (D, I, J, K, M; B is controversial); 3.  complex, wherein the proteins are laid down in patches or layers (A).  Some animal viruses have envelopes which enclose their nucleocapsid (D, G, I). The envelopes are embedded with viral proteins that secre their entry and exit in cells. Only bacteriophages have tails which  are used for adsorption and penetration of their host cell.


Figure 3. Gallery of electron micrographs of viruses illustrating diversity in form and structure. Clockwise: Human immunodeficiencyvirus (HIV); Aeromonas virus 31, Influenza virus, Orf virus, Herpes simplex virus (HSV), Smallopx virus.
 

The Bacteriophages

Viruses that attack bacteria were observed by Twort and d'Herelle in 1915 and 1917. They observed that broth cultures of certain intestinal bacteria could be dissolved by addition of a bacteria-free filtrate obtained from sewage. The lysis of the bacterial cells was said to be brought about by a virus which meant a "filterable poison" ("virus" is Latin for "poison").

Probably every known bacterium is subject to infection by one or more viruses or "bacteriophages" as they are known ("phage" for short, from Gr. "phagein" meaning "to eat" or "to nibble"). Most research has been done on the phages that attack E. coli, especially the T-phages and phage lambda.

Like most viruses, bacteriophages typically carry only the genetic information needed for replication of their nucleic acid and synthesis of their protein coats. When phages infect their host cell, the order of business is to replicate their nucleic acid and to produce the protective protein coat. But they cannot do this alone. They require precursors, energy generation and ribosomes supplied by their bacterial host cell.

Bacterial cells can undergo one of two types of infections by viruses termed lytic infections and lysogenic (temperate) infections. In E. coli, lytic infections are caused by a group seven phages known as the T-phages, while lysogenic infections are caused by the phage lambda.

Lytic Infections

The T-phages, T1 through T7,  are referred to as lytic phages because they always bring about the lysis and death of their host cell, the bacterium E. coli. T-phages contain double-stranded DNA as their genetic material. In addition to their protein coat or capsid (also referred to as the "head"), T-phages also possess a tail and some related structures. A diagram and electron micrograph of bacteriophage T4 is shown below. The tail includes a core, a tail sheath, base plate, tail pins, and tail fibers, all of which are composed of different proteins. The tail and related structures of bacteriophages are generally involved in attachment of the phage and securing the entry of the viral nucleic acid into the host cell.

Figure 4. Left. Electron Micrograph of bacteriophage T4. Right. Model of phage T4. The phage possesses a genome of linear ds DNA contained within an icosahedral head. The tail consists of a hollow core through which the DNA is injected into the host cell. The tail fibers are involved with recognition of specific viral "receptors" on the bacterial cell surface.

Before viral infection, the cell is involved in replication of its own DNA and transcription and translation of its own genetic information to carry out biosynthesis, growth and cell division. After infection, the viral DNA takes over the machinery of the host cell and uses it to produce the nucleic acids and proteins needed for production of new virus particles. Viral DNA replaces the host cell DNA as a template for both replication (to produce more viral DNA) and transcription (to produce viral mRNA). Viral mRNAs are then translated, using host cell ribosomes, tRNAs and amino acids, into viral proteins such as the coat or tail proteins. The process of DNA replication, synthesis of proteins, and viral assembly is a carefully coordinated and timed event. The overall process of lytic infection is diagrammed in the figure below. Discussion of the specific steps follows.
 

Figure 4. The lytic cycle of a bacterial virus, e.g. bacteriophage T4.

The first step in the replication of the phage in its host cell is called adsorption. The phage particle undergoes a chance collision at a chemically complementary site on the bacterial surface, then adheres to that site by means of its tail fibers.

Following adsorption, the phage injects its DNA into the bacterial cell. The tail sheath contracts and the core is driven through the wall to the membrane. This process is called penetration and it may be both mechanical and enzymatic. Phage T4 packages a bit of lysozyme in the base of its tail from a previous infection and then uses the lysozyme to degrade a portion of the bacterial cell wall for insertion of the tail core. The DNA is injected into the periplasm of the bacterium, and generally it is not known how the DNA penetrates the membrane. The adsorption and penetration processes are illustrated below.

Figure 5. Adsorption, penetration and injection of bacteriophage T4 DNA into an E. coli cell. T4 attaches to an outer membrane porin protein, ompC.
 
Immediately after injection of the viral DNA there is a process initiated called synthesis of early proteins. This refers to the transcription and translation of a section of the phage DNA to make a set of proteins that are needed to replicate the phage DNA. Among the early proteins produced are a repair enzyme to repair the hole in the bacterial cell wall, a DNAase enzyme that degrades the host DNA into precursors of phage DNA, and a virus specific DNA polymerase that will copy and replicate phage DNA. During this period the cell's energy-generating and protein-synthesizing abilities are maintained, but they have been subverted by the virus. The result is the synthesis of several copies of the phage DNA.

The next step is the synthesis of late proteins. Each of the several replicated copies of the phage DNA can now be used for transcription and translation of a second set of proteins called the late proteins. The late proteins are mainly structural proteins that make up the capsomeres and the various components of the tail assembly. Lysozyme is also a late protein that will be packaged in the tail of the phage and be used to escape from the host cell during the last step of the replication process.

