Bacterial Genetics and Mutations

Bacterial Genetics

Bacterial Resistance

  • Bacterial cells acquire resistance through horizontal gene transfer.

  • Horizontal gene transfer includes bacterial conjugation, transformation, and transduction.

Bacterial Conjugation

  • Involves the transfer of genetic material, often plasmids, via an F pilus (sex pilus).

  • The donor cell contains a plasmid with genes to create the F pilus.

  • The plasmid is copied and transferred to the recipient cell (acceptor cell).

  • If the plasmid contains resistance genes, the recipient cell becomes resistant.

Plasmids

  • Plasmids are extra-chromosomal, circular DNA in bacterial cells.

  • R plasmids carry resistance genes.

  • Example: PLW1043, a resistance plasmid, contains multiple resistance genes, including:

    • Beta-lactam genes (resistance to penicillin).

    • Trimethoprim resistance.

    • Genes for plasmid spread (F pilus creation).

    • Resistance to disinfectants.

    • Streptomycin family resistance.

    • Vancomycin resistance (a last line of defense against MRSA).

Vertical vs. Horizontal Gene Transfer

  • Vertical Gene Transfer: Passing on of genes from one generation to the next (e.g., parent to offspring).

  • Horizontal Gene Transfer: Transfer of genes between existing cells (donor to recipient), not necessarily from parent to offspring.

  • Vertical transfer occurs when a cell divides into two, replicating its DNA, including plasmids, into both daughter cells.

Mutation Definition

  • A mutation is any change to the nucleotide sequence of DNA.

  • Mutations can occur inside or outside of genes, but phenotypic changes are more likely when they occur within a gene.

Nucleotide Substitutions

  • These are spontaneous mutations that change the identity of one nucleotide into another.

  • Transitions: Interchange of pyrimidines (T and C) or purines (A and G).

    • T↔CT \leftrightarrow C

    • A↔GA \leftrightarrow G

  • Transversions: Change of pyrimidines to purines or vice versa.

    • T/C↔A/GT/C \leftrightarrow A/G

Perpetuation of Mutations

  • Wild Type Gene: The normal, unmutated gene with normal function.

  • During DNA replication, strands separate and serve as templates for new strands.

  • If a mutation occurs during replication (e.g., nucleotide substitution), the new strand will have the mutant sequence.

  • Cells with the mutant sequence will pass on the mutation to subsequent generations through vertical gene transfer.

  • If DNA replication is faithful, all progeny will be wild type.

Example of Mutation Perpetuation

  • Original DNA Sequence:

    • 5'-CGTTAG-3'

    • 3'-GCAATC-5'

  • Mutation occurs during replication:

    • 5'-CGTTAG-3' (wild type)

    • 3'-GCAGTC-5' (mutant - A changed to G)

  • The cell with the mutant sequence perpetuates the mutation in future generations.

Types of Mutations

  • Using the RNA sequence AUG GCA UAA as an example:

    • Corresponding DNA template sequence: TAC CGT ATT

    • Wild type amino acid sequence: Methionine - Alanine - Stop

  • Missense Mutation: Changes the identity of the encoded amino acid.

    • Example: Changing GCA (Alanine) to UUA. This alters the amino acid sequence.

    • Original RNA sequence: AUGGCAUAA (Met-Ala-Stop)

    • Mutated RNA sequence: AUUUCAUAA (Ile-Ala-Stop)

  • Nonsense Mutation: Changes the identity of an encoded amino acid into a stop codon.

    • Example: Changing AGA (Arginine) to UGA (Stop).

    • Original RNA sequence: AUG GCA AGA (Met-Ala-Arg)

    • Mutated RNA sequence: AUG GCA UGA (Met-Ala-Stop). This results in premature termination of translation.

  • Silent Mutation: A mutation that does not change the identity of the encoded amino acid.

    • Example: GCA (Alanine) mutating to GCU (also Alanine).

    • Mutations exist at the DNA level, but do not change the protein sequence.

