1 microbial genetics transduction,bacteriophages, and gene transfer mi 505 –
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Microbial GeneticsTransduction,Bacteriophages, and Gene Transfer
MI 505 –
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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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Classification of Bacteriophages
based on two major criteria phage morphology nucleic acid properties
4Figure 17.1
Major phage families and genera
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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
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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
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free viruses
no virions –either free orwithin host
latent period – noviruses releasedfrom host
rise period –viruses released
Figure 17.2
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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
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Plaque assay
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Focus on T4 replication complex process highly regulated
some genes expressed early some genes expressed late
early genes and late genes clustered separately
11Figure 17.7
earlygenes
lategenes
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adsorption andpenetration
Figure 17.6a
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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.
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Figure 17.3
penetration mechanismdiffers from that of otherbacteriophages
empty capsidremains outsideof host cell
tail tube may form porein host membrane throughwhich DNA is injected
T4
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Bacteriophage structure
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Phage Tour www.mansfield.ohio-state.edu
/.../bgnws020.htm
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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
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some byregularhost RNApolymerase
others bymodifiedhost RNApolymerase
some products needed for DNA replication
Figure 17.6
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Synthesis of T4 DNA contains
hydroxymethyl-cytosine (HMC) instead of cytosine synthesized by
two phage encoded enzymes
then HMC glucosylated
Figure 17.8
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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
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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
22Figure 17.9
sticky ends
units linked together
An example of terminal redundancy
23Figure 17.10
during assembly – concatemers arecleaved, generatingcircularlypermuted genomes
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synthesizedby host RNApolymeraseunder directionof virus-encodedsigma factor
encodecapsidproteinsandproteinsneededforassembly
Figure 17.6
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The Assembly of Phage Particles
Figure 17.11
scaffolding proteins –aid in construction ofprocapsid
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Figure 17.6b2
27Figure 17.6
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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
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Reproduction of Single-Stranded DNA Phages
focus on two phagesX174
filamentous phages
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X174
Figure 17.12
newvirionsreleasedby lysisof host
by usualDNA replicationmethod
by rolling-circlemechanism
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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)
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Bacteriophage PhiX174.
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Reproduction of RNA Phages
most are plus strand RNA viruses only one (6) is double-stranded
RNA virus also unusual because is envelope
phage
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ssRNAphages
Figure 17.14
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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
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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
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Induction process by which phage
reproduction is initiated results in switch to lytic cycle
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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
40Figure 17.17
rate of productionof cro and cI geneproducts determines iflysogeny or lytic cycleoccurs
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Focus on lambda phage double-
stranded DNA phage
linear genome with cohesive ends circularizes
upon entry into host
Figure 17.16
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Lambda repressor product of cI
gene blocks
transcription of lytic cycle genes, including cro gene
Figure 17.18
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Cro protein involved in
regulating lytic cycle genes
blocks synthesis of lambda repressor
Figure 17.20
44Figure 17.19
The choice
the race
lambda repressor wins lysogeny
cro wins lysis
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If lambda repressor wins… lambda genome inserted into E. coli
genome integrase
catalyzes integration
46Figure 17.21
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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
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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
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M 13 Filamentous Phage
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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)
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Gene VIII codes for the major structural protein of the bacteriophage particles
Gene III codes for the minor coat protein
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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
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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)
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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
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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
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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)
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Summary of Repliation
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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
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M13
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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
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M13 Cloning Vector
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Polylinker Cloning Region
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Medicine and Phages www.intralytix.com/sciencemag.htm
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Bacteriophage PhiX174.
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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.
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Adsorption and penetration by other phages PRD1)
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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.
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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).
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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
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M18 strain of GAS Significant part of genome contains
phage genes Difference in phage genes accounts
for differences in pathogenicity
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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
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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.
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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)
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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
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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
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Plaque assay
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Generalized transduction http://www.cat.cc.md.us/courses/bio
141/lecguide/unit4/genetics/recombination/transduction/gentran.html
http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/control/genrec/u4fg21a.html
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Specialized transduction http://www.cat.cc.md.us/courses/bio
141/lecguide/unit4/genetics/recombination/transduction/spectran.html