Horizontal Gene Transfer and Genetic Engineering Conjugation, Transformation, and Transduction

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Horizontal Gene Transfer and Genetic
EngineeringConjugation, Transformation, and
Transduction

• Genetics

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Gene Transfer

• Refers to the movement of genetic information
  between organisms
• When genes are transferred between two bateria
  or a bacteria and a virus it involves a
  combination of the DNA from two different
  sources.
• This is referred to as recombination
• This type of transfer is referred to as
  horizontal( lateral) gene transfer

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Horizontal Gene Transfer

• Horizontal gene transfer is a driving force in
  the development of drug resistance in bacteria
• This type of transfer is different from the
  transmission of genetic chracteristics from one
  generation to generation vertically

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Gene transfer can occur between bacteria and
plants

• Agrobacterium tumefaciens lives in the soil
• It is able to transfer a plasmid from its cells
  into roots or stems through a scratch or injury
  to the plant tissue
• The plasmid integrates in host DNA and affects
  the host to cause the growth of tumors called
  CROWN GALLS

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Gene transfer from Bacteria to plant
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Gene transfer can occur between viruses and
animals

• SV 40
• Simian virus
• Is a DNA virus
• Transforms or alters DNA and causes cancer
• Used to study cancer and HIV

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BCTERIAL CONJUGATION
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Bacterial Conjugation

• transfer of DNA by direct cell to cell contact
• Contact with the pili
• discovered 1946 by Lederberg and Tatum

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F x F Mating

• F donor( contains the plasmid with the gene
  for conjugation)
• This is referred to as the F factor or Fertility
  Factor
• F recipient
• does not contain F factor
• F factor replicated by rolling-circle mechanism
  and duplicate is transferred ACROSS the pilus
  from the to the -
• recipients usually become F after it receives a
  copy of the DNA
• donor remains F

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Gene transfer and recombination

• Genes are transferred in a linear manner
• The F factor integrates into chromosomes at
  different points

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Mating

• When two strains were mixed
• There were incubated.
• At intervals of 5 minutes, samples were taken of
  the F- cells
• The cells were centrifuged so that they would
  know which genes were transferred.
• The distance between genes was measured by the
  time that it took for the genes to be
  transferred.
• During the first five minutes, the strains were
  mixed there was no recombination

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F x F mating

• In its extrachromosomal state the factor has a
  molecular weight of approximately 62 kb

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Conjugative Proteins

• Key players are the proteins that initiate the
  physical transfer of ssDNA, the conjugative
  initiator proteins
• They nick the DNA and open it to begin the
  transfer
• Working in conjunction with the helicases they
  facilitate the transfer of ss RNA to the F- cell

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- Formation of Hfr
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Hfr - high frequency of recombination
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DNA Transformation

• Uptake of naked DNA molecule from the environment
  and incorporation into recipient in a heritable
  form
• Competent cell
• capable of taking up DNA
• May be important route of genetic exchange in
  nature

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Transformation

• Uptake of DNA can only occur at a certain cell
  density
• Cells need to be in the log phase of growth
• A competence factor is required for the uptake of
  DNA from the environment

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Streptococcus pneumoniae
nuclease nicks and degrades one strand
DNA binding protein
competence-specific protein
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Bacteria and transformation

• Not all bacteria can be transformed in nature
• Streptococcus pneumonia, Haemophilus influenza,
  and Neisseria gonorrhea

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Lab protocol
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Genetic recombination and transformation in the
laboratory

• Plasmids are designed to contain genes of
  interest
• Transformation done in laboratory with species
  that are not normally competent (E. coli)
• Variety of techniques used to make cells
  temporarily competent
• calcium chloride treatment
• makes cells more permeable to DNA

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Cloning vectors
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pAmp
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pGlo and transformation
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Microbial Genetics

• Bacteriophages

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Horizontal gene transfer

• Clearly this plays a central role in the
  diversity of E. coli
• The greatest contributors are the bacteriophages
• Among the 18 prophage remnants on O157 12
  resemble lambda phage
• They all contain a variety of deletions and or
  insertions
• Some of the phages are so similar that they
  contain a 20 kb segment tat is identical.

