The main stumbling block in early studies of lysogeny was its apparent lack of reproducibility. It was never very clear when a culture of a bacterial lysogen would lyse nor what factors governed the process. André Lwoff -- at the Pasteur Institute -- found the answer one day when he was studying lysogeny in Bacillus megaterium:

Until the day came when André exposed a culture for several seconds to a UV lamp which I [Monod] was using for mutagenizing coli bacilli. The culture lysed immediately. The culture lysed a half-hour later. The experiment was repeated immediately. The same result. Then the next day, the following day, and the next week. No more erratic results. The phenomenon was reproducible with the regularity of a metronome. André had discovered induction, exorcised the spectre of statistics, and at the same time proved beyond doubt that all the cells of a clone carried the latent virus. The news caused a tremendous uproar. Induction made it possible to analyze indisputably the lysogenic system. Max [Delbrück] himself immediately repeated the experiment and had to admit that even he was convinced.

A translation from Monod taken from The Emergence of Bacterial Genetics by Thomas D. Brock (1990), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Note the emphasis that Monod gives to two important parts of this result:

 

• The phenomenon was reproducible
  
• All of the cells in a culture carried the latent virus

 

Definitions

Once it was clear that lysogeny was a real phenomenon, two types of bacteriophage were defined:

VIRULENT

virulent phage always grow LYTICALLY

 

TEMPERATE

temperate phage are able to form LYSOGENS wherein the phage exist in a latent or quiescent state known as a PROPHAGE. This state continues until, as a result of some stimulus, INDUCTION occurs. The phage then switch to LYTIC growth.

 

BACTERIOPHAGE LAMBDA

André Lwoff, François Jacob and Jacques Monod recognized that the life cycle of bacteriophage lambda could be interpreted as a simple switch between two states and that it represented an example of genes turning ON and OFF:

[P1.2]

 

lambda genes ON -> LYTIC growth

lambda genes OFF -> LYSOGENIC growth

 

They also recognized that there were many parallels between the process of turning genes on which resulted in the induction of a lysogen and the process by which beta-galactosidase synthesis was induced in the lac operon. Many advances were made by isolating mutants in one of the two systems analogous to mutants that had been found in the other system.

 

Plaque Morphology

When bacteriophage lambda is used to infect E. coli, and the infected culture is then plated on an agar plate, bacteriophage plaques will be seen in the lawn of bacterial cells (if the phage concentration used was correct!). The plaques represent areas of bacterial lysis -- each plaque arising as a result of phage lysis from an initial infected cell, then infecting the adjacent cells, lysing them, infecting again, and so on...

[P4.1]

In the case of bacteriophage lambda, however, the appearance of the plaques is informative. Wild-type phage produce cloudy or TURBID plaques.

Turbid plaques arise because of the fact that bacteriophage lambda can grow lysogenically as well as lytically. Although most of the infected cells are lysed and killed by the infecting phage, some of the bacterial cells are not. They become lysogens and survive. It is the growth of bacterial lysogens within the plaque that causes the turbid phenotype.

There is one important implication of this fact. The bacterial lambda lysogens that cause the turbid appearance must be (and are) able to prevent any further productive infection by lambda. Although, the lysogens are growing in a plaque full of bacteriophage lambda, they survive and grow and are, therefore, IMMUNE TO SUPER-INFECTION.

[P1.3]

Both of these properties (the turbid plaque phenotype and immunity) are directly related to the mechanisms of bacteriophage lambda growth and to the switch between lytic and lysogenic growth.

 

The Meaning of Clear Plaques

Occasionally the plaques formed by bacteriophage lambda are clear instead of turbid. Since turbid plaques are due the formation of bacterial lysogens, clear plaques are an indication that bacterial lysogens do not and cannot form. Mutants of bacteriophage lambda that form clear plaques map into 3 complementation groups: cI, cII, and cIII.

Under normal conditions, bacterial lysogens cannot be found if the phage carries mutations in any one of these three genes. In rare circumstances, however, lysogens of cII^- or cIII^- phage can be found. It is never possible to obtain a lysogen of a lambda cI^- phage.

As we will see, the products coded by the cII and cIII genes are required to establish lysogeny but they are not required for maintaining lysogeny. So, if by some chance a cII^- or cIII^- lysogen did form, it could still be maintained. On the other hand, the product of the cI gene is required for maintaining lysogeny. So, even if a cI^- phage lysogen did form, it would not be able to maintain itself and lytic growth would ensue.

