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Transduction,Bacteriophages, and Gene Transfer. Microbial Genetics. MI 505 –. Bacteriophages. Bacterial viruses Obligate intracellular parasites Inject themselves into a host bacterial cell Take over the host machinery and utilize it for protein synthesis and replication.

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Transduction bacteriophages and gene transfer

Transduction,Bacteriophages, and Gene Transfer

Microbial Genetics

MI 505 –


Bacteriophages
Bacteriophages

  • Bacterial viruses

  • Obligate intracellular parasites

  • Inject themselves into a host bacterial cell

  • Take over the host machinery and utilize it for protein synthesis and replication


Classification of bacteriophages
Classification of Bacteriophages

  • based on two major criteria

    • phage morphology

    • nucleic acid properties



Reproduction of double stranded dna phages the lytic cycle
Reproduction of Double-Stranded DNA Phages: The Lytic Cycle

  • lytic cycle

    • phage life cycle that culminates with host cell bursting, releasing virions

  • virulent phages

    • phages that lyse their host during the reproductive cycle


The one step growth experiment
The One-Step Growth Experiment

mix bacterial host and phage

brief incubation

(attachment occurs)

dilute greatly

(released viruses can’t infect new cells)

over time, collect sample and enumerate viruses


latent period – no

viruses released

from host

no virions –

either free or

within host

rise period –

viruses released

free viruses

Figure 17.2


Plaque assay
Plaque assay

  • Phage infection and lysis can easily be detected in bacterial cultures grown on agar plates

  • Typically bacterial cells are cultured in high concentrations on the surface of an agar plate

  • This produces a “ bacterial lawn”

  • Phage infection and lysis can be seen as a clear area on the plate. As phage are released they invade neighboring cells and produce a clear area



Focus on t4 replication
Focus on T4 replication

  • complex process

  • highly regulated

    • some genes expressed early

    • some genes expressed late

  • early genes and late genes clustered separately


early

genes

late

genes

Figure 17.7


adsorption and

penetration

Figure 17.6a


Adsorption to the host cell and penetration
Adsorption to the Host Cell and Penetration

  • receptor sites

    • specific surface structures on host to which viruses attach

    • specific for each virus

    • can be proteins, lipopolysaccharides, techoic acids, etc.


T4

empty capsid

remains outside

of host cell

tail tube may form pore

in host membrane through

which DNA is injected

Figure 17.3

penetration mechanism

differs from that of other

bacteriophages



Phage tour
Phage Tour

  • www.mansfield.ohio-state.edu/.../bgnws020.htm


Synthesis of phage nucleic acids and proteins
Synthesis of Phage Nucleic Acids and Proteins

  • sequential process

    • early mRNA synthesis

    • synthesis of proteins that enable T4 to take over host cell

    • phage DNA replication

    • late mRNA synthesis

      • encode capsid proteins and other proteins needed for phage assembly


some by

regular

host RNA

polymerase

others by

modified

host RNA

polymerase

some products needed for DNA replication

Figure 17.6


Synthesis of t4 dna
Synthesis of T4 DNA

  • contains hydroxymethyl-cytosine (HMC) instead of cytosine

    • synthesized by two phage encoded enzymes

  • then HMC glucosylated

Figure 17.8


Hmc glucosylation
HMC glucosylation

  • protects phage DNA from host restriction endonucleases

    • enzymes that cleave DNA at specific sequences

  • restriction

    • use of restriction endonucleases as a defense mechanism against viral infection


Post synthesis events
Post synthesis events

  • T4 DNA is terminally redundant

    • base sequence repeated at both ends

    • allows for formation of concatamers

      • long strands of DNA consisting of several units linked together


An example of terminal redundancy
An example of terminal redundancy

sticky ends

units linked together

Figure 17.9


during assembly –

concatemers are

cleaved, generating

circularly

permuted

genomes

Figure 17.10


synthesized

by host RNA

polymerase

under direction

of virus-encoded

sigma factor

encode

capsid

proteins

and

proteins

needed

for

assembly

Figure 17.6


The assembly of phage particles
The Assembly of Phage Particles

scaffolding proteins –

aid in construction of

procapsid

Figure 17.11




Release of phage particles
Release of Phage Particles

  • T4

    • lysis of host brought about by several proteins

      • e.g., endolysin – attacks peptidoglycan

      • e.g., holin – produces lesion in cell membrane

  • other phages

    • production of enzymes that disrupt cell wall construction


Reproduction of single stranded dna phages

focus on two phages

X174

filamentous phages

Reproduction of Single-Stranded DNA Phages


X174

by usual

DNA replication

method

by rolling-circle

mechanism

new

virions

released

by lysis

of host

Figure 17.12


M13

  • M13 is a filamentous bacteriophage which infects E. coli host. The M13 genome has the following characteristics:

