Chapter 10 continued

1
Chapter 10 (continued)

• Bacterial Genetics

2
Transformation vs. Transduction vs. Conjugation
3
Genetic Transformation

• Genetic Transformation process by which free
  DNA is incorporated into a recipient cells and
  brings about genetic change.
• A number of prok. are naturally transformable,
  including gram-pos. and gram-neg. Bacteria and
  some Archaea.
• Only a small number of genes from one cell can be
  transferred to another by a single transformation
  event.

4
Competent Cells

• Competent cells able to take up a molecule of
  DNA via transformation.
• Only certain strains are competent and this
  ability is genetically determined.
• Competence is regulated with special proteins
  playing a role in DNA uptake and processing.
• ssDNA or dsDNA may taken up by cells, though it
  must be in ssDNA form to be incorporated into the
  genome by recombination.
• Competent cells bind up to 1000X more DNA than
  noncompetent cells (dsDNA binds better to cells).
• DNA fragments compete with each other for uptake.
  
• While the max. frequency of transformation 20
  of the population, actual values 0.1-1.0.
• Min. conc. of DNA yielding detectable
  transformants 0.00001 ?g/ml.

5
Integration of Transforming DNA

• DNA is either taken up single-stranded or dsDNA
  is taken up and one strand is degraded ? ssDNA.
• Next, the ssDNA associates with
  competence-specific protein that remains attached
  to the DNA to protect it from nuclease attack
  until it reaches the chromosome, where RecA takes
  over.
• DNA is then integrated into the genome of the
  recipient by recombination.
• During replication of this heteroduplex DNA, one
  parental and one recombinant DNA molecule are
  formed. On segregation at cell division, the
  recombinant DNA molecule is present in the
  transformed cell.

6
Transfection

• Transfection Bacteria transformed by
  bacteriophage DNA instead of DNA from another
  bacterium.
• Transfection by a lytic bacteriophage leads to
  normal virus production.

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Artificially Induced Competence/Electroporation

• Only a few bacteria exhibit natural
  transformation. Other bacteria can be made
  competent through artificially induced
  competence.
• High conc. of cold Ca2 ions causes E. coli to
  become competent at low efficiency.
• Electroporation a technique in which cells are
  exposed to pulsed electric fields to open small
  pores in their membranes. DNA present outside
  the cells can enter through these pores. This
  method works for a variety of prok. and euk.
  Plasmids can be transferred directly from one
  cell to another because DNA can exit as well as
  enter through these pores.

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Transduction

• Transduction DNA is transferred from cell to
  cell via viruses.
• A variety of prok. can undergo transduction and a
  variety of phages can transduce.
• 2 types of transduction ? virus ends up defective
  and homologous recomb. can occur in either case
  
  (1) generalized transduction
  host DNA derived from virtually any portion of
  the host genome becomes a part of the DNA of the
  mature virus particle in place of the virus
  genome.
  (2) specialized transduction
  occurs only in some temperate viruses DNA from
  a specific region of the host chromosome is
  integrated directly into the virus genome
  usually replacing some of the virus genes.

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Generalized Transduction
10
Specialized Transduction
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Phage Conversion

• Phage conversion phenotypic alterations made in
  a lysogenized cell, can be acquisition of
  immunity to further infection by the same type of
  phage or can be some other change.
• ex. toxin production in Corynebacterium
  diphtheriae.

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Plasmids

• Plasmids genetic elements that replicate
  independently of the host chromosome.
• Thousands of different types of plasmids are
  known, almost all of which are dsDNA, most of
  which are supercoiled and circular, are vary in
  size from 1-1000 kbp.
• Different plasmids are present in cells in a
  particular number of plasmid molecules per cell
  copy number, which can vary from 1-100.
• Most gram-neg. plasmids replicate similar to the
  chrom., although some replicate unidirectionally.
  Most gram-pos. plasmids replicate by the rolling
  circle mechanism similar to a phage.

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Plasmids (cont.)

• Cells can contain different types of plasmids. A
  cell in which two plasmids cannot be maintained
  together are said to be incompatible.
• Curing elimination of a plasmid from a cell.
  Curing can occur spontaneously or with the help
  of chemicals or electroporation.
• Plasmids lack extracellular form.
• The main mechanism of cell-to-cell plasmid
  transfer conjugation.
• Plasmids that govern their own transfer by
  cell-to-cell contact conjugative.

