Chapter 8: Microbial Genetics

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Chapter 8Microbial Genetics
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Terminology

• Genome All of the genetic material (DNA) in a
  cell
• Gene Segment of DNA that encodes a
  functional product (protein)
• Genotype Genetic makeup of an organism
• Phenotype Expressed properties
• Genetically determined characteristics
• Manifestation of genotype

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Flow of Genetic Information
Horizontal gene transfer
(Vertical gene transfer)
Figure 8.2
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DNA

• Polymer of nucleotides adenine, thymine,
  cytosine, guanine
• Double helix associated with proteins
• "Backbone" is deoxyribose-phosphate
• Strands held together by hydrogen bonds between
  base pairs (A-T and C-G)
• Strands are antiparallel

Figure 8.4
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DNASemiconservative Replication

• Unwinding
• Free nucleotides are linked to the growing strand
  by DNA polymerase
• Nucleotide addition occurs only in the 5?3
  direction
• The two daughter strands must grow in different
  directions

Figure 8.3
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DNA
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5
3
3
3
DNA polymerase
3
5
5
Figure 8.5
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DNASemiconservative Replication

• Daughter DNA strands are extended by DNA
  polymerase enzyme
• In the 5? ? 3? direction
• Initiated by an RNA primer
• Leading daughter strand synthesized continuously
• Lagging daughter strand synthesized
  discontinuously
• Okazaki fragments
• RNA primers are removed (by DNA polymerase) and
  Okazaki fragments joined (by DNA ligase)

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DNASemiconservative Replication
Figure 8.6
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DNAReplication of Bacterial DNA

• DNA replication in bacteria is often bidirectional

Figure 8.7
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Prokaryotic TranscriptionDNA?RNA

• DNA is transcribed to make RNA
• mRNA
• rRNA
• tRNA
• Transcription begins when RNA polymerase binds to
  the promotor sequence of a gene
• Transcription proceeds in the 5? ? 3? direction
• Transcription stops when it reaches
  theterminator sequence
• mRNA No further processing is necessary before
  translation in prokaryotic organisms

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RNA processing in Eukaryotes

• Eukaryotic organisms mRNA must be processed
  before leaving the nucleus to be translated
• Introns must be removed (spliced out)

Figure 8.12
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Translation

• Site of translation Ribosomes
• mRNA is translated in groups of 3 nucleotides
  called codons
• Translation of mRNA begins at the start codon
  AUG
• Prokaryotes Formylmethionine
• Eukaryotes Methionine
• Translation ends at a STOP codon UAA, UAG, UGA

Figure 8.2
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Translation
mRNA codon sequences Figure 8.9
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Translation
Figure 8.10.1
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Translation
Figure 8.10.2
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Translation
Figure 8.10.3
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Translation
Figure 8.10.4
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Translation
Figure 8.10.5
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Translation
Figure 8.10.6
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Translation
Figure 8.10.7
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Translation
Figure 8.10.8
http//www-class.unl.edu/biochem/gp2/m_biology/ani
mation/gene/gene_a3.html
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Regulation of Bacterial Gene Expression

• Constitutive proteins are expressed at a fixed
  rate
• Regulated proteins are expressed only as needed
• Repressible genes
• Regulatory mechanism to inhibit a genes
  transcription
• Default transcription status is on
• Inducible genes
• Regulatory mechanism to permit a genes
  transcription
• Default transcription status is off

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Operon Model of Gene Expression

• Operon promoter operator structural genes
• Operator DNA sequence that interacts with
  regulatory proteins (i.e. a repressor)
• Gives stop or go signal for transcription
• Structural genes are transcribed as one unit
• i.e. a single, polycistronic mRNA

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Operon

• A regulatory gene (upstream of the operon)
  encodes a repressor protein

Figure 8.12
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Operon Model of Gene ExpressionTryptophan
synthesis operon

• Genes for the enzymes responsible for tryptophan
  synthesis are organized in an operon
• Repressible operon

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Tryptophan Synthesis OperonNormal (Default)
Cellular Conditions

• Tryptophan (a common amino acid) is constantly
  synthesized
• Default Repressor inactive operon on

Figure 8.12
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Tryptophan Synthesis OperonPresence of excess
tryptophan

• Tryptophan acts as a corepressor (activates
  repressor protein)
• Repressor active operon off

Figure 8.12
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Operon Model of Gene ExpressionThe lac operon

• lac operon three enzymes necessary for lactose
  catabolism in E. coli
• Inducible operon

Operator
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The lac operonAbsence of lactose

• Default Repressor active operon off

(Operator)
Figure 8.12
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The lac OperonPresence of lactose

• Some lactose enters the cell and is converted to
  allolactose
• Allolactose isomer of lactose, acts as an
  inducer
• Repressor cannot bind the operator RNA pol
  transcribes the operon
• Inactive repressor operon on

