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Bacterial Genetics
Overview
Two general mechanisms of genetic change in bacteria:
Mutation - alteration in existing DNA sequence
Spontaneous
Induced (caused by mutagens)
DNA transfer - acquisition of DNA from another source
Overview
Two general mechanisms of genetic change in bacteria:
Mutation - alteration in existing DNA sequence
Spontaneous
Induced (caused by mutagens)
DNA transfer - acquisition of DNA from another source
Why study bacterial genetics?
Model system
•Spontaneous mutations occur in all cells at a very low frequency (≈one
per billion nucleotides)
•Bacteria quickly grow to high concentrations (109/ml) in culture,
making it possible to study rare occurrences
•Test chemicals for potential carcinogens
Understand bacterial adaptation
•Resistance to antimicrobial drugs
Agency Urges Change in Antibiotics for Gonorrhea
By LAWRENCE K. ALTMAN
Published: NY Times April 13, 2007
The rates of drug-resistant gonorrhea in the United States have
increased so greatly in the last five years that doctors should now
treat the infection with a different class of antibiotics, the last line
of defense for the sexually transmitted disease, officials said
yesterday…..
No new antibiotics for gonorrhea are in the pipeline, officials of the
centers told reporters by telephone.
“Now we are down to one class of drugs,” said Dr. Gail Bolan, an
expert in sexually transmitted diseases at the California
Department of Health Services. “That’s a very perilous situation to
be in.”
Overview
Two general mechanisms of genetic change in bacteria:
Mutation - alteration in existing DNA sequence
Spontaneous
Induced (caused by mutagens)
DNA transfer - acquisition of DNA from another source
Why study bacterial genetics?
Model system
•Spontaneous mutations occur in all cells at a very low frequency (≈one
per billion nucleotides)
•Bacteria quickly grow to high concentrations (109/ml) in culture,
making it possible to study rare occurrences
•Some mutagens are carcinogens
Understand bacterial adaptation
•Resistance to antimicrobial drugs
•Acquisition of disease-causing traits
Terms
Phenotype
Genotype
Terms
Phenotype - the observable characteristics of an organism
Genotype - the sequence of nucleotides in the DNA of an organism
Wild type - characteristics similar to the organism as it occurs in nature.
Prototroph - requires the same nutrients as the wild type.
Auxotroph - a strain that has lost the ability to synthesize a specific
compound; as a consequence, that compound must be supplied as a nutrient in
the growth medium.
disrupt gene required for histidine synthesis
Prototroph
When studying mutations, you only see what you look for
His- auxotroph
Part I Mutation
•How mutations occur, and their
consequences
•How cells can repair errors/damage
•How we can select (and therefore,
study) mutants
Spontaneous Mutation
Mistakes during replication
Base substitution
TGT
cysteine
Silent mutation
Missense mutation
Nonsense mutation
TGC
cysteine
TGG
tryptophan
TGA
Stop codon
No consequence
Consequence varies
Truncated protein;
generally non-functional
Spontaneous Mutation
Mistakes during replication
Base substitution
Removal or addition of nucleotides
TGTTTGACCTAGGT
Spontaneous Mutation
Mistakes during replication
Base substitution
Removal or addition of nucleotides
TGTTTGACCTAGGT
TGT TTG ACC TAG GT
TGTTGACCTAGGT
TGT TGA CCT AGG T
Frameshift mutation
• Generates an entirely different set of triplets
• Often, a stop codon is generated
Spontaneous Mutation
Mistakes during replication
Base substitution
Removal or addition of nucleotides
Spontaneous Mutation
Transposons “jumping genes”
• Insertional inactivation of the gene in which the transposon lands
• A transposon can insert elsewhere in the same DNA molecule, or
into an entirely different DNA molecule
• Some transposons simply “hop”; others replicate then hop
Summary
Mutations
spontaneous
mistakes during replication
base substitution
addition/removal of nucleotides
transposable elements
induced
Induced Mutation
Chemical mutagens (potential carcinogens)
Chemicals that modify purines and pyrimidines
Alter the base-pairing properties
Induced Mutation
Chemical mutagens
Chemicals that modify purines and pyrimidines
Alter the base-pairing properties
Example: nitrous acid strips the amino group from nucleotides
:G
:A
Induced Mutation
Chemical mutagens
Chemicals that modify purines and pyrimidines
Alter the base-pairing properties
Example: nitrous acid strips the amino group from nucleotides
Base analogs
Resemble nucleotide bases; erroneously incorporated into DNA
Analog base-pairs with a different nucleotide