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Types of Plasmids

• While all plasmids carry genes that ensure their
  own replication, some carry genes for
  conjugation, as well as other unique properties
  conferred upon the cell.
• Resistance (R) plasmids confer resistance to
  antibiotics and other inhibitors of growth.
  These plasmids often transfer resistance to other
  cells via cell-to-cell contact, resulting in
  antibiotic resistant populations. R plasmids
  with genes for resistance to most antibiotics are
  known.
• The following virulence factors of pathogenic
  bacteria can be encoded on plasmids
  
  (1) ability of
  microorganisms to attach and colonize specific
  sites in the host
  
  (2) formation of substances
  (ex. toxins, etc.) that cause damage to the host.
  What is this similar to that we just discussed?

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Bacteriocins

• Bacteriocins agents produced by bacteria that
  inhibit or kill closely related species or
  different strains of the same species. They are
  different from antibiotics, which have a wider
  spectrum of activity.
• Bacteriocins are often plasmid-encoded.
• Bacteriocins are named according to the organism
  that produces them.
• They can interfere with another cells proton
  motive force and, thus, have practical uses such
  as food preservatives.

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Conjugation and Chromosome Mobilization

• What is bacterial conjugation also known as?
• Conjugation is a plasmid-encoded mechanism, but
  can mobilize host chromosome as well.
• The F plasmid of E. coli first confirmed the
  occurrence of conjugation.
• Conjugation involves a donor cell containing a
  conjugative plasmid and a recipient cell, which
  does not. What are these cells also known as?
• Sex pilus may be specified by the plasmid,
  allowing for specific pairing between donor and
  recipient. The pilus formed by the F plasmid is
  called the F pilus.

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DNA Transfer During Conjugation

• DNA synthesis is necessary for DNA transfer to
  occur.
• Rolling circle replication model best explains
  DNA transfer during conjugation. This process is
  triggered by cell-to-cell contact.
• At the end of the process, both donor and
  recipient possess plasmids and the recipient can
  become a donor, spreading the plasmids between
  populations like infectious agents.

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Transfer of Plasmid DNA by Conjugation
19
Hfr (High Frequency of Recombination) Strains

• F plasmid conjugative, can integrate into host
  chromosome ( episome), and can also mobilize
  chromosome transfer.
• Cells with an unintegrated F plasmid F, while
  those having a chromosome-integrated F plasmid
  Hfr, and cells without and F plasmid F-.
• Conjugation with Hfr donor ? transfer of host
  chromosome.
• After transfer, an Hfr strain remains Hfr since
  it retains a copy of the F plasmid in the
  chromosome.
• Note ori origin, ex. of replication or of
  transfer

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F Plasmid

• Cells having an F plasmid are able to synthesize
  and F pilus, mobilize DNA for transfer to another
  cell, and alter surface receptors so that the
  cell can no longer serve as a recipient.
• The F plasmid can integrate into the host
  chromosome at sites called insertion sequences
  (IS) . Once integrated, the F plasmid no longer
  controls its own replication.
• Usually, because of breakage of the DNA strand
  during transfer, only part of the donor
  chromosome is transferred. Although Hfr strains
  transmit chromosomal genes at high frequency,
  they usually do not convert F- strains to F or
  Hfr because the entire F plasmid is rarely
  transferred. However, F strains can convert F-
  strains to F because the entire F plasmid is
  transferred.

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Interrupted Mating

• Recombinants from conjugation can be selected
  for.
• In an Hfr strain, the transfer of chromosomal
  genes will always occur in the same order from a
  fixed site on a given Hfr strain.
• Interrupted mating interrupt mating pairs by
  agitation after a certain time that conjugation
  has occurred. Genes present closer to the origin
  enter the F- cell first.
• This technique leads to genetic mapping since you
  can determine the order in which the genes occur
  by the order in which they are transferred.
  Genes at certain points can be referred to as
  positioned at so many minutes.
• Genetic recombination is dependent on the
  occurrence of homologous recombination and is not
  a result of genetic transfer alone.
• F cells in which the F plasmid has been
  excised from the chromosome and takes some
  chromosomal DNA with it.

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Interrupted Mating Experiment
23
Other Conjugation Systems

• Conjugative transposons can be transferred from
  the chromosome of a donor to a recipient and can
  mobilize other genetic elements.
• Conjugative transposons are common to gram-pos.
  cells.

