The document discusses three bacteriophages - Lambda phage, M13 phage, and φX174 phage. It describes the structure and genome of each phage. It then outlines the life cycle of each phage, including adsorption to host cells, DNA replication, gene expression, assembly of new virions, and release from the host cell. Applications of each phage in research are also mentioned, such as using Lambda and M13 for DNA cloning and φX174 being the first genome sequenced.

1. Plasmids and Bacteriophages
(Part 2 of 3)
MBH-201: Microbial Genetics

2. Lysogenic Phages
 Lambda
 M13
 X174

3. Lambda Phage
 A lambda phage has a head measuring around 50-60 nanometers in
diameter and a flexible tail that is around 150 nanometers long and
may contain tail fibers.
 The head functions as a capsid that contains its genome, which contains 48,490
base pairs of double-stranded linear DNA.
 When E. coli is infected with a lambda phage, two cycles may
happen: lytic or lysogenic.

4. Lambda Phage: Life cycle

5. Lambda Phage: Life cycle
 Adsorption
 Differs from T4 bacteriophage in that Lambda uses the outer
membrane protein necessary for maltodextrin transport.
 Lysogeny
 The genetic structure of λ is structurally simple but functionally
complex.
 Early transcripts are involved in replication, integration, and
excision.
 Late transcripts are involved with capsid protein assembly and cell
lysis.
 The cos site is the point at which the linear ends of the genome
come together to form a circular genome structure within the host-
cell.
 The att site is involved with host-cell genome interactions, the ori
site is the origin of circular replication, and the cI site is involved with
controlling gene expression.

6. Lambda Phage: Life cycle
 The lambda promoter initially expresses cro and cII proteins. If cro
concentration is higher than cII, the virus enters lytic cycle and
replication ensues. If cII is higher than cro, the genome enters
latency.
 cII activates the promoter Pi which transcribes the integrase mRNA.
Lambda genome is integrated at attB site in the bacterial genome.
 During latency, only cI is expressed, shutting off all viral promoters
at high concentration.
 Upon cellular stress, cI is degraded.
 Most temperate phages encode an integrase for both integration
and excision. Excision may require an accessory protein called
excisionase or recombination directionality factor to promote
excision. Integrase (int) and excisionase (xis) together promote viral
genome excision.
 The lytic cycle is again restored.

7. Lambda Phage: Life cycle
 Replication
 Replication of λ occurs through rolling circle replication, in
bidirectional manner starting at the ori site. One strand of circular
DNA is cut and the exposed 3` end acts as a primer. The first
several replication cycles occur in circular form.
Then, sigma replication leads
to the elongation of the viral
genome creating
concatamers. Elongation of
the phage occurs using
several different host proteins
including: DnaB helicase,
DnaG primase, Pol III
holoenzyme, and the host
RNA polymerase.

8. Lambda Phage: Life cycle
 Assembly
 Concatamers of replication are simultaneously cut at cos sites
between each complete genome using the viral encoded protein
terminase. The λ phage packages two proteins which help
activate the origin of replication and direct host replicative
enzymes.
 Release
 Lambda is released through lysis of the host-cell which is
mediated by two viral encoded proteins. One of the proteins is a
holoenzyme (S107) which becomes embedded in the
cytoplasmic membrane. When activated, the holoenzyme
creates a hole in the cytoplasmic wall and allows the release of
lysozymes into the periplasm. Lysozymes break apart the
peptidoglycan cell wall which leads to cell lysis.

9. Lambda Phage: Applications
 The lambda phage has different applications, most of which are related to
DNA cloning. This is because lambda phage can be used as a vector for
generating recombinant DNA, which are combined DNA sequences that
result from using laboratory techniques like molecular cloning to assemble
genetic material from several sources. The site-specific recombinase of
lambda phage can be used for shuffling cloned DNAs via the gateway
cloning system, a molecular biology technique that ensures the effective
transfer of DNA fragments between plasmids.
 The lambda phage’s ability to mediate genetic recombincation is due to its
red operon, which is a functioning unit of genomic DNA that has a cluster of
genes controlled by a promoter or a single regulatory signal. This red
operon can be expressed to yield the proteins red alpha (or exo), beta, and
gamma, which can be used in recombination-mediated genetic engineering,
a method commonly employed in bacterial genetics, generation of target
vectors, and DNA modification.
 Undoubtedly, the lambda phage is a powerful genetic tool that can be used
in many important studies.

