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Chapter 8:
Microbial Genetics
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
Flow of Genetic Information
Horizontal gene
transfer
(Vertical gene
transfer)
Figure 8.2
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
DNA:
Semiconservative 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
DNA
5’
3’
5’
3’
3’
DNA polymerase
3’
5’
5’
Figure 8.5
DNA:
Semiconservative 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)
DNA:
Semiconservative Replication
Figure 8.6
DNA:
Replication of Bacterial DNA
• DNA replication in bacteria is often bidirectional
Figure 8.7
Prokaryotic Transcription:
DNA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 the
terminator sequence
• mRNA: No further processing is necessary before
translation in prokaryotic organisms
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
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
Translation
mRNA codon sequences
Figure 8.9
Translation
Figure 8.10.1
Translation
Figure 8.10.2
Translation
Figure 8.10.3
Translation
Figure 8.10.4
Translation
Figure 8.10.5
Translation
Figure 8.10.6
Translation
Figure 8.10.7
Translation
Figure 8.10.8
http://www-class.unl.edu/biochem/gp2/m_biology/animation/gene/gene_a3.html
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 gene’s transcription
◦ Default transcription status is on
─ Inducible genes
◦ Regulatory mechanism to permit a gene’s transcription
◦ Default transcription status is off
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
Operon
• A regulatory gene (upstream of the operon) encodes a repressor
protein
Figure 8.12
Operon Model of Gene Expression:
Tryptophan synthesis operon
• Genes for the enzymes responsible for tryptophan
synthesis are organized in an operon
─ Repressible operon
Tryptophan Synthesis Operon:
Normal (Default) Cellular Conditions
• Tryptophan (a common amino acid) is constantly synthesized
• Default: Repressor inactive = operon on
Figure 8.12
Tryptophan Synthesis Operon:
Presence of excess tryptophan
• Tryptophan acts as a corepressor (activates repressor protein)
• Repressor active = operon off
Figure 8.12
Operon Model of Gene Expression:
The lac operon
• lac operon: three enzymes necessary for lactose
catabolism in E. coli
─ Inducible operon
Operator
The lac operon:
Absence of lactose
• Default: Repressor active = operon off
(Operator)
Figure 8.12
The lac Operon:
Presence 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
The lac Operon:
Presence 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
The lac Operon:
Presence of lactose AND absence of glucose
• Cellular levels of glucose and cAMP are inversely
proportional
─ As glucose is depleted, cAMP accumulates
Mechanisms for bacteria to acquire
new genotypes:
• Mutation
• Horizontal gene transfer
• Plasmids
• Transposons
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
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
Sickle cell anemia: missense mutation (GluVal) in hemoglobin
http://www.huck.psu.edu
http://www.nhlbi.nih.gov
Types of DNA Mutations
• Nonsense mutation
Point mutation that results in a
nonsense (stop) codon
Figure 8.17a, c
Types of DNA Mutations
• Frameshift mutation
Insertion or deletion of one or more
nucleotide pairs
- shift in translational reading frame
Figure 8.17a, d
Mutagens:
Radiation
• UV Radiation: causes
thymine dimers
Nucleotide excision repair
• Ionizing radiation: free
radicals modify nucleotides or
break sugar-phosphate
backbone
• Cells have DNA repair
mechanisms
─ Nucleotide excision repair
Figure 8.19
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
The Ames Test for Chemical
Carcinogens
Selecting
for
revertants
(cells that
were mutated
by the
chemical)
Figure 8.22
Plasmids
Examples:
• Conjugative plasmid
(F factor)
Carries genes for sex
pili and transfer of the
plasmid itself
• R factors
Encode antibiotic
resistance
• Virulence factors
Encode factors that
increase the pathogenicity
of an organism (toxins)
Plasmids:
Virulence factors
• Infant diarrhea and traveler’s 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
Transposons
• Segments of DNA
that can jump
around
http://fire.biol.wwu.edu/trent/trent/index.html
Figure 8.30a, b
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…)
Horizontal gene transfer:
Transformation
• Transformation:
genes are
transferred as
“naked” DNA in
solution
Recombinant DNA
Recombinant cell
Figure 8.25
Horizontal gene transfer:
Conjugation
• Requires direct cell-to-cell contact
• Conjugating cells must be of opposite mating types
─ Donors: F+
─ Recipients: F-
Figure 8.26a
Horizontal gene transfer:
Conjugation
Figure 8.26
Horizontal gene transfer:
Transduction
• Transduction: Bacterial DNA is transferred from a
donor cell to a recipient cell inside a bacteriophage
─ Bacteriophage: virus that infects bacteria
Horizontal gene transfer:
Transduction
Phage protein coat
Bacterial
chromosome
Recombinant
1 A phage infects the
donor bacterial
cell.
2 Phage DNA and proteins
are made, and the
bacterial chromosome is
broken down into pieces.
Bacterial
DNA
Donor
bacterial
DNA
Phage
DNA
3 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.
Recipient cell
4 A phage carrying bacterial
DNA infects a new host cell,
the recipient cell.
Recipient
bacterial
DNA
Recombinant cell
5 Recombination can occur,
producing a recombinant
cell with a genotype
different from both the
donor and recipient cells.
Figure 8.27
Mechanisms for bacteria to acquire
new genotypes:
• Mutation
• Plasmids
• Transposons
• Transformation
• Conjugation
• Transduction
Horizontal gene transfer