Bacteria Viruses Flashcards

1
Q

Phage genetics

A

• Genetic characteristics may be either DNA or RNA,
single or double stranded, circular or linear, and usually
present as a single copy

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2
Q

Phage morphology

A

Morphology diversity in phages differs from simple,
icosahedral and filamentous phages to more complex
phages with a tail and an icosahedral head.

• Most phages are have tails

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3
Q

Phage environments

A

• Phages are more common in environments where their

bacteria host are more abundan

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4
Q

Classification of phages

A

• Phages can be classified into two broad categories:

  1. Virulent phages:
  2. Temperate phages
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5
Q

Virulent phages

A

Virulent phages convert the cell replication machinery of
their bacteria host for viral gene replication resulting in
cell lysis, especially in obligately lytic phages, and release
of progeny virions. Most members undergo the phage
lytic lifecycle as opposed to the lysogenic lifecycle.
In exceptional cases, the filamentous ssDNA phage M13
releases progeny virions continously from cells without
lysis and is commonly known as a ‘’chronically infecting’’
phage.

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6
Q

Temperate phages

A

. Temperate phages exhibit alternating
replication cycles involving:
i. Productive (lytic) infection
ii. Reductive (lysogenic) infection: occurs when
the phage lies latent or dormant in the
bacterial host usually due to unfavourable
conditions. The phage at the latent stage is known as a
lysogen.

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7
Q

the lysogeny cycle

A

• In lysogeny, the phage genome is repressed for lytic
functions and most time integrates into the chromosome of
the bacteria.

• An example is the phage lambda (λ) capable of existing
extrachromosomally.

• In phage P1, the prophage replicates together with the host
and remain dormant until the lytic cycle is initiated usually
under conditions that result in the disruption of host DNA.

• Inactivation of the repressor gene follows the damage of
host DNA, initiating a lytic cycle.

last superinfection immunity?

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8
Q

Superinfection immunity

A

• Superinfection immunity: prophages in bacterial host
(resident phages) can prevent superinfection by the same
or other phages by repressing the incoming phage genome
resulting in protection of the host.

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9
Q
  1. RNA phages genotypes
A

ssRNA phages

• dsRNA phages

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10
Q
  1. DNA phages genotypes and examples
A

ssDNA phages
• icosahedral ssDNA phages
• Filamentous ssDNA phages

dsDNA phages
• Phage T4
• Phage T7
• Phage lambda

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11
Q

Single-stranded RNA phages

taxonomy

A

• They are grouped into the family Leviviridae

• They belong to one of the smallest group of
viruses and possess a high rate of mutation.

• Members are small phages with icosahedral
shape.

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12
Q

Single-stranded RNA phages

Genome/ infection

A

• ssRNA phages have (+) strand RNA, hence
there genome function as a mRNA.

• They possess only a few genes, capable of
infecting gram-negative bacteria such as,
Pseudomonas spp, E. coli and Caulibacter spp.

• ssRNA phages infect enterobacteria via the sex
pilus during conjugation.

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13
Q

The virion of ssRNA phages

A
  • The virion of RNA phages is composed of the following:
  • 180 molecules of major capsid protein (CP) or coat per virion.

• One molecule of maturation (A) protein (minor virion protein),
acquired for infectivity by reconition of sex pilus and maturation.

• Typically, ssRNA phages possess
linear ssRNA genome of
approximately 3500-4200
nucleotides.

• The genome exhibits significant
amount of secondary structures
(around 80%), which affects
access of the ribosome binding
sites (RBCs) to host ribosome
during translation.
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14
Q

The genome of a representative phage (MS2) is composed
of 3569 nucleotides.

4 orfs

A

MS2 was the first RNA virus identified by genome

MS2 genome has four open reading frames (ORFs) which
codes for the following proteins, characterized by intergenic
spaces:
1. Major coat/capsid protein (coats each virus particle)
2. Maturation (A) protein (maturase)
3. Replicase (RNA-dependent RNA replication)
4. Lysis protein (overlapping the coat and replicase genes)

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15
Q

Figure 3: The genetic map

A

• The solid blue boxes represent intergenic regions involved with ribosome binding and regulation of translation.