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Recombinant phages

• It is believed that the phages have undergone
  recombination and diversification
• They have been a major force in developing
  resistance and pathogenicity in bacteria such as
  E. coli and Streptococcus pyogenes
• Recombination could occur with in a single cell
• It could occur as the result of recombination

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Bacteriophages
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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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T- 4 Bacteriophage

• Ds DNA virus
• 168, 800 base pairs
• Phage life cycles studied by Luria and Delbruck

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Bacteriophage structure
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Bacteriophage structure(con)

• Most bacteriophages have tails
• The size of the tail varies.
• It is a tube through which the nucleic acid is
  injected as a result of attachment of the
  bacteriophage to the host bacterium
• In the more complex phages the tail is surrounded
  by a contractile sheath for injection of the
  nucleic acids

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Bacteriophage structure

• Many bacteriophages have a base plate and tail
  fibers
• Some have icosahedral capsids
• M13 has a helical capsid

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Capsid

• The base plate requires 12 protein products
• The head or capsid requires 10 genes
• The capside requires scaffolding proteins for
  assembly
• DNA packaging a mysterious process
• Many phages lyse their host cells at the end of
  the intracellular phase

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T even phagesLuria and Delbruck

• Four distinct periods in the release of phages
  from host cells
• Latent period- follows the addition of phage( no
  release of virions)
• Eclipse period virions were detectable before
  infection and are now hidden or eclipsed
• Rise or burst period Host cells rapidly burst
  and release viruses
• The total number of phages released can be
  determined by the burst size the number of
  viruses produced per
• infected cell

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General Steps
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Steps in the life cycle

• Adsorption of the virus to the host
• This is mediated by tail fibers or some analagous
  structure
• When the tail fibers make contact, the base plate
  settles to the surface
• This connection which is maintianed by
  electrostatic attraction and the ions Mg and
  Ca

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Attachment

• There is host specificity in the attachment and
  adsorption of the bacteriophage
• There are receptors for the attachment. They
  vary from bacteria to bacteria
• The receptors are on the bacteria for other
  purposes the bacteriophages evolved to utilize
  them for their invasion

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T even phages - Injection

• The phage sheath shortens from 24 rings to 12
  rings
• The sheath becomes shorter and wider
• This causes the central tube to push through the
  bacterial cell wall

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Gp5

• The baseplate contains the protein gp5 with
  lysozyme activity which made aid in the
  penetration of the host

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Early Genes

• E. coli RNA polymerase starts transcribing
  genes( phage genes) within minutes of entering
  the bacterial cell
• The early m RNA direct the synthesis of proteins
  and enzymes that are needed for hostile tack over
• Some early virus specific enzymes degrade host
  DNA to nucleotides so that virus DNA synthesis
  can commence

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Late mRNA

• Phage structural structural proteins
• Proteins that help with phage assembly
• Proteins involved in cell lysis and release

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Release

• When the bacteriophages are released from the
  bacteria they can lyse the bacterial cell and
  break it open
• They can be released through the cell membrane

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Irreversible attachment

• The attachment of the tail fibers to the
  bacterium is a weak attachment
• The attachment of the bacteriophage is also
  accompanied by a stronger interaction usually by
  the base plate

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Sheath contraction

• The irreversible binding results in the sheath
  contraction

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Injection

• When the irreversible attachment has been made
  and the sheath contracts, the nucleic acid passes
  through the tail and enters the cytoplasm

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Phage Multiplication Cycle Lytic phages

• Lytic phages or virulent phages enter the
  bacterial cell, complete protein synthesis,
  nucleic acid replication, and then cause lysis of
  the bacterial cell when the assembly of the
  particles has been completed.

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Eclipse Period

• The bacteriophages may be seen inside or outside
  of the bacterial cells
• The phages take over the cells machinery and
  phage specific mRNAs are made
• Early mRNAs are generally needed for DNA
  replication
• Later mRNAs are required for the synthesis of
  phage proteins

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Intracellular accumulation phase

• The bacteriophage sub units accumulate in the
  cytoplasm of the bacterial cell and are assembled

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Lysis or Release Phase

• A lysis protein is released
• The bacterial cell breaks open
• The viruses escape to invade other bacterial cells

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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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Lambda and Plaques

• The plaque produced by Lambda had a different
  appearance on the Petri Dish.
• It is considered to be turbid rather than clear
• The turbidiy is the result of the growth of phage
  immune lysogens in the plaque
• The agar surface contains a ratio of about a
  phage /107 bacteria

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MOI

• Average number of phages /bacterium
• After several lytic cycles the MOI gets higher
  due to the release of phage particles

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Transduction

• Transfer of bacterial genes by viruses
• Virulent bacteriophages
• reproduce using lytic life cycle
• Temperate bacteriophages
• reproduce using lysogenic life cycle

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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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Generalized transduction

• E. coli phage P21 or P22.
• As a part of the lytic cycle, the phage cuts the
  bacterial DNA into fragments
• This fragmentation prevents the expression of
  bacterial genes
• Nucleotides can be used to make phage DNA
• Occasionally these DNA fragments are about the
  same size as phage DNA
• They become mistakenly packaged into phage
  capsids in place of phage DNA

