Bacterial GeneticsChapter 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

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

8.10 Conjugation8.10 Conjugation

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

8.11 Transposons8.11 Transposons

A B C TP D E

C TP D

X Y Z

C TP D

Jump

X Y ZC TP D

Integration