• The MS2 genome is composed of: genes for maturase (maturation protein); coat (capsid) protein; replicase protein for
RNA-dependent RNA replication; and lysis protein (from overlapping coat and replicase genes).

  • The Qβ genome is composed of: genes for maturase (A); coat protein; replicase protein; and read-through protein.
  • Note that the MS2 genome is smaller than that of Qβ,

• The maturation protein in the Qβ genome perform additional role of mediating lysis, as the genome lacks a separate
lysis protein

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16
Q

The Qβ genome of ssRNA phages

A

• The Qβ genome is composed of four genes encoding
proteins namely: genes for maturase (A); coat protein;
replicase protein; and read-through protein. It lacks a
lysis gene.

• The genome for ssRNA phages code efficiently for
some of its needed proteins and determines specific
secondary and tertiary structures that control
functions such as, translation and replication. • ssRNA phages manipulate host proteins for activation
of its proteins; some of which perform multiple
functions.

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17
Q

attachment of ssRNA phages

A

• Attachment: Infection of a susceptible phage in coliphage
M2 involves attachment to pilin (pilus singular), along the
length of the sex pilus through the maturase (A protein). In
E. coli, binding between phages can prevent conjugation.

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18
Q

gene expression and translation of ssRNA phages

A

• Gene expression and Translation: Proceeds immediately
after infection of a new phage.

• The (+) strand RNA phage genome directs protein synthesis
using the host (bacterial) ribosomes.

• This results in the translation of phage virion proteins such
as replicase, coat (capsid) protein, A protein (maturase) and
lysis protein.

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19
Q

genome replication in ssRNA phages

A

• Genome replication: Immediately the replicase is
translated, the ssRNA genome of phages switches from
protein synthesis to template for gene replication.

• Replicase is associated with several host proteins to
form a phage RNA specific polymerase.

• Genome replication occurs in two phases (replication
intermediates [RI]); the (+) strand RNA serves as a
template for synthesis of the (-) RNA strand, while the
(-) RNA strand in turn serves as template for the
synthesize of the (+) strand RNA.

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20
Q

Figure 4: Replication in ssRNA phages

A

Replication of the single-stranded phage
genome occurs through production of two
replicative intermediates, RI-1 and RI-2.

• First, the (–) strand RNA is synthesized 5′ to 3′ antiparallel and complementary to the (+)
sense template strand by replicase, in a multi- branched structure.

• The (–) strand RNA then serves as template for
formation of new (+) strand genomic RNA. This
is the only role for the (–) strands. Newly
replicated (+) strands can be recycled in
replication, translated to yield the capsid
proteins, or encapsidated in the formation of
progeny phages.

• The inset shows the activation of the replicase
protein (REP) by associating with host proteins:
for MS2 two elongation factors (EFTs and EFTu)
and the ribosomal S1 protein.

• Replicase is an RNA-templated RNA polymerase, synthesizing both plus (+) and minus (–) strands
of phage RNA 5′ to 3′ through specificity of the
replicase for the 3′ end of both template
strands.

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21
Q

Assembly and exit in ssRNA phages

A

• Assembly and exit: ssRNA phages assembly
occurs by spontaneous accumulation,
whereby protein ‘A’ (maturase) forms
association with (+) strand RNA.

• The association is surrounded by coat (capsid)
proteins forming an icosahedral-shaped shell.

• Exit: Progeny virions exit cells by lysis,
following accumulation of about 10,000
progeny virions.

22
Q

Figure 5: Replication cycle in ssRNA phages

A

.