10. M13 Phage
 M13 is a filamentous
bacteriophage composed of
circular single stranded DNA
(ssDNA) which is 6407
nucleotides long
encapsulated in
approximately 2700 copies
of the major coat protein
PVIII, and capped with 5
copies of two different minor
coat proteins (PIX, PVI, PIII)
on the ends. The minor coat
protein P3 attaches to the
receptor at the tip of the F
pilus of the host Escherichia
coli.
M13 is unusual because phage continually exit from a
bacterium without killing it. For this reason, M13 is not
considered to have a true lysogenic state.

11. M13 Phage: Life cycle
? inner
membrane
protein TolA
gpIII
SSB

12. M13 Phage: Life cycle
 M13 Adsorption and Infection
 M13 adsorbs to the tip of the F pilus, a hair-like structure on the
surface of some bacteria. It can only infect bacteria that carry an
F or F-like conjugative plasmid that encodes the proteins that
make up the F pilus. For the filamentous phage, it is known that
infection is initiated by the binding of gpIII to the tip of the F pilus.
GpIII then interacts with the inner membrane protein TolA.
 Two additional facts about gpIII suggest a mechanism for phage
DNA entry. GpIII contains amino acid sequences that are fusogenic
or promote localized fusion of two membranes and gpIII is capable
of forming pores in membranes that are large enough for DNA to go
through. If each of these properties of gpIII are important for phage
entry, then the phage could bind to the F pilus, promote fusion of the
membranes, and use gpIII to form holes in the membrane to gain
entry into the cytoplasm.

13. M13 Phage: Life cycle
 Protection of the M13 genome
 The M13 DNA that ends up in the cytoplasm is a circular single-
stranded DNA molecule. The strand present in phage particle is
known as the plus or + strand. After entry into the cytoplasm, the
+ strand DNA is immediately coated with an E. coli single
stranded DNA binding protein known as SSB. The SSB coating
protects the DNA from degradation.

14. M13 Phage: Life cycle
 M13 DNA replication
 The M13 plus strand is
converted to a double-stranded
molecule immediately upon
entry into E. coli. Synthesis of
the complementary strand is
carried out entirely by E. coli’s
DNA synthesis machinery. The
complementary strand is called
the minus or - strand. Only the
minus strand is used as the
template for mRNA synthesis
and ultimately it is the template
for the translation of the
encoded M13 gene products.
The conversion of the M13 plus strand to a doublestranded DNA
molecule. The plus strand enters the cell (a and b) with gpIII
attached. It is immediately coated with host SSB (c).
RNA polymerase synthesizes a short primer (d) and DNA
polymerase synthesizes the minus strand.

15. M13 Phage: Life cycle
 The SSB that coats the plus strand upon entry of the DNA into
the E. coli cytoplasm fails to bind to ~60 nucleotides of the
molecule (Fig c). These nucleotides form a hairpin loop that is
protected from nuclease degradation. M13 gpIII from the phage
is found associated with the hairpin loop. The hairpin loop is
recognized by E. coli RNA polymerase as a DNA replication
origin and is used to initiate transcription of a short RNA primer
(Fig d). The RNA primer is extended by E. coli DNA polymerase
III to create the minus strand (Fig e). The RNA primer is
eventually removed by the exonuclease activities of E. coli DNA
polymerase I. the 5’ to 3’ polymerizing activity of the same DNA
polymerase. E. coli ligase forms the final phosphodiester bond
resulting in a covalently closed double-stranded circular M13
chromosome. The double-stranded form of M13 chromosome is
called the replicative form (RF) DNA.

16. M13 Phage: Life cycle
 The RF form is replicated by rolling circle replication similar to
the mechanism used by the l chromosome (Fig b). The M13
gene II encoded protein is an endonuclease that nicks the plus
strand of the RF DNA at a specific place to initiate the replication
process for M13 RF DNA. Approximately 100 copies of M13 RF
DNA are made. While the M13 chromosome is being replicated,
the genes encoding the coat proteins are being transcribed and
translated. When M13 gpV protein accumulates to sufficient
levels, a switch from synthesizing RF DNA to synthesizing the
plus strand occurs. GpV blocks the synthesis of the minus
strand, presumably by displacing SSB on the plus strand and
preventing the plus strand from being used as a template. The
plus strand is circularized.

17. M13 Phage: Life cycle
 M13 phage production and release
from the cell
 M13 phage particles are assembled and
released from E. coli cells through a
process that does not involve lysing E. coli
or disrupting cell division. The gpV coated
plus strand makes contact with the
bacterial inner membrane (Fig a). This
interaction requires a specific packaging
sequence on the DNA and gpVII and gpIX.
The protein-coated DNA traverses the
membrane and gpV is replaced by gpVIII
in the process (Fig b). GpVIII is found in
the membrane. When the last of the phage
particle crosses the membrane, gpIII and
gpVI are added. M13 phage are
continually released from actively growing
infected E. coli.
M13 is released from the cell without lysing the
bacterium. (a) The plus strand, coated with gpV
interacts with the membrane through gpVII and gpIX.
(b) As the DNA traverses the membrane, the gpV
is replaced by gpVIII.