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Complementation

• Complementation is used to determine whether or
  not two mutations are in the same gene by
  restoring function of a gene by complementing the
  defective (mutant) gene with a normal (nonmutant)
  copy of that gene. Homologous recombination can
  restore gene function (unless both of the
  mutations include changes in exactly the same
  base pairs) but cannot reveal whether or not the
  mutations were in the same gene.
• The two mutations are said to complement each
  other.
• Bacterial gene transfer must be done in order to
  conduct this test.
• Complementation does not involve recombination.
• Cistron gene two mutations in the same
  cistron cannot complement each other.
• In diploid organisms Cis 2 mutations from the
  same parent, Trans 2 mutations from different
  parent.

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Complementation (cont.)
26
Transposons and Insertion Sequences

• Some genes are capable of moving under certain
  conditions. The process by which a gene moves
  from one place to another in the genome
  transposition.
• Transposition is relatively rare.
• Not all genes are capable of transposition.
  Transposition of genes is linked to the presence
  of special genetic elements called transposable
  elements.
• There are 3 types of transposable elements in
  Bacteria (1)
  insertion sequences
  
  (2) transposons
  
  (3) some special viruses (ex.
  Mu)
• Transposable elements have 2 features in common
  (1)
  transposase enzyme necessary for transposition
  (2) inverted
  terminal repeats and the ends of their DNA.

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Mechanisms of Transposition

• Two mechanisms of transposition
  (1) Conservative
  the transposable element is excised from one
  location in the chromosome and becomes reinserted
  at a second location. The copy number of a
  conservative transposon remains at one. Direct
  repeats are formed in the target site at the ends
  of the transposon.
  (2)
  Replicative (ex. bacteriophage Mu) transposons
  are duplicated and a new copy is inserted at
  another location. A composition structure
  called a cointegrate is formed.
• Transposition is essentially a recombination
  event, but one that does not occur between
  homologous sequences or use the general
  recombination system of the cell. It is called
  site-specific recombination and involves
  transposase instead of RecA.

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Mechanisms of Transposition (cont.)
29
Mutagenesis with Transposable Elements

• If the insertion site for a transposable element
  is within a gene, insertion of the transposon
  will result in loss of linear continuity of the
  gene, leading to mutation transposon
  mutagenesis means of creating mutants
  throughout the chromosome.

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Integrons

• Integrons genetic elements that can capture and
  express genes from other sources.
• Integrons code for integrase, which catalyzes a
  type of site-specific recombination.
• Integrase can integrate gene cassettes and a
  promoter that can then express the newly
  integrated gene cassette.
• The genes in the gene cassette that are captured
  are not captured randomly, but have specific DNA
  sequences recognized by the integrase and genes
  that are not expressed until they become part of
  an integron.

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Restriction Enzymes

• Protect prok. from foreign DNA, ex. viruses.
• Restriction enzymes recognize certain sequences
  of DNA and cut the DNA.
• Palindrome sequence of bases that reads the
  same when read from either right or left.
  Palindromes are often the target of REs.
• Introduce double stranded breaks.
• In a random DNA molecule, one would expect any
  4-bp sequence to occur once every 256 bps based
  on the probability of 1/4 X 1/4 x 14 x 1/4.
• A 6 bp sequence would appear every 4096 bps in
  random DNA and a 8 bp sequence would appear once
  every 1000 bps, so NotI cuts the E. coli genome
  (4600 bps) 21x, therefor the recognition sequence
  for NotI occurs more often than predicted.
• There are over 2000 REs known with over 200
  specificities.

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Protection from Restriction

• Cells protect their own DNA from their REs by
  methods such as methylation of their own
  sequences that would be targeted by their REs.

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RE Analysis of DNA

• RE analysis is done by gel electrophoresis -
  whats the procedure for this?
• Can be used to generate a physical map of DNA.
• Nucleic acids can be purified from gels and used
  to transform cells or for nucleic acid
  hybridization as nucleic acid probes to find
  similar sequences from different genetic elements
  Southern blot (RNA hybridization Northern
  blot, protein hybridization Western blot).