  1. Attachment along length of sex
    (F) pilus
  2. Entry of RNA genome
  3. Translation: Host ribosomes attach to phage RNA (+) strand for synthesis of some coat protein and replicase.
 4/5. Replication of RNA: Replicase binds to (+) strand
and synthesises (-) strands. (Replicase switches off coat protein
(CP) gene early to allow replication). (-) strand acts as template for (+) strand synthesis. A gene translated
during nascent (+) strand synthesis. Translation of coat protein
gene continues from (+) strands. 
  1. Assembly of Progeny virions:
    ‘A’ protein associates with RNA (+) strand and coat protein assembles around it to form an icosahedral shell. 7. Exit: Lysis occurs after ab
23
Q

dsRNA Phages taxonomy

A

• Members belong to the family Cystoviridae, derived

from the Greek word Kystic meaning ‘’bladder sac’’

24
Q

dsRNA phages RNA

A

• They contain a dsRNA which is segmented and
packaged in a polyhedral linear core with a lipid
containing envelope.

25
Q

Phi6

A

• Phi6 (φ6) was one of the first member of the dsRNA
phages to be studied; its genome consists of three
linear segment as follows:
1. RNA L (large): has about 6400 nucleotides
2. RNA M (medium): has about 4000 nucleotides
3. RNA S (small): has about 3000 nucleotides.

26
Q

Transcription in dsRNA phages

A

• Is controlled by RNA-dependent RNA polymerase;
leading to the synthesis of (+) strand RNA which
serves as template for mRNA.

• Translation of L segment results in early proteins
that assemble to form the polymerase complex.

• M segments produce structural proteins for
membrane (spike especially).

• S segment is translated into structural proteins
for capsid, membrane assembly, cell lysis and
entry and a non-structural protein surrounding
the capsid.

27
Q

Infection of bacterium by Phi6 (φ6)

A

• Infection begins by attachment of the Phi6 (φ6) to its host
Pseudomonas syringe via the pilus.

• Cell entry: The pilus withdraws and attach the phage to the
cell surface of the bacterium, thereby gaining entrance into
its cell.

• Phage uncoating results in release of polymerase which
induces gene transcription, translation and assembly in the
cytoplasm.

  • Phage envelope is derived from the bacterium cell.
  • Packaging of virion genome (+ RNA) occurs in the order S- M-L.

• Replication of the complementary strand (-) RNA occurs in
the capsid leading to matured dsRNA phage.

• Cell lysis occurs following the accumulation of about 100
virions.

28
Q

two catagories of ssDNA phages

A

• Two categories of the single stranded DNA
phages exist: filamentous and Icosahedral.

• The icosahedral phages (φX174 and S-13) are
amongst the first group of phages studied. • Years after, the filamentous phages were
identified in 1960 by two group of
researchers: Don Marvin and Hartmut
Hoffman-Berling; and Norton Zinder and
colleagues.

29
Q

taxonomy of ssDNA phages

A

• They belong to the virus family: Microviridae,
derived from the Greek word micros meaning
small.

30
Q

what has the study of ssDNA phages led to

A

• Knowledge derived from the study of these
group of phages led to the understanding of
genome economy and coding efficiency, which
is characteristic of small viruses such as
hepatitis B virus and ssRNA phages.

31
Q

The virion of phage φX174

what are the 4 proteins, and what are their functions

A

• It is a ssDNA filamentous phage. • The virion contains four major proteins
represented by F, G, H and J.

• Protein F: forms the main shell (capsid).

• Protein G forms the spike, as
projections on the icosahedral 5-fold
axis. Projection function as attachment
to host and release of phage genome
into the host cell.

• Protein H: spans the inside and outside
of the capsid and associated with
protein G.

• Protein J: Is a phage DNA binding
protein forming a core around the
ssDNA genome.

32
Q

The genome of φX174

A

• The genome was the first complete DNA genome to
be sequenced.

• It is a circular DNA molecule composed of 5386
nucleotides, which codes for 11 proteins.

