Bacterial Genetics
Chapter 8
Diversity in Bacteria
 Bacteria use three different mechanism to
adapt to changing environments



Mutation
Gene transfer
Regulation of gene expression
Spontaneous Mutation
 Spontaneous mutations occur in natural
environment

Occur infrequently and randomly
 Rate of mutation


probability that a mutation will be observed in
a given gene each time the cell divides
Rate is generally between 1 in 10,000 and 1 in
a trillion

Low rate is due to cellular repair mechanisms
Spontaneous Mutation
 Mutations are stable heritable changes in the
base sequence of DNA
 Can occur from



Base substitutions
Removal or addition of nucleotides
Transposable elements
Spontaneous Mutation
 Base substitutions


Most common type of mutation
Results from mistakes during DNA
synthesis

Incorrect base is incorporated
into DNA
 Point mutations
 Occur when one base pair is
changes
 Missense mutation
 Mutation resulting from amino
acid substitution is called
 Nonsense mutation
 Mutation that changes a
amino acid codon to a stop
codon
Spontaneous Mutation
 Removal and addition of
nucleotides


Shifts the translational
reading frame
 Shifts the codons
Mutation called frameshift
mutation
 Affects all amino acids
downstream from
addition or deletion
 Mutations frequently
result in premature stop
codons
Spontaneous Mutation
 Transposable elements

Special segments of DNA that move
spontaneously from gene to gene


Elements called transposons
Transposons disrupt proper function of gene

Gene or gene product generally non functional
Induced Mutations
 Mutations are essential for understanding genetics
 Mutations can be intentionally produced to
demonstrate function of particular gene or set of genes
 These mutations are termed induced
 Mutations can be induced via
 Chemical mutagens
 Transposition
 Radiation
Induced Mutations
 Chemical mutagens

Nitrous acid

Converts amino group to a keto group
 Changes cytosine to uracil
 Uracil binds to adenine while cytosine binds to
guanine

Alkylating agents

Largest group of chemical mutagens
 Alters hydrogen bonding of bases
 Nitrosoguanine is common alkylating agent
Induced Mutations
 Chemical mutagens
 Base analogs
 Chemicals that are
structurally similar to the
nitrogenous bases but have
slightly altered base pairing
properties
 Base analogs include:
 2-aminopurine which
incorporates in the place
of adenine but binds with
cytosine
 5-bromouracil which
incorporates in the place
of thymine but binds with
guanine
Induced Mutations
 Chemical mutagens

Intercalating agents

Molecules that insert themselves between
adjacent bases
 Creates space between bases
 Extra base is often added to fill space

Ethidium bromide is common intercalating agent
 Potential carcinogen
Induced Mutations
 Transposition


Common procedure used to induce mutation
in laboratory
Gene that receives transposon will undergo a
knockout mutation

Termed insertion mutation
Induced Mutations
 Radiation
 Two types
 Ultraviolet light
 Causes covalent bonding between
adjacent thymine bases
 Forms thymine dimers which
distorts DNA

X rays
 Cause breaks and alterations in
DNA
 Breaks that occur on both
strand are often lethal
Repair of Damaged DNA
 Repair of base substitution
 Cells develop two methods of
repair
 Proofreading
 DNA polymerase has
proofreading function
 Able to excise incorrect base
and replace with correct one

Mismatch repair
 Endonuclease enzyme
removes short stretch of
nucleotide
 DNA polymerase fills gap
 DNA ligase joins ends
Mismatch repair
Repair of Damaged DNA
 Repair of thymine dimers

Thymine dimer repair
Two mechanisms

Light repair
 A.k.a photoreactivation
 Enzyme uses visible light to break covalent bonds between bases

Dark repair
 A.k.a excision repair
 Endonuclease excises damages section
 New section replicated and joined to original strand
Repair of Damaged DNA
 Repair of modified bases
 Enzyme cuts DNA backbone and removes
base
 DNA polymerase incorporates new base
 SOS repair
 Last ditch effort to bypass damage
 Damage induces SOS system

Produces new DNA polymerase
 Highly error prone
 Mutations can arise from synthesis with new
enzyme
Mutations and Their
Consequences
 Mutation provide organism way to respond to
changing environments
 Environment selects for cells suited to survive

Environment does not cause mutation
Mutation Selection
 Major problem in induced mutation is
identifying bacteria with desired mutation
 Techniques used include
 Direct selection