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Sequencing and Synthesizing DNA

• 2 procedures (1) Maxim and Gilbert method (2)
  Sanger dideoxy method
• Both methods generate DNA fragments that end at
  each of the four bases (G, A, T, C) and that are
  radioactive.
• The fragments are subjected to gel
  electrophoresis in which 4 sample lanes are
  featured, one for each base.
• Maxim-Gilbert method used chemicals that break
  the DNA preferentially at each of the four
  nucleotides.
• Sanger dideoxy method sequence is determined by
  making a copy of the ssDNA using DNA pol., which
  used dNTPs as substrates, adding them to a
  primer. The dNTPs feature a dideoxy sugar analog
  that prevent lengthening of the chain and acts as
  a specific chain-termination reagent. Fragments
  of variable length are obtained. Either the
  dNTPs or primer are radioactive. This method can
  be used to sequence RNA as well.
• Sequencing by the Sanger method is now automated
  and fluorescent labels have replaced radioactive
  ones.

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Molecular Cloning

• Gene cloning
• Purpose isolate large quantities of specific
  genes or chromosomal fragments in pure form.
• Basic strategy move the desired gene or region
  from a large, complex genome to a small, simple
  one.
• Tools used restriction enzymes, DNA ligase,
  synthetic DNA (see 2 below).
• Steps
• 1. Isolation and fragmentation of the source DNA.
• 2. Joining the DNA fragments to a cloning vector
  (ex. plasmid or virus) with DNA ligase. Blunt or
  sticky ends may be created on the ends of the
  source and/or vector DNA - what does this mean
  and how do you deal with each?
• 3. Introduction and maintenance in a host
  organism. What types of organisms are used as
  host organisms (what are their characteristics)?

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Molecular Cloning (cont.)

• What makes plasmids good cloning vectors?
• What is plasmid pBR322 a good cloning vector?
• How does insertional inactivation work?

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Polymerase Chain Reaction (PCR)

• PCR requires that the nucleotide sequence of a
  portion of the desired gene be known because
  short oligonucleotide (- what does this mean?)
  primers complementary to sequences in the gene or
  genes of interest must be available for PCR to
  work.
• Steps
• 1. Two oligonucleotide primers flanking the
  target DNA are made (how?) and added to excess to
  heat-denatured target DNA.
• 2. As the mixture cools, the target strands
  anneal mostly to a primer, which are in excess,
  and not to each other.
• 3. DNA pol. then extends the primers using target
  strands as template.
• 4. After an appropriate incubation period, the
  mixture is heated again to separate the strands.
  The mixture is then cooled to allow the primers
  to hybridize with complementary regions of newly
  synthesized DNA, and the whole process is
  repeated.

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Polymerase Chain Reaction (PCR)
39
PCR (cont.)

• Taq pol. is stable at 95C and is unaffected by
  the denaturation step. However, it has no
  proofreading activity.
• Pfu pol. is stable at 100C and has proofreading
  activity, and is therefore more accurate.
• PCR is often conducted in automated thermocycling
  machines.
• It can be used to amplify very small quantities
  of DNA present in a sample.
• It is not necessary for the organism to be grown
  in the lab, so it is important for environmental
  studies.
• It can also be used for DNA fingerprinting, a
  powerful forensic tool permitting ID of
  individuals (crime scene/suspects) or
  relationships between individuals (paternity
  testing).

40
In Vitro and Site-Directed Mutagenesis

• In Vitro in glass, i.e. in the lab external
  to the organism, as opposed to in vivo in the
  living organism. In other words, you can remove
  genes from and organism, manipulate them,
  engineer in mutations and put them back into an
  organism.
• Site-directed mutagenesis Mutations can be
  introduced at precisely determined sites on
  genes.
• Mutagenesis studies are often done on at the gene
  level to make amino acid changes to study protein
  structure.
• Cassette mutagenesis segments of DNA can be
  manipulated in which synthetic fragments of DNA
  have replaced the wild-type sequence. These
  cassettes can be used for insertion inactivation,
  causing gene disruption. How is this done?

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The Bacterial Chromosome

• The entire genome of E. coli K-12 has been
  sequenced and has been found to be 4,639,221 bp
  with 4288 open reading frames, corresponding to
  88 of the genome (what are these and what is the
  rest of the DNA used for?).
• The map distances on this genome are given in
  minutes of transfer, in which 0 time origin of
  transfer and 100 min. time the whole chromosome
  takes to be transferred from an Hfr strain to an
  F- strain.

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The Bacterial Chromosome