T
C
Induced Mutation
Chemical mutagens
Chemicals that modify purines and pyrimidines
Alter the base-pairing properties
Example: nitrous acid strips the amino group from nucleotides
Base analogs
Resemble nucleotide bases; erroneously incorporated into DNA
Analog base-pairs with a different nucleotide
Intercalating agents
Insert between base-pairs, pushing
nucleotides apart; extra nucleotide
may then be erroneously added
during replication
Induced Mutation
Transposons
Intentional use of an agent that naturally creates spontaneous
mutations
Induced Mutation
Radiation
Ultraviolet irradiation
Causes formation of covalent bonds
(thymine dimers) between adjacent
thymine bases
Distorted DNA can be repaired, but
the process (SOS repair) may
introduce errors
High doses are used to sterilize
surfaces, lower doses to introduce
mutations
X rays
Causes double- and single-stranded
breaks in DNA
Summary
Mutations
spontaneous
mistakes during replication
base substitution
removal or addition of nucleotides
transposable elements
induced
chemical mutagens
radiation
transposons
DNA Repair
Repair of errors in base incorporation
DNA polymerase
proofreading
Mismatch repair
excision/replacement
Repair of thymine dimmers
DNA Repair
Repair of errors in base incorporation
DNA polymerase
proofreading
Mismatch repair
excision/replacement
Repair of thymine dimmers
Light reactivation (photorepair)
DNA Repair
Repair of errors in base incorporation
DNA polymerase
proofreading
Mismatch repair
excision/replacement
Repair of thymine dimmers
Light reactivation (photorepair)
Excision repair (dark repair; lightindependent repair)
DNA Repair
Repair of errors in base incorporation
DNA polymerase
proofreading
Mismatch repair
excision/replacement
Repair of thymine dimers
Light reactivation (photorepair)
Excision repair (dark repair; lightindependent repair)
Repair of Modified Bases
Glycosylase removes oxidized guanine
SOS repair
Induction of SOS system
New polymerase (tolerates “slop”)
Mutant Selection
Direct selection
Obtain resistant mutants
(ex. antibiotic resistant)
Obtain prototrophs that have
reverted from auxotrophs
Prototroph
(revertant)
Auxotrophs
Application of direct selection
Ames Test - screens for mutagens
(used to narrow down list of
possible carcinogens)
Minimal medium
(glucose-salts)
Enriched complex
medium
The Ames Test
Also do expt.
with liver
extract added
Mutant Selection
Indirect selection (replica plating)
Obtain auxotrophs
106 prototrophs
1 auxotroph
Indirect selection (replica plating)
Obtain auxotrophs
Indirect selection (replica plating)
Obtain auxotrophs
Joshua and Esther Lederberg
Summary
Mutations
spontaneous
mistakes during replication
transposons
induced
chemical mutagens
radiation
transposons
Repair
repair of errors in base incorporation
repair of thymine dimmers
SOS repair
Selecting mutants
direct - obtain antibiotic resistant mutants, Ames test
indirect - obtain prototrophs
Part II DNA Transfer
Donor
Recipient
Horizontal (lateral) transfer
DNA Transfer
1920s; Frederick Griffithstrains of Streptococcus
pneumoniae that produce
capsules kill mice
“transforming principle”
(DNA)
DNA Transfer
1/109 =10-9
10-9 x 10-9 = 10-18
DNA Transfer
Donor
Recipient
To be stably maintained, transferred DNA must either be a plasmid (has an
origin of replication), or integrate into the host cell’s genome
DNA Transfer
Donor
Recipient
To be stably maintained, transferred DNA must either be a plasmid (has an
origin of replication), or integrate into the host cell’s genome
DNA Transfer
Donor
Recipient
Integrate into host genome by
Homologous recombination (site-specific recombination)
DNA Transfer
Donor
Recipient
Integrate into host chromosome by
Homologous recombination (site-specific recombination)
DNA Transfer
Donor
Recipient
Integrate into host chromosome by
Homologous recombination (site-specific recombination)
DNA Transfer
Donor
Recipient
Integrate into host chromosome by
Homologous recombination (site-specific recombination)
heteroduplex
replication
DNA Transfer
Donor
Recipient
Integrate into host chromosome by
Homologous recombination (site-specific recombination)
DNA Transfer
Donor
Recipient
Horizontal (lateral) gene transfer
A+, B-
A-, B+
B-
AA+, B+
A-, BA+, B-
DNA Transfer
Donor
DNA-mediated transformation
Transduction
Conjugation
Recipient
DNA-Mediated Transformation
Uptake of naked DNA
Process is sensitive to the addition of DNAse
DNA-Mediated Transformation
Uptake of naked DNA
Process is sensitive to the addition of DNAse
Recipient cell must be competent
Natural competence
Observed in only certain species
Example - Streptococcus pneumoniae (GPC)
•Becomes competent in late log phase
