Microbial Genetics

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Microbial Genetics

• Transduction,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

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Major phage families and genera
Figure 17.1
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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 cant infect new
cells) ? over time, collect sample and enumerate
viruses
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latent period no viruses released from host
no virions either free or within host
rise period viruses released
free viruses
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

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early genes
late genes
Figure 17.7
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adsorption and penetration
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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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
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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 by regular host RNA polymerase
others by modified host RNA polymerase
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

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An example of terminal redundancy
sticky ends
units linked together
Figure 17.9
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during assembly concatemers are cleaved,
generating circularly permuted genomes
Figure 17.10
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synthesized by host RNA polymerase under
direction of virus-encoded sigma factor
encode capsid proteins and proteins needed for ass
embly
Figure 17.6
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The Assembly of Phage Particles
scaffolding proteins aid in construction
of procapsid
Figure 17.11
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Figure 17.6b2
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Figure 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 phages
• ??X174

• filamentous phages

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?X174
by usual DNA replication method
by rolling-circle mechanism
new virions released by lysis of host
Figure 17.12
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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

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rate of production of cro and cI gene products
determines if lysogeny or lytic cycle occurs
Figure 17.17
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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
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The choice
the race
lambda repressor wins
lysogeny
cro wins
lysis
Figure 17.19
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If lambda repressor wins

• lambda genome inserted into E. coli genome
• integrase
• catalyzes integration

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Figure 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. colis 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/bio141/lecguide/un
  it4/genetics/recombination/transduction/gentran.ht
  ml
• http//www.cat.cc.md.us/courses/bio141/lecguide/un
  it1/control/genrec/u4fg21a.html

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Specialized transduction

• http//www.cat.cc.md.us/courses/bio141/lecguide/un
  it4/genetics/recombination/transduction/spectran.h
  tml