  • Circular single-stranded DNA

  • 6400 base pairs long

  • The genome codes for a total of 10 genes (named using Roman numerals I through X)



Reproduction of rna phages
Reproduction of RNA Phages

  • most are plus strand RNA viruses

  • only one (6) is double-stranded RNA virus

    • also unusual because is envelope phage


Ssrna phages
ssRNAphages

Figure 17.14


6 reproduction
6 reproduction

  • icosahedral virus with segmented genome

    • capsid contains an RNA polymerase

    • three distinct double-stranded RNA (dsRNA) segments

      • each encodes an mRNA

  • mechanism of synthesis of dsRNA genome is not known


Temperate bacteriophages and lysogeny
Temperate Bacteriophages and Lysogeny

  • lysogeny

    • nonlytic relationship between a phage and its host

    • usually involves integration of phage genome into host DNA

      • prophage – integrated phage genome

    • lysogens (lysogenic bacteria)

      • infected bacterial host

    • temperate phages

      • phages able to establish lysogeny


Induction
Induction

  • process by which phage reproduction is initiated

  • results in switch to lytic cycle


Lysogenic conversion
Lysogenic conversion

  • change in host phenotype induced by lysogeny

    • e.g., modification of Salmonella lipopolysaccharide structure

    • e.g., production of diphtheria toxin by Corynebacterium diphtheriae


rate of production

of cro and cI gene

products determines if

lysogeny or lytic cycle

occurs

Figure 17.17


Focus on lambda phage
Focus on lambda phage

  • double-stranded DNA phage

  • linear genome with cohesive ends

    • circularizes upon entry into host

Figure 17.16


Lambda repressor
Lambda repressor

  • product of cI gene

  • blocks transcription of lytic cycle genes, including cro gene

Figure 17.18


Cro protein
Cro protein

  • involved in regulating lytic cycle genes

  • blocks synthesis of lambda repressor

Figure 17.20


The choice
The choice

the race

lambda repressor wins

lysogeny

cro wins

lysis

Figure 17.19


If lambda repressor wins
If lambda repressor wins…

  • lambda genome inserted into E. coli genome

    • integrase

      • catalyzes integration



Induction1
Induction

  • triggered by drop in levels of lambda repressor

    • caused by exposure to UV light and chemicals that cause DNA damage

  • excisionase

    • binds integrase

    • enables integrase to reverse integration process


M13

  • Among the simplest helical capsids are those of the well-known bacteriophages of the family Inoviridae, such as M13 and fd - known as Ff phages. These phages are about 900nm long and 9nm in diameter and the particles contain 5 proteins. All are similar and are known collectively as Ff phages - they require the E.coli F pilus for infection



M13

  • M13 is a filamentous bacteriophage which infects E. coli host. The M13 genome has the following characteristics:

  • Circular single-stranded DNA

  • 6400 base pairs long

  • The genome codes for a total of 10 genes (named using Roman numerals I through X)


  • Gene VIII codes for the major structural protein of the bacteriophage particles

  • Gene III codes for the minor coat protein


Infection
Infection

  • The gene VIII protein forms a tubular array of approx. 2,700 identical subunits surrounding the viral genome

  • Approximately five to eight copies of the gene III protein are located at the ends of the filamentous phage (i.e. genome plus gene VIII assembly)

  • Allows binding to bacterial "sex" pilus

    • Pilus is a bacterial surface structure of E. coli which harbor the "F factor" extrachromosomal element


Infection1
Infection

  • Single strand genome (designated '+' strand) attached to pilus enters host cell

    • Major coat protein (gene VIII) stripped off

    • Minor coat protein (gene III) remains attached

  • Host components convert single strand (+) genome to double stranded circular DNA (called the replicative or "RF" form)


Transcription
Transcription

  • Transcription begins

    • Series of promoters

      • Provides a gradient of transcription such that gene nearest the two transcription terminators are transcribed the most

    • Two terminators

      • One at the end of gene VIII

      • One at the end of gene IV

    • Transcription of all 10 genes proceeds in same direction


Part one
Part One

  • Gene II protein introduces 'nick' in (+) strand

  • Pol I extends the (+) strand using strand displacement (and the '-' strand as template)

