Lecture for microbiology students.

1. Chapter Six
Viruses
Topic: Multiplication of Bacteriophages
Outline:
• One step growth curve
• Lytic cycle
• Lysogenic cycle

2. Teaching and learning methods: Lectures, visual aid, interactive forms,
questions- answer session and group discussion.
At end of the lecture student should be able to answer the following
questions:
• Explain the one step growth curve of virus.
• Summarize how virus multiply using lytic cycle.
• Define lysogenic cycle. Compare lytic and lysogenic cycle.
Reference books:
1. Microbiology: An Introduction, 12th edition- Tortora GJ & Funke BR, Chapter
Thirteen; Page: 369

3. Multiplication of Bacteriophages
• Although the means by which a virus enters and exits a host cell
may vary, the basic mechanism of viral multiplication is similar for
all viruses. The best understood viral life cycles are those of the
bacteriophages. Phages can multiply by two alternative
mechanisms: the lytic cycle or the lysogenic cycle.
• The lytic cycle ends with the lysis and death of the host cell,
whereas the host cell remains alive in the lysogenic cycle. The T-
even bacteriophages (T2, T4, and T6) have been studied most
extensively; we will describe the multiplication of T-even
bacteriophages in their host, E. coli, as an example of the lytic
cycle.

4. Multiplication & one-step growth curve of bacteriophage
One Step Growth Curve:
• The growth response during virus replication is
illustrated in the figure. The response takes the
form of a one-step growth curve. This is so named
because a time course of virion numbers in the
culture medium shows essentially no increase
during the replication cycle until cells burst and
release their newly synthesized virions.
• Following adsorption, infectious virions cannot be
detected in the growth medium, a phenomenon
called eclipse.
• During the latent period, which includes the
eclipse and early maturation phases, viral nucleic
acid replicates and protein synthesis occurs.
• During the maturation period, virus nucleic acid
and protein are assembled into mature virions
and then released.

5. The Lytic Cycle
• The virions of T-even bacteriophages are large, complex, and nonenveloped,
with a characteristic head and tail structure. The length of DNA contained in
these bacteriophages is only about 6% of that contained in E. coli, yet the
phage has enough DNA for over 100 genes. The multiplication cycle of these
phages, like that of all viruses, occurs in five distinct stages: attachment,
penetration, biosynthesis, maturation, and release.
1. Attachment
• After a chance collision between phage particles and bacteria, attachment, or
adsorption, occurs. During this process, an attachment site on the virus
attaches to a complementary receptor site on the bacterial cell. This
attachment is a chemical interaction in which weak bonds are formed
between the attachment and receptor sites. T-even bacteriophages use fibers
at the end of the tail as attachment sites. The complementary receptor sites
are on the bacterial cell wall.

6. The Lytic Cycle
2. Penetration:
• After attachment, the T-even bacteriophage injects its DNA into the
bacterium. To do this, the bacteriophage’s tail releases phage lysozyme, which
breaks down a portion of the bacterial cell wall. During the process of
penetration, the tail sheath of the phage contracts and the tail core is driven
through the cell wall. When the tip of the core reaches the plasma membrane,
the DNA from the bacteriophage’s head passes through the tail core, through
the plasma membrane, and enters the bacterial cell. The capsid remains
outside the bacterial cell. Therefore, the phage particle functions like a
hypodermic syringe to inject its DNA into the bacterial cell.

8. 3. Biosynthesis:
• Once the bacteriophage DNA has reached the cytoplasm of the host cell, the
biosynthesis of viral nucleic acid and protein occurs. Host protein synthesis is
stopped by virus-induced degradation of the host DNA, viral proteins that interfere
with transcription, or the repression of translation.
• Initially, the phage uses the host cell’s nucleotides and several of its enzymes to
synthesize many copies of phage DNA. Soon after, the biosynthesis of viral proteins
begins. Any RNA transcribed in the cell is mRNA transcribed from phage DNA for the
biosynthesis of phage enzymes and capsid proteins. The host cell’s ribosomes,
enzymes, and amino acids are used for translation. Genetic controls regulate when
different regions of phage DNA are transcribed into mRNA during the multiplication
cycle. For example, early messages are translated into early phage proteins, the
enzymes used in the synthesis of phage DNA. Also, late messages are translated into
late phage proteins for the synthesis of capsid proteins. For several minutes following
infection, complete phages cannot be found in the host cell. Only separate
components DNA and protein can be detected. The period during viral multiplication
when complete, infective virions are not yet present is called the eclipse period.
The Lytic Cycle

9. 4. Maturation:
• In the next sequence of events, maturation occurs. In this process,
bacteriophage DNA and capsids are assembled into complete virions. The
viral components essentially assemble into a viral particle spontaneously,
eliminating the need for many non-structural genes and gene products. The
phage heads and tails are separately assembled from protein subunits, and
the head is filled with phage DNA and attached to the tail
5. Release:
• The final stage of viral multiplication is the release of virions from the host
cell. The term lysis generally used for this stage in the multiplication of T-
even phages because in this case, the plasma membrane actually breaks open
(lysis). Lysozyme, which is coded for by a phage gene, is synthesized within
the cell. This enzyme causes the bacterial cell wall to break down, and the
newly produced bacteriophages are released by the host cell. The released
phages infect other susceptible cells in the vicinity, and the viral multiplication
cycle is replaced within those cells.
The Lytic Cycle

10. Bacteriophage Lambda (l): The lysogenic cycle:
• In contrast to T-even bacteriophages, some viruses do not cause
lysis and death of the host cell when they multiply. Some phages
can either proceed through a lytic cycle or begin a lysogenic cycle
by incorporating their DNA into the host cell's DNA. In the latter
state, called lysogeny, the phage remains latent (inactive). Phages
that can multiply by either mechanism are called lysogenic phages
or temperate phages. The participating bacterial host cells are
known as lysogenic cells.
• We will use the bacteriophage l (lambda), a well-studied
temperate phage, as an example of the lysogenic cycle.

11. Figure-8: The lysogenic cycle of bacteriophage l in E. coli.

12. (1) l-phage penetrates into an E. coli cell,
(2) The originally linear phage DNA forms a circle,
(3A) this circle can multiply and be transcribed,
(3B) alternatively, the circle can recombine with and become part of the circular
bacterial DNA (the lysogenic cycle). The inserted phage DNA is now a prophage. Most of
the prophage genes are repressed by two repressor proteins produced by the phage
genes. These repressors bind operator and stop transcription of all the other phage
genes. Thus, the phage genes that would otherwise direct the synthesis and release of
new virions are turned off, in much the same way that the genes of the E. coli lac
operon are turned off by the lac repressor. Every time the host cell's machinery
replicates bacterial chromosome,
(4A) leading to the production of new phage and to cell lysis (the lytic cycle).
(4B) it also replicates the prophage DNA. The prophage remains latent within the
progeny cells.
(5) However, a rare spontaneous event, or action of UV light or certain chemicals, can
lead to excision (popping-out) of the phage DNA, and to initiation of the lytic cycle.