18. M13 Phage: Applications
 M13 phages are useful for a number of applications:
 sequencing
 mutagenesis
 probes
 lambda-ZAP subcloning
 phage display libraries
 M13 was developed into a useful cloning vector by
inserting the following elements into the genome:
 a gene for the lac repressor (lac I) protein to allow
regulation of the lac promoter
 the operator-proximal region of the lac Z gene (to
allow for a-complementation in a host with operator-
proximal deletion of the lac Z gene).
 a lac promoter upstream of the lac Z gene
 a polylinker (multiple cloning site) region inserted
several codons into the lac Z gene

19. M13 Phage: Applications
 M13 plasmids are used for many recombinant DNA
processes, and the virus has also been studied for its
uses in nanostructures and nanotechnology.
 George Smith, among others, showed that fragments of
EcoRI endonuclease could be fused in the unique Bam site of f1
filamentous phage and thereby expressed in gene III whose protein pIII
was externally accessible. M13 does not have this unique Bam site in
gene III. M13 had to be engineered to have accessible insertion sites,
making it limited in its flexibility in handling different sized inserts.
Because the M13 phage display system allows great flexibility in the
location and number of recombinant proteins on the phage, it is a
popular tool to construct or serve as a scaffold for nanostructures. For
example, the phage can be engineered to have a different protein on
each end and along its length. This can be used to assemble structures
like gold or cobalt oxide nano-wires for batteries or to pack carbon
nanotubes into straight bundles for use in photovoltaics.

20. X174 Phage
 Non-enveloped, round, T=1 icosahedral symmetry, about
30 nm in diameter. The capsid consists of 12 pentagonal
trumpet-shaped pentomers. The virion is composed of
60 copies each of the F, G, and J proteins, and 12 copies
of the H protein. There are 12 spikes which are each
composed of 5 G and one H proteins.

21. X174 Phage
 Genome
 Circular, ssDNA(+)
genome of 4.4 to
6.1kb. replication
occurs via dsDNA
intermediate
and rolling circle.
 Gene Expression
 Early and late
genes promoters
tightly regulate the
timing of gene
expression, which
is crucial for the
replication cycle.

22. X174 Phage: Life Cycle
Host Polymerase
RFI
RFII
RFIII
Assembly
Release
Viral protein A
Viral protein C

23. X174 Phage: Life Cycle
 Pilus-mediated adsorption of the virus onto host cell
 The proteins of the capsid perform Injection of the
viral DNA through bacterial membranes into cell
cytoplasm.
 Host polymerase convert the (+) ssDNA viral genome
into a covalently closed dsDNA called replicative
form DNA I (RF-I).
 Early viral genes are transcribed by
host RNA polymerase, producing viral replication
proteins.
 Viral protein A cleaves RF-I(+) DNA strand at the origin
of replication and covalently attaches itself to the DNA.

24. X174 Phage: Life Cycle
 (+) strand replication occurs by rolling circle, which is
converted to dsDNA by host polymerase, generating RF-
II molecules (amplification of RF-I).
 Late viral genes are transcribed by host RNA
polymerase.
 Procapsid assembly in the cytoplasm.
 Viral protein C binds to replication complex, inducing
synthesis and packaging of neo-synthesized (+) ssDNA
genomes (RF-III) into procapsids.
 Procapsids maturation occurs in host cytoplasm
 Mature virions are released from the cell by lysis.

25. X174: Applications
 The X174 bacteriophage was the first DNA-based genome to be
sequenced. This work was completed by Fred Sanger and his team
in 1977.
 In 1962, Walter Fiers and Robert Sinsheimer had already
demonstrated the physical, covalently closed circularity of X174.
 Nobel prize winner Arthur Kornberg used X174 as a model to first
prove that DNA synthesized in a test tube by purified enzymes could
produce all the features of a natural virus, ushering in the age of
synthetic biology.
 In 2003, it was reported by Craig Venter's group that the genome of
X174 was the first to be completely assembled in vitro from
synthesized oligonucleotides.
 The X174 virus particle has also been successfully assembled in
vitro.
 Recently, it was shown how its highly overlapping genome can be
fully decompressed and still remain functional.