Involves inoculating population of
bacteria on medium on which only
mutants will grow
 Used to select antimicrobial resistant
organisms

Indirect selection

Required to isolate organisms that
require growth factor that parent
strain does not
 Replica plating
Replica Plating
Mutation Selection
 Testing for cancer causing
chemicals
 Many mutagens are also
carcinogens


Cancer causing agents
Microbes used to test potential
carcinogenic activity


Tests are based on effect
chemical has on microbial
DNA
Ames test common chemical
carcinogen test
 Test rate of reversion of
Salmonella auxotroph
 Also test potential lethality
Mechanisms of Gene Transfer
 Genes are naturally transferred between
bacteria using three mechanisms



DNA-mediated transformation
Transduction
Conjugation
Mechanisms of Gene Transfer
 Gene exchange in bacteria

Two event occur in gene exchange

1.
Donor DNA is transferred and accepted
by the recipient cell
 Donor DNA is transferred one of three ways
 Transformation
 Transduction
 Conjugation

2.
Donor DNA is integrated onto the
recipient cell’s chromosome
Mechanisms of Gene Transfer
 DNA-mediated transformation


Definition: the transfer of naked DNA from
one bacterium to another
Discovered by Fredrick Griffith in 1928 while
working with Streptococcus pneumoniae
Mechanisms of Gene Transfer
 Griffith realized S. pneumonic existed in two forms
 Encapsulated, virulent form
 Smooth in appearance
 Nonencapsulated, avirulent form
 Rough in appearance
 Griffith wondered if injections with the smooth strain
could be used as a vaccine against pneumonia
 He injected mice with the different strains and recorded
his results
Griffith Experiment
Mechanisms of Gene Transfer
 DNA-mediated transformation
 Involves the transfer of naked DNA from the
environment to the recipient cell

Cells rupture during the stationary and death phase
 The chromosome breaks into small pieces and explode
through the ruptured cell wall


Recipient cell picks up piece of the naked DNA
The naked DNA is integrated onto the recipient
chromosome

Replaces the homologous gene on the chromosome
of the recipient cell
Mechanisms of Gene Transfer
 DNA-mediated transformation

Natural transformation occurs when bacterial
cells are “competent”


Competence is a condition in which bacterial cells
are capable of taking up and integrating larger
fragments of DNA
Competence occurs during the late log, early
stationary phase
 Natural transformation occurs in four stages

Entry of the DNA


Integration of the donor DNA



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Donor DNA is integrated by hydrogen
bonding
Enzymes cleave recipient DNA
Donor DNA is put in place
Mismatch repair



Only single strands enter, double
strands are degraded
Repair mechanism remove either
donor or recipient DNA that doesn’t
match
Repairs with correct nucleotides
Cell multiplication

Transformed cells multiply under
selective conditions in which nontransformed cell will not grow
Transformation
Mechanisms of Gene Transfer
 Transduction


Bacterial DNA that is transferred from donor to
recipient via a bacterial virus (bacteriophage)
Two types of transduction

Generalized
 Any gene of donor can be transferred

Specialized
 Only specific genes can be transferred
 Transduction is a mis-packaging
of DNA during viral replication


The mis-packaged phage infects
a new bacterial cell and insert
the donor DNA into the recipient
cell
The donor DNA is integrated and
mismatched pairs are repaired
Transduction
Mechanisms of Gene Transfer
 Conjugation – only form of gene exchange in which
donor survives

Conjugation is mediated by a plasmid
 Plasmid is self replicating extrachromosomal piece of
DNA
 Can code for traits that give bacteria advantage


Conjugation requires direct contact between cells
Cells must be of opposite mating types
 Donor cells carry a plasmid that codes for fertility
factor or “F factor”
 This cell is designated F+

Recipient cell does not carry a plasmid
 This cell is designated F-
 During conjugation, the
plasmid is replicated in
the donor cell and is
transferred to the
recipient

After plasmid is
transferred, F- cell
becomes F+
Conjugation
 In some F+ cells, the F
factor integrates onto the
host chromosome


Converts F+ to Hfr
 Hfr= High frequency of
recombination
Conjugation between Hfr
and F- cell results in only
a portion of the F factor
being replicated and
transferred
 F remain F
 F- has new
information but may
not have the F factor
gene
Conjugation - Hfr