  • After one trip around the genome the gene II protein nicks again to release a completed (linear) '+' genome

  • Linear (+) genome is circularized


Part two
Part Two

  • During first 15-20 minutes of DNA replication the progeny (+) strands are converted to double stranded (RF) form

  • These serve as additional templates for further transcription

  • Gene V protein builds up

  • This is a single stranded DNA binding protein

  • Prevents conversion of single (+) strand to the RF form

  • Now get a buildup of circular single stranded (+) DNA (M13 genome)



Phage packaging
Phage Packaging

  • Phage packaging

  • Major coat protein (Gene VIII) present in E. coli membrane

  • M13 (+) genome, covered in ss binding protein - Gene V protein, move to cell membrane

  • Gene V protein stripped off and the major coat protein (Gene VIII) covers phage DNA as it is extruded out

    • Packaging process is therefore not linked to any size constraint of the M13 genome

    • Length of the filamentous phage is determined by size of the DNA in the genome

    • Inserts of up 42 Kb have been introduced into M13 genome and packaged (7x genome size)

  • ~8 copies of the Gene III protein are attached at the end of the extruded genome



M13 cloning vector
M13 Cloning Vector

  • 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

  • The vectors were named according to the specific polyliner region they contained

  • The vectors were typically constructed in pairs, with the polylinker regions in opposite orientations




Medicine and phages
Medicine and Phages

  • www.intralytix.com/sciencemag.htm



Prd1 phages
PRD1 phages

  • Virions consist of a capsid and an internal lipid membrane.

  • Virus capsid is not enveloped.

  • Virions are tail-less, but can produce tail-like tubes

  • Capsid/nucleocapsid is round and exhibits icosahedral symmetry.



Structural characteristics
Structural characteristics

  • The isometric capsid has a diameter of 63 nm. The capsid shells of virions are composed of two layers.

  • The outer capsid consists of a smooth, rigid 3 nm thin protein shell and appear to have a hexagonal in outline.

  • Surface projections are distinct 20 nm long spikes protruding from each apex

  • Inner capsids consist of a 5-6 nm flexible shell made from a thick lipoprotein vesicle.

  • The genome forms a tightly packed coil.


Genome
Genome

  • The genome is not segmented and contains a single molecule of linear double-stranded DNA.

  • The complete genome is 147000-157000 nucleotides long, is fully sequenced and encodes gene 8 for DNA terminal proteins and genes for protein P15 (lytic enzyme).


Group a streptococci gas
Group A Streptococci( GAS)

  • Genes activated when macrophages engulf bacterial cells

  • These phage genes are part of the ability of bacterial cells to avoid destruction


M18 strain of gas
M18 strain of GAS

  • Significant part of genome contains phage genes

  • Difference in phage genes accounts for differences in pathogenicity


Streptococcus canis
Streptococcus canis

  • Normally a bacterium that harmlessly infects dogs

  • Treatment with antibiotics for other infections especially fluoroquinolones, causes the activation of phage genes

  • Induces flesh eating infections and toxic shock


Listeria phages
Listeria phages

  • The Gram-positive bacterium Listeria monocytogenes can be found in raw food and causes human disease, The immune-compromised are particularly susceptible, and infection leading to listeric meningitis can be deadly.

  • Listeria transducing bacteriophage CU153, shown on the left has a very long tail with two disk-like structures at the distal end (DNA content is about 42Kbp).

  • Phage P35 shown on the right has a much shorter tail with a single disk-like structure at the distal end.


Fluoroquinolones
Fluoroquinolones

  • Cipro that fights Anthrax belongs to this group

  • Triggers phage genes

  • Can increase the amount of toxin released

  • ( Shiga toxin can be released by a variety of bacteria)


E coli
E. Coli

  • Shiga toxin is integrated into E. coli DNA – the gift of a phage

  • When it becomes active – E. coli’s food poisoning becomes more severe


Plaque assay2
Plaque assay

  • Phage infection and lysis can easily be detected in bacterial cultures grown on agar plates

  • Typically bacterial cells are cultured in high concentrations on the surface of an agar plate

  • This produces a “ bacterial lawn”

  • Phage infection and lysis can be seen as a clear area on the plate. As phage are released they invade neighboring cells and produce a clear area



Generalized transduction
Generalized transduction

  • http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/transduction/gentran.html

  • http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/control/genrec/u4fg21a.html


Specialized transduction
Specialized transduction

  • http://www.cat.cc.md.us/courses/bio141/lecguide/unit4/genetics/recombination/transduction/spectran.html


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