Figure 8.12
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The lac OperonPresence of lactose AND absence
of glucose

• Preferential carbon source is always glucose
• For maximal lac operon transcription
• Lactose is present
• Glucose is absent

Figure 8.13
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The lac OperonPresence of lactose AND absence
of glucose

• Cellular levels of glucose and cAMP are inversely
  proportional
• As glucose is depleted, cAMP accumulates

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Mechanisms for bacteria to acquire new genotypes

• Mutation
• Horizontal gene transfer
• Plasmids
• Transposons

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Mutation

• Mutation Change in DNA sequence
• Mutations may be neutral, beneficial, or harmful
• Mutagen Agent (chemical, radiation, etc.) that
  causes mutations
• Spontaneous mutations Occur in the absence of a
  mutagen

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Types of DNA Mutations

• Point mutation Change in one
  nucleotide
• Missense mutation Point mutation that
  results in an amino acid change

Ser
Figure 8.17a, b
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Sickle cell anemia missense mutation (Glu?Val)
in hemoglobin
http//www.huck.psu.edu
http//www.nhlbi.nih.gov
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Types of DNA Mutations

• Nonsense mutation Point mutation that
  results in a nonsense (stop) codon

Figure 8.17a, c
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Types of DNA Mutations

• Frameshift mutation Insertion or deletion
  of one or more nucleotide pairs
• - shift in translational reading frame

Figure 8.17a, d
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MutagensRadiation
Nucleotide excision repair

• UV Radiation causes thymine dimers
• Ionizing radiation free radicals modify
  nucleotides or break sugar-phosphate backbone
• Cells have DNA repair mechanisms
• Nucleotide excision repair

Figure 8.19
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The Frequency of Mutation

• Low rate, random mutations are necessary for
  adaptation and evolution
• Spontaneous mutation rate 1 in 109 replicated
  base pairs
• Mutagens increase the mutation rate by 10 to 1000
  times
• Ames test for chemical carcinogens
• Tests for the ability of a chemical to increase
  the rate of mutation (i.e. Is X a mutagen?)
• Selects for mutated bacteria
• Revertant a cell that contains a mutation that
  corrects its original mutation

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The Ames Test for Chemical Carcinogens
Selecting for revertants (cells that were
mutated by the chemical)
Figure 8.22
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Plasmids

• Examples
• Conjugative plasmid Carries genes for sex(F
  factor) pili and transfer of the plasmid itself
• R factors Encode antibiotic resistance
• Virulence factors Encode factors that increase
  the pathogenicity of an organism (toxins)

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PlasmidsVirulence factors

• Infant diarrhea and travelers diarrhea are
  caused by a plasmid-carrying strain of E. coli
• Otherwise, this strain is harmless
• Clostridium tetani neurotoxin (causes tetanus) is
  encoded in a plasmid

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Transposons

• Segments of DNA that can jump around

http//fire.biol.wwu.edu/trent/trent/index.html
Figure 8.30a, b
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Genetic Transfer

• Vertical gene transfer
• Occurs during reproduction, between generations
  of cells
• Animals, plants, bacteria
• Horizontal gene transfer
• Transfer of genes between cells of the same
  generation
• Bacteria (3 mechanisms)

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Horizontal gene transferTransformation

• Transformation genes are transferred as naked
  DNA in solution

Recombinant DNA
Recombinant cell
Figure 8.25
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Horizontal gene transferConjugation

• Requires direct cell-to-cell contact
• Conjugating cells must be of opposite mating
  types
• Donors F
• Recipients F-

Figure 8.26a
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Horizontal gene transferConjugation
Figure 8.26
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Horizontal gene transferTransduction

• Transduction Bacterial DNA is transferred from a
  donor cell to a recipient cell inside a
  bacteriophage
• Bacteriophage virus that infects bacteria

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Horizontal gene transferTransduction
Phage protein coat
Bacterial chromosome
Recombinant
Phage DNA and proteins are made, and the
bacterial chromosome is broken down into pieces.
A phage infects the donor bacterial cell.
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Bacterial DNA
Donor bacterial DNA
Recipient bacterial DNA
Phage DNA
Recipient cell
Recombinant cell
Occasionally during phage assembly, pieces of
bacterial DNA are packaged in a phage capsid.
Then the donor cell lyses and releases phage
particles containing bacterial DNA.
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A phage carrying bacterial DNA infects a new host
cell, the recipient cell.
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Recombination can occur, producing a recombinant
cell with a genotype different from both the
donor and recipient cells.
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Figure 8.27
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Mechanisms for bacteria to acquire new genotypes

• Mutation
• Plasmids
• Transposons
• Transformation
• Conjugation
• Transduction

Horizontal gene transfer
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