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Types of Lysogenic Cycle

• The most common type is the classic model of the
  Lambda phage
• The DNA molecule is injected into a bacterium
• In a short period of time, after a brief period
  of transcription, an integration factor and a
  repressor are synthesized
• A phage DNA molecule typically a replica of the
  injected molecules is inserted into the DNA
• As the bacterium continue to grow and multiply
  and the phage genes replicate as part of the
  bacterial chromosome

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Temperate

• A bacteriophage that can exist as a lytic or
  lysogenic phage is referred to as a temperate
  phage
• A bacterium containing a full set of phage genes
  is a lysogen
• The process of infecting a bacterial culture with
  a temperate phage is called lysogenization

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Immunization

• A bacterial cell or lysogen cannot be reinfected
  by a phage of the same type
• This is resistance to superinfection is called
  immunity
• More than 90 of the bacteriophages are temperate
• These are unable to produce bursts such as T4 and
  T7

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Lysogenic Phage
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Lambda Phage

• Temperate phage
• Alternate life cycle
• Ds DNA linear then circularizes when it enters
  the host
• 48,502 base pairs
• Molecular biology workhorse because of its life
  cycle

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Genes

• Lambda genes
• 46 genes have been identified
• 14 are non esswential to the lytic cycle
• Only 7 are nonessential to both the lytic and
  lysogenic cycles

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Life cycle of ? Phage
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Latency

• Lysogenic conversion can lead to virulence
• Botulism, cholera,and diptheria toxins are
  encoded by prophages that convert their host into
  a pathogenic bacterium

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Terminology

• LEGEND
• att an E.coli seqence for the "attachment" or
  integration of lambda's circular chromosome.
• oriC E.coli's origin of Chromosome replication
  (given here for orientation only)
• gal E.coli's gene for galactose utilization
• peprophage ends (site of integration)
• cos joined sticky ends of vegetative DNA
  sometimes called ve ("vegetative ends")
• int gene for the enzyme integrase
• c gene for lambda repressor to maintain
  lysogeny
• Q another gene concerned with lysogeny
• h the last of the many capsomer genes.

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Bacteriophages
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Specialized transduction
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Attachment site

• The E. coli chromosome contains one site at which
  lambda integrates. The site, located between the
  gal and bio operons, is called the attachment
  site and is designated attB since it is the
  attachment site on the bacterial chromosome.
• The site is only 30 bp in size and contains a
  conserved central 15 bp region where the
  recombination reaction will take place.
• The structure of the recombination site was
  determined originally by genetic analyses and is
  usually represented as BOB', where B and B'
  represent the bacterial DNA on either side of the
  conserved central element

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Recombination site

• The bacteriophage recombination site - attP - is
  more complex. It contains the identical central
  15 bp region as attB.
• The overall structure can be represented as POP'.
  However, the flanking sequences on either side of
  attP are very important since they contain the
  binding sites for a number of other proteins
  which are required for the recombination
  reaction. The P arm is 150 bp in length and the
  P' arm is 90 bp in length.

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Integration

• Integration of bacteriophage lambda requires one
  phage-encoded protein - Int, which is the
  integrase - and one bacterial protein - IHF,
  which is Integration Host Factor.
• Both of these proteins bind to sites on the P and
  P' arms of attP to form a complex in which the
  central conserved 15 bp elements of attP and attB
  are properly aligned.
• The integrase enzyme carries out all of the steps
  of the recombination reaction, which includes a
  short 7 bp branch migration.

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Normal Excision
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Generalized Transduction

• Any part of bacterial genome can be transferred
• Occurs during lytic cycle
• During viral assembly, fragments of host DNA
  mistakenly packaged into phage head
• generalized transducing particle

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Generalized transduction
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Specialized Transduction

• Also called restricted transduction
• carried out only by temperate phages that have
  established lysogeny
• only specific portion of bacterial genome is
  transferred
• occurs when prophage is incorrectly excised

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Specialized transduction
Figure 13.20
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Figure 13.20
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Recombination and Genome Mapping in Viruses

• viral genomes can also undergo recombination
  events
• viral genomes can be mapped by determining
  recombination frequencies
• physical maps of viral genomes can also be
  constructed using other techniques

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Specialized transduction mapping

• provides distance of genes from viral genome
  integration sites
• viral genome integration sites must first be
  mapped by conjugation mapping techniques

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Recombination mapping

• recombination frequency determined when cells
  infected simultaneously with two different viruses

Figure 13.24