  • The genes are highly clustered with minimal non- coding sequence.
  • Thus, enhancing gene coding efficiency.
33
Q

Functions of the gene components: of The genome of φX174

A

ene A (3981–136) protein, viral strand synthesis
and RF replication;
gene A∗ (4497–136) protein, shutting down host
DNA synthesis;
gene B (5075–51) protein, capsid morphogenesis;
gene C (133–393) protein, DNA maturation;
gene D (390–848) protein, capsid morphogenesis;
gene E (568–843) protein, host cell lysis;
gene F (1001–2284) protein, capsid
morphogenesis—major coat protein;
gene G (2395–2922) protein, capsid
morphogenesis—major spike protein;
gene H (2931–3917) protein, capsid
morphogenesis—minor spike protein;
gene J (848–964) protein, capsid morphogenesis—
core protein (DNA condensing
protein);
gene K (51–221) protein, function not clear;
appears to enhance phage yield (burst size).
IG: intergenic region at borders of genes A, J, F, G, H
contains a ribosome binding site
and other features.

34
Q

Filamentous ssDNA Phages taxonomy

A

• Members belong to the virus family- Inoviridae;
derived from the Greek word ina meaning fiber or
filament.

35
Q

Filamentous ssDNA phage infection

A

• Members of the F-specific filamentous family have
been well studied such as M13, fd, and f1.

The F-specific filamentous phages are ‘’male
specific’’ capable of infecting E. coli strains
containing the conjugative plasmid F.

• Infection is by adsorbtion via the tip of the F pilus
which encodes the bacteria plasmid.

36
Q

How are Filamentous ssDNA phages unique

A

• Filamentous ssDNA phages are unique from other
DNA phages as they do not inject their DNA into the
host cell.

• Instead, the whole phage particle is ingested.

• Another unique feature is that progeny phages are
not released for infected cells by lysis, rather,
progeny are released regularly through the cell
membrane.

37
Q

The virion structure of F-specific
filametous phages

What are their capsid proteins, and what are their functions

A

• The capsid is composed of flexible protein
filament of varying length, depending on
the size of the genome. E.g., the size of the
wild type capsid is about 900 nm.

• The capsid comprises five proteins: pIII, pVI, pVII, pVIII and pIX.

• pVIII forms the major coat protein along
the length of the filament; • pIII and pVI: forms the minor coat
proteins that form an adsorption knob at
one end of the filament;

• pVII and pIX: forms the minor coat
proteins that form at the other end. Note: Arrangement of both pVII and pIX
proteins isn’t yet clealy defined: pVII may
interact with pVIII and be shielded from the
environment, whilst pIX may interact with
pVII and be exposed.

38
Q

Replication in F-specific filamentous phages

A
  1. Attachment. The phage binds to the tip of the
    F pilus via pIII, 2. Entry: Retraction of the pilus brings the phage
    particle to the periplasm to interact with Tol
    proteins. Phage coat proteins disassemble into
    the cytoplasmic membrane; the phage ssDNA is
    translocated into the cytoplasm. 3. Transcription: using (–) strand DNA of
    replicative form (RF) as template. 4. Translation: for synthesis of all phage proteins. 5. Genome replication occurs in three stages:
    i. ssDNA plus (+) strand is converted to the
    dsDNA replicative form (RF).
    ii. Amplification of RF by rolling circle replication
    involving pII and pX. Amplification of RF continues until DNA binding
    protein (pV) reaches sufficient concentration to
    bind to (+) strand DNA and prevent its conversion
    to RF.
    iii. pV switches most of the DNA replication from
    dsDNA RF to ssDNA (+) strand. 6. Assembly. The pV:DNA complex migrates to
    the cell membrane. pV is removed and
    replaced by the capsid proteins
39
Q

dsDNA Phages

most studied members

A

T phages and phage lambdi

denoted by numbers

40
Q

Which family are phages T2, T4, and T6 in

A

• Phages T2, T4 and T6 are grouped in the family Myoviridae; derived from
the Greek word myos meaning muscle, implying phages with contractile
tails

41
Q

Which family are phages T1, T5 in

A

• Phages T1, T5 and phage lambdi belong to the family Siphoviridae, from
the Greek word siphon meaning tube, implying phages with long, flexible,
non-contractile tail.

