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Microbial Genetics
Chapter 9
(p. 251-265)
Copyright © The McGraw-Hill Companies, Inc) Permission required for reproduction or display.
Learning Objectives
• Explain the regulation of gene expression in
bacteria by induction and repression
• Classify mutations by type, define mutagen.
• Discuss two ways mutations can be repaired
• Outline the methods of direct and indirect
selection of mutants
• Identify the purpose and outline the procedure
for Ames test
• Compare the mechanisms of genetic
recombination in bacteria: transformation,
conjugation, and transduction
Regulation of Bacterial Gene Expression
• Constitutive enzymes are expressed at a fixed
rate (i.e, they are on all the time)
• Other enzymes are expressed only as needed.
They are usually under tight control
– Repressible enzymes
– Inducible enzymes
Regulation of Transcription
• Groups of coordinately expressed and
regulated genes are called operons
• Regulatory proteins bind to operators
• Transcription can be turned on or off
Inducible Operons:
Lac operon
a. In absence of the
substrate (lactose) the
operon is off.
b. When substrate is
present the operon is
on.
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Repressible Operons:
Arg operon
a. Nutrient product
(arginine) is being
used by the cell. The
operon is on.
b. Nutrient product
builds up, the operon
is off.
Mutations
• Change in the genetic code
• Wild type: strain expressing natural, non-mutated
characteristic
• Mutant: strain expressing mutated gene
• Mistakes during replication or damage to DNA
• Wrong bases incorporated
– Transition
AG or CT
– Transversion
A(C or T) or G(C or T)
• Insertion or deletion of bases
• Most are lethal, some are beneficial (e.g. drug
resistance)
Replica Plating
Point Mutations
A single base is altered in the sequence
• Silent mutation:
TAT to TAC >> Tyr to Tyr
• Missense mutation: TAT to TTT >> Tyr to Phe
• Nonsense mutation: TAT to TAA >> Tyr to stop
Potential Reading Frames
• Since triplet codons are read, there are three
reading frames in the forward direction.
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Frame Shift Mutations
• Addition or deletion of 1 or 2 bases knocks the
sequence out of frame
• The whole amino acid sequence changes, usually
results in a truncated (shortened) protein
• If the gene is essential, the mutation is lethal.
• Insertions or deletions in multiples of three may
be tolerated
• These are back in frame again
• Can still be a big problem (Cystic Fibrosis, ∆508)
Causes of Mutations
• Spontaneous mutations during replication (1 in 1051010)
• Induced mutations
• Physical: Electromagnetic radiation
– X-rays, gamma rays nick DNA
– UV light causes T-T dimers to form
• Chemicals
– Analogs of bases
– Base-modifying chemicals
• Nitrosoguanidine, nitrous acid
– Intercalators insert between bases
• Cause frameshift mutations
Ames Test
• Quick test for
mutagen strength.
• His- Salmonella
typhimurium
• Mutagen reverts cells
to His+
• The degree of
mutagenicity is
calculated
Repair of Mutations
• Proofreading by DNA
polymerase III
• Photoactivation or
light repair by DNA
photolyase
• Excision repair by
DNA polymerase I
and ligase.
DNA Recombination
• Horizontal gene transfer:
Transformation (chromosomal DNA fragments)
Conjugation (plasmids)
Transduction (bacterial viruses)
Transformation
• Transfer of “naked” DNA.
• Griffith worked with Streptococcus pneumoniae
 Encapsulated: smooth colony appearance (S),
virulent
 Lacking a capsule: rough colony appearance
(R), non-virulent
• Dead virulent bacteria were able to “transform”
live non-virulent bacteria
The Transforming Principle
Strain of
Colony
Cell Type
Effect
Strain of
Colony
Effect
Cell Type
Capsule
Smooth
(S)
(a)
No capsule
LiveS
strain
Rough (R)
Dies
(b)
Heat-killed
S strain
Survives
Live R strain
Heat-killed S
Live S and R strains
strain
Survives
(c)
LiveR
strain
(d)
isolated from dead
mouse
Dies
Competence
• Non-specific ability to take up exogenous
soluble fragments of DNA
• Some bacteria are always naturally competent
• Others regulate competence
• Still others need to be coaxed
– Calcium chloride, chemicals, heat-shock, or
electroporation
Physical
Conjugation
F factor
Bacterial
chromosome
F+
Sex pilus makes
contact with F*
recipient cell.
F–
F+
F–
Sex pilus
contracts,
bringing cells
together.
Conjugation
• Plasmid-directed
transfer of plasmid DNA
• Requires cell contact via
pilus
F Factor
• Donor (F+) cell makes a
copy of the F factor
• F factor is transferred
into the recipient (F+)
cell via pilus
• Medical importance: R
factors transfer
resistance to antibiotics
Hfr Strains
• High frequency of
recombination
• Integrated F factor
(episome)
• Conjugal transfer
• Incorporation of new genes
into the chromosome
• Used earlier to map the
genome.
Transduction
• Bacterial virus (bacteriophage) serves as the
carrier of DNA from the donor cell to a
recipient cell
• Generalized transduction
• Specialized transduction
Generalized
Transduction
• Phage injects its DNA
into a cell
• Phage DNA serves as a
template for new
phage DNA and protein
synthesis
• Packaging of a random
fragment of bacterial
DNA, and transfer to
newly infected
bacterial cells
Specialized
Transduction
• Only certain genes
transferred.
• Medical importance:
toxins of Corynebacteria
diphtheriae, Clostridium
spp., and Streptococcus
pyogenes are transferred
by specialized
transduction
Telling Them Apart
• Transformation – involves competency, transfers
naked DNA (chromosomal or plasmid)
• Conjugation - involves cell contact through a
pilus, transfers plasmid DNA
• Transduction - involves bacteriophage, transfers
chromosomal DNA (specific or non-specific)
• DNase sensitivity (transformation)
• 0.2 µm membranes (conjugation)
Transposons
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
• Jumping genes
• Mobile genetic
elements
• Move from place to
place in the genome,
plasmids, and viral
genomes
• Disrupt genes when
they land
• May mobilize other
genes (like antibiotic
resistance)
(1)
(2)
(3)
(4)
Pathogenicity Islands
• Found in pathogens, improve pathogenicity:
Yersinia pestis: ability to scavenge iron
Staphylococcus aureus: ability to produce exotoxin
• Discovered because of the different G+C
concentration and presence of bacteriophage
or transpozon sequences.