•Competent cell binds ds DNA
•Enzymes cut DNA into smaller fragments (5 - 15 kb)
•Single strand is taken up by cell
Example - Haemophilus influenzae (GNR)
•Cell binds DNA only from related species
•Takes up ds DNA
Artificial competence
In the laboratory, treat cells with specific chemicals (plasmids taken up)
Conjugation
Requires cell-to-cell contact
Involves a conjugative plasmid
F plasmid (fertility plasmid) serves as a model
Three types of donors:
F+
Hfr
F’
Conjugation: F+ donor
“male”
“female”
Conjugation: F+ donor
Conjugation: F+ donor
Conjugation: F+ donor
In donor cell, replication replaces strand
that’s being transferred
In recipient cell, complement to
transferred strand is synthesized
Conjugation: F+ donor
Note: some R plasmids (encode
resistance to one or more antibiotics)
are conjugative
F+ + F-  F+ + F+
In donor cell, replication replaces strand
that’s being transferred
In recipient cell, complement to
transferred strand is synthesized
Conjugation
Formation of an Hfr cell
Figure 8.25
Hfr = High-frequency recombination
Conjugation: Hfr donor
Conjugation: Hfr donor
• Some F plasmid DNA is transferred
first, followed by chromosomal DNA
• In donor cell, replication replaces
strand that’s being transferred
• In recipient cell, complement to
transferred strand is synthesized
Cells inevitably separate before entire
chromosome is transferred
Conjugation: Hfr donor
• Some F plasmid DNA is transferred
first, followed by chromosomal DNA
• In donor cell, replication replaces
strand that’s being transferred
• In recipient cell, complement to
transferred strand is synthesized
Cells inevitably separate before entire
chromosome is transferred
Conjugation: Hfr donor
Hfr + F-  Hfr + F-
Significance of Hfr strains:
•Chromosomal transfer
•Allowed mapping of E. coli
chromosome
Recombinant DNA and
Biotechnology
DNA
RNA
protein
Preview
• Fundamental tools of biotechnology
• Molecular Cloning
• PCR
Fundamental Tools Used in Biotechnology
•Restriction Enzymes - used to cut DNA at specific sequences
•Gel Electrophoresis - used to separate nucleotide (or protein) fragments
•DNA Probes - used to “find” specific nucleotide sequences
•Primers - used to initiate DNA synthesis at a specific location
Restriction Enzymes - cut DNA
1
reflects name of org. from which enz.
was first isolated
palindrome
Restriction Enzymes - cut DNA
Restriction Enzymes - cut DNA
Gel Electrophoresis - separates fragments
Note: millions of each “player”
Gel Electrophoresis - separates fragments
DNA Probes - “find” sequences
Primers - dictate sites of synthesis initiation
Techniques Used in Genetic
Engineering
DNA
Techniques Used in Genetic
Engineering
DNA
Techniques Used in Genetic
Engineering
Self-replicating DNA
(ex. plasmid)
insert
vector
recombinant molecule
Cloning Overview
•
•
•
•
Cut out the gene of interest from donor
Put the gene into a vector
Transfer the vector into a recipient
Select for the recipient from a mixed
population
Cloning Vectors





1) Plasmids
2) Bacteriophage lambda
3) P1 Phage
4) Cosmids
5) Yeast artificial chromosomes (YAC)
Characteristics of cloning vectors



1) Should have it’s own replicon i.e., be
capable of autonomous replication in the
host cell
2) Should carry one or more selectable
markers that permit identification of parent
and recombinant vectors
3) Restriction sites in non-essential regions
of DNA into which foreign DNA can be
inserted
Molecular Cloning
Genetically Engineering
Bacterial Cells
Note: millions of each “player”
Applications of molecular cloning
• Medical application
– gene therapy
– production of drugs (insulin, antibiotics, hormones)
• Agricultural application
– Nutrients enriched food
– Nitrogen fixing plants
• Scientific research
(PCR) Polymerase chain reaction
Amplifies target sequence
ds DNA containing the target
Taq polymerase (Thermus aquaticus)
nucleotides
primers
thermocycler
PCR
• Medical application:
– Genetic diseases
– Infectious disease
• Forensic science:
– Identify criminals
– parental identification
• Research application
DDC: DNA Test Sets Inmate Free After 18 Years
Forensic Resources
DNA Diagnostics Center’s Forensics Division provided
the DNA testing that eventually resulted in the release of
inmate Robert McClendon. McClendon had spent 18
years in prison, convicted of a child rape that he has
always maintained he did not commit.
McClendon’s is 1 of 30 cases in Ohio that were identified
to have “legitimate claims of innocence” in an
investigation conducted by The Columbus Dispatch
together with the Ohio Innocence Project (OIP). DDC
has volunteered to provide DNA testing for the OIP's
post-conviction cases free of charge.