bacterial genetics chapter 8. the problem of antibiotic resistance staphylococcus aureus common gram...
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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
Organisms that acquire mutations are termed mutants
Mechanisms of mutagenesis
Spontaneous mutations of DNA
Horizontal gene transfer
8.1 Diversity in Bacteria8.1 Diversity in Bacteria
8.2 Spontaneous MutationsCauses 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”)
Mutations are a numbers game
Rates of mutations vary from 10-4 to 10-12
Cells have DNA repair mechanisms that repair most mutations
8.2 Spontaneous Mutations8.2 Spontaneous Mutations
Base Substitution
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 Mutations8.2 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 Mutations8.2 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
8.2 Spontaneous Mutations8.2 Spontaneous Mutations
8.3 Induced MutationsMutagens 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
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 Mutations8.3 Induced Mutations
Radiation
Ultraviolet radiation induces thymine dimers
X-rays induce double strand DNA breaks
8.3 Induced Mutations8.3 Induced Mutations
8.4 Repair of Damaged DNACells 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
Repair of thymine dimers
Photoreactivation
Recognizes bulges in DNA
Harness light energy to break covalent bond between adjacent thymines
8.4 Repair of Damaged DNA8.4 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 DNA8.4 Repair of Damaged DNA
Repair of modified bases in DNA
Lesion-specific glycosylases recognize modified bases and remove them
Endonucleases then remove the deoxy-phosphate 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
8.4 Repair of Damaged DNA8.4 Repair of Damaged DNA
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
A common method for producing an antibiotic-resistant bacterium
8.6 Mutant Selection8.6 Mutant Selection
Inoculate entire surface with susceptible
bacteria
1 week
2 weeks
Antibiotic-containing
medium
Normalmedium
Antibiotic gradient
8.7 DNA-Mediated TransformationCompetence
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 TransductionBacteriophages (aka. phages) are bacterial viruses
They frequently incorporate genes from previously-infected 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 PlasmidsPlasmids 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 ConjugationCompetent 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
8.11 TransposonsTransposons (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