42
Q

Which family are the T3 and T7 phage in

A

• T3 and T7 are in the family Podoviridae, derived from the Greek word
podos meaning foot, implying dsDNA phages with short, non-contractile
tail.

43
Q

Phage T4

A

• T4 is a dsDNA phage infecting Escherichia coli
bacteria.

  • It belong to the virus family- Myoviridae.
  • T4 is one of the largest known phage.
  • It has a linear dsDNA genome of around 169 kbp.

• T4 is virulent phage capable of inducing only the lytic
lifecycle in bacteria (bacteria cell lysis), but not the
lysogenic cycle.

44
Q

Phage T4 virion morphology

A

• Members have a complex morphology, consisting of:
• an elongated icosahedral head,
• several types of protein, including two accessory
proteins (HOC: Higher antigen capsid protein; and
SOC: small outer capsid protein). • a contractile tail,
• baseplate,
• tail fibers
• pins

45
Q

Figure 10: Structure of phage T4

A

The capsid is composed of many proteins, spheroid icosahedral head separated from
the tail by the neck. The tail is made up of two helical
arrangements of protein: one forming the
sheath, the other the tube, with a collar and
a baseplate with pins and tail fibers. The pins at the baseplate vertices anchor
the phage to the host cell. Proteins are labeled with their corresponding
gene name or number. Virus assembly requires a protein scaffold. The dsDNA is densely packed as concentric
layers in the head

46
Q

Phage T7

A

• Phage T7 is a dsDNA phage belonging to the family- Podoviridae

• Phage T7 infects most strains of E. coli, which it
depends upon for replication.

• It is a virulent phage capable of inducing the lytic
lifecycle, leading to destruction of infected cells
releasing progeny phages.

The tail is attached to the head via
a structure composed of one or
more distinct proteins; referred to as
a “portal” or a “connector.

47
Q

Phage Lambdi ( λ)

structural physiology

A

• The virion has an icosahedral head, containing a
dsDNA genome of around 48.5 kbp.

• It has a long flexible tail, with central tail tip fiber
and side tail fiber, similar to that of T4.

• The genes are arranged in clusters according to
functions in the genome and encodes some
enzymes and proteins needed for replication.

• Examples are the capsid proteins, protein kinase,
helicase, DNA ligase, etc.

48
Q

Phage Lambdi ( λ) replication cycle

A

hage λ infect E. coli bacteria.

• Phage λ is temperate, thus, can either:

(a) establish a lysogenic cycle: remains as a prophage
integrated into the bacterial chromosome, or

(b) establish a lytic cycle: undegoes gene multiplication
after bacterial infection to produce progeny virions
which are released to infect new cells. The type of cycle undergone by Phage λ is determined
by several factors involving genetic, physiological and
environmental factors.

49
Q

Application of phage biology in science

cloning vectors

A
  1. Cloning vectors: Filamentous ssDNA phages confer
    advantage as vectors due to their small-size, induces nonlytic infection and ease of manipulation.

• Discovery of restriction enzymes was derived from the
study of phages and laid the foundation for gene cloning.

• ssDNA phages have very useful application as vectors in
gene sequencing, DNA probes and detecting site-directed
mutagenesis.

• Also, ssDNA phages have applied in phage display
technology for the engineering of antibodies.

• Phages such as T7 have potential application as expression
vectors for specific proteins and promoters.

50
Q

Application of phage biology in science

Therapeutic

A

Phages are been considered as a potential
therapeutic agent against bacteria owing to increasing bacterial
resistance to antibiotics and emergence of new diseases.

• E.g., T4 phages have been proposed as possible therapeutic
agent for the treatment of E. coli-induced diarrhea.

51
Q

Application of phage biology in science

Dignostic tool

A

Phages are important in diagnostic systems as
biological indicators for characterizing bacteria in contaminated
food, polluted environment and hospital.