Point Mutations

  • Point mutations are nucleotide substitutions involving a single nucleotide.

  • Missense, nonsense, and silent mutations are all types of point mutations

Insertions and Deletions (Indels)

  • Insertions: addition of a nucleotide.

  • Deletions: deletion of a nucleotide.

  • Indels cause a frameshift, changing the reading frame of the mRNA.

  • Alters the amino acid sequence of the encoded protein.

  • Example: Insertion of a 'U' in AUG GCA UAA:

    • Original RNA sequence: AUG GCA UAA (Met-Ala-Stop)

    • Mutated RNA sequence: AUG UGC AUA A (Met-Cys-Ile-)

Spontaneous Mutations

  • Base substitutions (nucleotide substitutions) are the most common.

  • During DNA replication, the wrong nucleotide may be incorporated.

  • Leads to silent, missense, or nonsense mutations.

  • Deletion and addition of nucleotides can lead to a frameshift.

Jumping Genes and Barbara McClintock

  • Mobile DNAs (transposable elements) can be excised from one chromosome and insert into another.

  • Discovered by Barbara McClintock.

  • Can act as a large insertion mutation, altering the amino acid sequence of a gene.

Induced Mutations

  • Caused by mutagens, such as carcinogenic chemicals (e.g., in cigarettes).

  • Chemical agents modify nucleotide bases or insert into the DNA double helix.

  • Radiation (e.g., UV radiation) can damage DNA.

  • Examples of Chemical Mutatgens:

    • Alkylating agents

    • Base analogs

    • Intercalating agents (insert between base pairs)

Specific Examples of Induced Mutations

  • Deamination: Removal of an amino group.

    • Cytosine loses its amino group and becomes uracil.

    • C is converted to U, altering pairing during replication.

  • Alkylation: Addition of an alkyl group (−CH3-CH_3).

    • Guanine becomes O6-methylguanine, which pairs with T instead of C.

    • Disrupts normal DNA replication.

  • Oxidation: Changes guanine to 8-oxo-guanine, which pairs differently.

  • Base Analogs: Molecules that resemble normal bases but cause mispairing.

    • Example: 5-bromouracil (looks like uracil) pairs with G instead of A.

    • Example: 2-aminopurine (looks like adenine) pairs with C instead of T.

  • Intercalating Agents: Insert between DNA base pairs, causing frameshifts.

    • Example: Ethidium bromide.

Radiation-Induced Mutations

  • UV radiation causes thymine dimers: adjacent thymines on the same strand stick together.

  • Thymine dimers create a bulge in the DNA backbone, stalling DNA polymerase.

  • Photolyase: An enzyme in bacteria that uses energy from light to break thymine dimers, correcting the error. (Humans do not have this enzyme)

  • Excision Repair (XPD): in Humans recognizes damage and recruits other proteins to remove the damaged area and repair the gap

  • Xeroderma Pigmentosum: A genetic disorder caused by mutations in genes involved in DNA repair (e.g., XPD gene).

    • Individuals with this disorder are extremely sensitive to UV radiation and prone to skin lesions and cancer.

Repair of Errors in Nucleotide Incorporation

  • Occurs when DNA polymerase adds the wrong nucleotide during replication.

  • An enzyme cuts the sugar-phosphate backbone of the new strand near the mismatch.

  • Another enzyme removes the stretch of DNA with the incorrect nucleotide.

  • DNA polymerase inserts the correct nucleotides.

  • Ligase seals the gap in the phosphodiester backbone.

Repair of Oxidized Guanines

  • Glycosylase removes the damaged base (oxidized guanine).

  • The backbone is cut, and surrounding bases are removed.

  • DNA polymerase lays down the new sequence.

  • Ligase seals the gap.

Repair of Thymine Dimers

  • Photolyase breaks the thymine dimer, removing the bulge (in bacteria).

  • Excision repair: enzymes cut on either side of the dimer, removing the damaged section.

  • DNA polymerase adds the correct nucleotides.

  • Ligase seals the gap.