Transcript chapter08

Bacterial Genetics
Chapter 8
The Problem of Antibiotic Resistance
Staphylococcus aureus
Common Gram+ bacterium
Multi-drug resistant strains are prevalent in hospitals
These are described as methicillin-resistant
Staphylococcus aureus (MRSA)
Antibiotic resistance is a genetic event
8.1 Diversity in Bacteria
Bacterial diversity
Bacteria have haploid genomes
Mutations in bacterial genes alters genotype
Genotype is the DNA-level information
encoded by the genome
Mutations also alter phenotype
Phenotype is principally controlled by
proteins by alteration of amino acid
composition
8.1 Diversity in Bacteria
Organisms that acquire mutations are
termed mutants
Mechanisms of mutagenesis
Spontaneous mutations of DNA
Horizontal gene transfer
8.2 Spontaneous Mutations
Causes of spontaneous mutations
Chemicals and radiation can induce mutations of
DNA
Mutases are enzymes that are expressed during
times of stress that increase rates of mutations
Types of mutations
Nucleotide base substitution (aka, point mutation)
Deletion or insertion of nucleotides
Transposable elements (”jumping genes”)
8.2 Spontaneous Mutations
Mutations are a numbers game
Rates of mutations vary from 10-4 to 10-12
Cells have DNA repair mechanisms that
repair most mutations
Base Substitution
8.2 Spontaneous
Mutations
Silent mutation has no effect on amino acid
content
CCC, CCT, CCA, CCG all encode proline
Missense mutation results in an amino acid
change
CCC➙CTC = proline to leucine
Can alter 3D shape of protein or compromise
critical amino acid
Nonsense mutation results in a premature stop
codon
TTG➙TAG = leucine to termination
8.2 Spontaneous Mutations
Removal or addition of nucleotides
Deletions (removal) or insertion (addition)
mutations alter the reading frame of DNA
These mutations are termed frameshift
mutations
Frameshift mutations are usually intolerant
for the bacterium
Spurious amino acid sequences
Premature stop codons
8.2 Spontaneous Mutations
Transposable elements (aka, jumping genes,
transposons)
Mobile genetic elements
Can “jump” from species to species
Often carry genes that alter the phenotype of
recipient bacteria, including antibiotic
resistance
8.3 Induced Mutations
Mutagens are used to discover gene functions
Alterations in genes often result in changes in
phenotype
Chemical mutagens
Base modifiers change bases that are misread
during DNA replication
Nitrous acid converts NH2 groups to C=O groups
8.3 Induced Mutations
Base analogs resemble normal bases, but have
different H-bond characteristics and are
mismatched during DNA replication
Intercalating agents, such as ethidium bromide,
insert between adjacent bases on a strand, which
can lead to the insertion of a base pair by DNA
polymerases
This results in a frameshift mutation
8.3 Induced Mutations
Radiation
Ultraviolet radiation
induces thymine dimers
X-rays induce double
strand DNA breaks
8.4 Repair of Damaged DNA
Cells possess systems
that can repair mutated
DNA
Repair of errors in base
incorporation
Proofreading: Some DNA
polymerases can step
backward and remove a
misincorporated base
Mismatch repair:
Endonucleases are enzymes
that recognize inappropriate 3D
structures of DNA and remove
mutant bases, which are
corrected by DNA polymerases
8.4 Repair of Damaged DNA
Repair of thymine dimers
Photoreactivation
Recognizes bulges in DNA
Harness light energy to break covalent bond between
adjacent thymines
8.4 Repair of Damaged DNA
Repair of thymine
dimers (cont.)
Excision repair
removes several
adjacent bases
DNA polymerases fill in
the gap
DNA ligase forms
phosphodiester bonds
8.4 Repair of Damaged DNA
Repair of modified bases in DNA
Lesion-specific glycosylases recognize modified
bases and remove them
Endonucleases then remove the deoxyphosphate backbone (i.e., excision repair)
DNA polymerase adds the appropriate base
SOS repair
System of 30+ genes for repair of highly
damaged DNA
System of desperation
Highly prone to error
8.5 Mutations and Their Consequences
Mutations are a natural biological process
All DNA polymerases have inherent mutation rates
In times of stress, mutational rates increase (mutases)
Without mutations, evolution cannot occur and
organisms will be ill-equipped to adjust to changes
in their environment
Evolution requires three events
Genetic variation, which is mostly random (e.g., mutation)
The variations must be heritable
Natural selection of those traits most suitable for an
environment
8.6 Mutant Selection
Isolating a mutant is a statistically-unlikely event
without selection
In nature, natural selection favors the outgrowth of mutant
microbes
In the laboratory, artificial selection is employed to find these
unlikely events
Many important strains of microbes have been
developed using artificial selection
Oil-consuming bacteria
Heavy metal decomposition
Wines
8.6 Mutant Selection
A common method for producing an
antibiotic-resistant bacterium
Inoculate entire
surface with
susceptible bacteria
Antibiotic
-containing
medium
Normal
medium
Antibiotic gradient
1 week
2 weeks
8.7 DNA-Mediated Transformation
Competence
Cells that are receptive to DNA transfer are
termed competent
The process of becoming competent is largely
unknown, but requires protein synthesis
Competent cells permit DNA to pass through their
cell walls and membranes
This process is termed transformation and can
lead to the acquisition of new genes
Competent cells can be induced artificially and
play an important role in biotechnology
8.8 Transduction
Bacteriophages (aka. phages) are bacterial viruses
They frequently incorporate genes from previouslyinfected host cells
When the progeny viruses infect other bacteria, the
new genetic information can be recombined with
the host’s genome
This process is termed transduction
8.9 Plasmids
Plasmids are circular molecules of DNA
They can be hundreds to thousands of nucleotides
long
They frequently contain virulence factors that
contribute to disease susceptibility
Antibiotic resistance
Toxins
They are considered promiscuous because they
can disseminate between species of bacteria
Plasmids are routinely used in biotechnology for
gene cloning and recombinant protein production
8.10 Conjugation
Competent cells acquire plasmids by random
chance
Conjugation is the direct transfer of plasmids (or
chromosomes) between bacteria
It is a four-step process
Contact between a donor cell (F+) and recipient cell (F-) is
mediated by a sex pilus, a tubular structure
The plasmid becomes mobilized by an enzyme that cleaves the
plasmid
One strand of the plasmid is transferred to the recipient,
presumably through the pilus
The copies of the plasmids are used as templates for DNA
synthesis
8.10 Conjugation
8.11 Transposons
Transposons (transposable elements, jumping
genes) are self-replicating DNA molecules
They occur in virtually all organisms
When they jump, they frequently take adjacent
genes with them, such that integration in another
cell leads to the introduction of novel genetic
information
Trans-species transposition has been observed
in nature
All transposons encode DNA and/or RNA
polymerase, termed transposases, that mediate
jumping
8.11 Transposons
A
B
C
TP
D
C
TP
D
E
Jump
TP
C
X
Y
D
Z
Integration
X
Y
C
TP
D
Z