Having replicated all of their parts, there follows an assembly process. The proteins that make up the capsomeres assemble themselves into the heads and "reel in" a copy of the phage DNA. The tail and accessory structures assemble and incorporate a bit of lysozyme in the tail plate. The viruses arrange their escape from the host cell during the assembly process.

While the viruses are assembling, lysozyme is being produced as a late viral protein. Part of this lysozome is used to escape from the host cell by lysing the cell wall peptiodglycan from the inside. This accomplishes the lysis of the host cell and the release of the mature viruses, which spread to nearby cells, infect them, and complete the cycle. The life cycle of a T-phage takes about 25-35 minutes to complete. Because the host cells are ultimately killed by lysis, this type of viral infection is referred to a lytic infection.

Lysogenic Infections

Figure 6. Bacteriophage Lambda, the lysogenic phage that infects E. coli. Bock laboratories. University of Wisconsin-Madison.



Lysogenic or temperate infection rarely results in lysis of the bacterial host cell. Lysogenic viruses, such as lambda which infects E. coli, have a different strategy than lytic viruses for their replication.  After penetration, the virus DNA integrates into the  bacterial chromosome and it becomes replicated every time the cell duplicates its chromosomal DNA during normal cell division. The life cycle of a lysogenic bacteriophage is illustrated below.



Figure 7. The lysogenic cycle of a temperate bacteriophage such as lambda.

Temperate viruses usually do not kill the host bacterial cells they infect. Their chromosome becomes integrated into a specific section of the host cell chromosome. Such phage DNA is called prophage and the host bacteria are said to be lysogenized. In the prophage state all the phage genes except one are repressed. None of the usual early proteins or structural proteins are formed.

The phage gene that is expressed is an important one because it codes for the synthesis of a repressor molecule that prevents the synthesis of phage enzymes and proteins required for the lytic cycle. If the synthesis of the repressor molecule stops or if the repressor becomes inactivated, an enzyme encoded by the prophage is synthesized which excises the viral DNA from the bacterial chromosome. This excised DNA (the phage genome) can now behave like a lytic virus, that is to produce new viral particles and eventually lyse the host cell (see diagram above). This spontaneous derepression is a rare event occurring about one in 10,000 divisions of a lysogenic bacterium., but it assures that new phage are formed which can proceed to infect other cells.

Usually it is difficult to recognize lysogenic bacteria because lysogenic and nonlysogenic cells appear identical. But in a few situations, the prophage supplies genetic information such that the lysogenic bacteria exhibit a new characteristic (new phenotype), not displayed by the nonlysogenic cell, a phenomenon called lysogenic conversion. Lysogenic conversion has some interesting manifestations in pathogenic bacteria that only exert certain determinants of virulence when they are in a lysogenized state. Hence, Corynebacterium diphtheriae can only produce the toxin responsible for the disease if it carries a temperate virus called phage beta. Only lysogenized streptococci  produce the erythrogenic toxin (pyrogenic exotoxin) which causes the skin rash of scarlet fever; and some  botulinum toxins are synthesized only by lysogenized strains of C. botulinum.


Figure 8. Corynebacterium diphtheriae only produces diphtheria toxin when lysogenized by beta phage. C.  diphtheriae strains that lack the prophage do not produce diphtheria toxin and do not cause the disease diphtheria. Surprisingly, the genetic information for production of the toxin is found  to be on the phage chromosome, rather than the bacterial chromosome.

A similar phenomenon to lysogenic conversion exists in the relationship between an animal tumor virus and its host cell. In both instances, viral DNA is incorporated into the host cell genome, and there is a coincidental change in the phenotype of the cell. Some human cancers may be caused by viruses which establish a state in human cells analogous to lysogeny in bacteria.

Phage Therapy

Phage therapy is the therapeutic use of lytic bacteriophages to treat pathogenic bacterial infections. Phage therapy is an alternative to antibiotics being developed for clinical use by research groups in Eastern Europe and the U.S. After having been extensively used and developed mainly in former Soviet Union countries for about 90 years, phage therapies for a variety of bacterial and poly microbial infections are now becoming available on an experimental basis in other countries, including the U.S. The principles of phage therapy have potential applications not only in human medicine, but also in dentistry, veterinary science, food science and agriculture.

An important benefit of phage therapy is derived from the observation that bacteriophages are much more specific than most antibiotics that are in clinical use. Theoretically, phage therapy is harmless to the eucaryotic host undergoing therapy, and it should not affect the beneficial normal flora of the host. Phage therapy also has few, if any, side effects, as opposed to drugs, and does not stress the liver. Since phages are self-replicating in their target bacterial cell, a single, small dose is theoretically efficacious. On the other hand, this specificity may also be disadvantageous because a specific phage will only kill a bacterium if it is a match to the specific subspecies. Thus, phage mixtures may be applied to improve the chances of success, or clinical samples can be taken and an appropriate phage identified and grown.

Phages are currently being used therapeutically to treat bacterial infections that do not respond to conventional antibiotics, particularly in the country of Georgia. They are reported to be especially successful where bacteria have constructed a biofilm composed of a polysaccharide matrix that antibiotics cannot penetrate.