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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.

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the problem of antibiotic resistance
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
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 bacteria4
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
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 mutations6
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
8 2 spontaneous mutations7
8.2 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
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 mutations9
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
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 mutations11
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 mutations12
8.3 Induced Mutations
  • Radiation
    • Ultraviolet radiation induces thymine dimers
    • X-rays induce double strand DNA breaks
8 4 repair of damaged dna
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 dna14
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 dna15
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 dna16
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 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 5 mutations and their consequences
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
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 selection19

Antibiotic

-containing

medium

Inoculate entire surface with susceptible bacteria

2 weeks

1 week

Normal

medium

Antibiotic gradient

8.6 Mutant Selection
  • A common method for producing an antibiotic-resistant bacterium
8 7 dna mediated transformation
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
8.8 Transduction
  • Bacteriophages (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 plasmids
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
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 11 transposons
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 transposons26

Integration

TP

TP

TP

D

D

D

TP

D

C

C

C

A

X

B

Y

C

Z

E

Jump

X

Y

Z

8.11 Transposons