 

Zygotic Induction

In 1954, François Jacob and Elie Wollman did an important experiment that:

 

• demonstrated the polarity of bacterial mating
  
  
• laid the groundwork for the idea of REPRESSORS
  
  
• helped to explain the physical nature of the bacteriophage lambda PROPHAGE in a lysogen
  

 

The experiment involved the mating of a lambda lysogen with a non-lysogen:

Hfr l⁺ x F^- l^-

 

 

When the bacteria from the mating mixture were plated on agar plates, a number of bacteriophage plaques were observed:

 

	No. of lambda plaques per 100 lysogenic bacteria
Controls	Hfr l+	0.41
	F- l+	2.6
Experiments	Hfr l+ x F- l-	52.1
	Hfr l- x F- l+	3.3

 

When they devised their repressor model to explain induction of gene expression in the lac operon, Jacob & Monod realized that they could explain zygotic induction in almost exactly the same way.

[P4.3]

If the prophage state is governed by the presence of a REPRESSOR protein then only cells containing prophage will synthesize this protein. In the conjugation of a Hfr l⁺ strain with a F^- l^- strain, the recipient is not making any repressor and, therefore, genes on the donor DNA will be expressed once they enter the cell. Hence, lysis occurs. In the conjugation of a Hfr l⁺ strain with a F^- l⁺ strain, the recipient contains repressor proteins which prevent expression of the phage genes on the donor DNA.

This result also shows that as long as repressor protein is present, lysogeny is maintained. We have already seen that as long as the cI gene is wild-type, lysogeny can be maintained. In fact, the cI gene codes for the bacteriophage lambda repressor protein.

 

Immunity

Immunity to superinfection is just another manifestation of the same phenomenon observed in zygotic induction and fundamentally the same fundamental explanation applies. The only real difference is the source of phage DNA. In the case of zygotic induction, it is a bacterial cell; in the case of immunity, it is another phage.

[P1.3]

A bacterial lysogen synthesizes a repressor which turns off all of the prophage genes (except one). Any phage of the same type as the prophage that tries to infect the cell will also be repressed -- its genes will not be expressed.

Immunity is both easier to study and more informative than zygotic induction. It can be and has been analyzed genetically so that the bacteriophage loci that are responsible for immunity have been identified.

Immunity in bacteriophage lambda (and related bacteriophages) is conferred by a short and specific region of the bacteriophage chromosome. It consists of 2 genes: cI and cro; and 2 operators: OR and OL. The cI and cro gene products are both DNA-binding proteins -- both are repressors. CI and Cro both bind to DNA at both OR and OL. The interaction of two different proteins at the same DNA binding site is complex and is at the heart of the control of lysogenic growth in bacteriophage lambda.

[P4.2]

Immunity is also a phenomenon that can be used to discriminate between different lambdoid phages. Bacteriophage 21, bacteriophage 434 and bacteriophage P22 (which infects Salmonella) are all closely related and have similar regulatory systems. An E. coli cell that is lysogenic for bacteriophage lambda will be immune to super-infection by another bacteriophage lambda but it will not be immune to super-infection by bacteriophage 434. Conversely, an E. coli cell that is lysogenic for bacteriophage 434 will be immune to super-infection by another bacteriophage 434 but it will not be immune to super-infection by bacteriophage lambda. The following table illustrates when cells are immune, when they are not and, if not, what the plaque morphology is:

[PT4.1]

	l	l cI-	434	l imm434
E. coli K	turbid	clear	turbid	turbid
E. coli K(l)	no plaques	no plaques	turbid	turbid
E. coli K(l imm434)	turbid	clear	no plaques	no plaques

 

• Bacteriophage l can infect E. coli but not a lambda lysogen of E. coli. It can, however, infect a lysogen of E. coli if that lysogen carries a prophage with a different immunity. Where it can infect, it will produce turbid plaques.
  
  
• Bacteriophage 434 can infect E. coli but not a lysogen of E. coli if that lysogen carries a prophage with the same immunity. It can, however, infect a lambda lysogen of E. coli. Where it can infect, it will produce turbid plaques.
  
  
• Bacteriophage  l imm434 is a variant of bacteriophage lambda in which the normal immunity region has been replaced with that of bacteriophage 434. This phage behaves as if it were bacteriophage 434. It can infect E. coli but not a lysogen of E. coli carrying a prophage with the same immunity. It can infect a lambda lysogen of E. coli. Where it can infect, it will produce turbid plaques.
  
  
• Bacteriophage  l cI^- is mutated in the repressor gene. This phage can never form a lysogen; its plaques are always clear. However, it too cannot infect a lambda lysogen of E. coli because the lysogen will still contain repressor which will prevent it from growing.
  

Different bacteriophage lambda containing the immunity regions of related lambdoid phage were very useful in the genetic studies of the immunity region.

One other bacteriophage lambda immunity region mutant should be mentioned. Some lambda mutants are virulent -- i.e. they will infect an E. coli cell whether it is a lysogen or not. These phage have defective operators to which repressor does not bind. These phage can only undergo lytic growth.