Microbio Exam 3

T8

Microbial Genetics

What does “wild-type” mean?

An organism obtained from nature

What kinds of things make mutations in cells?

• UV light

• Ethidium bromide

• Transposons

• A genomic rearrangement in the DNA due to stress

• UV light

• Ethidium bromide

• Transposons

What kinds of things make mutations in cells?

• Infrared light

• A mistake made by RNA polymerase in bacterium

• A mistake made by DNA polymerase in bacterium

Mistakes made by RNA polymerase are not heritable

Correct: A mistake made by DNA polymerase in bacterium

Which of the following is an example of a spontaneous mutation?

• UV light causing T-T dimers in a bacterium

• Ethidium bromide causing DNA breaks in a bacterium

• Transposons jumping into the chromosome and disrupting a gene

• A mistake made by DNA polymerase in a bacterium

• A genomic rearrangement in the DNA due to stress

A mistake made by DNA polymerase in a bacterium

Which of the following describes a nonsense mutation?

• It must occur inside an open reading frame

• It does not change the amino acid sequence in a gene

• It changes an amino acid sequence encoded in a gene

• It prematurely terminates a protein

• Open reading frame (codes for proteins)

• It changes the amino acid sequence

• It prematurely terminates a protein

Artificially inserts stop codon

Mutation

A heritable change in genome that can lead to a change in phenotype

Mutant

A strain of any cell or virus differing from parental strain in genotype

Wild-type strain

Strain isolated from nature

Spontaneous mutations

• Occur without external intervention

• Inherent mistakes made by polymerases during genome replication

Induced mutations

• Those made environmentally or deliberately

• Can result from exposure to natural radiation or oxygen radicals

Direct reversal

Mutated base is still recognizable and can be repaired without referring to other strand

• Error free or error-prone

Repair of single strand damage

Damaged bases or DNA strand is removed and repaired using opposite strand as template

Repair of double strand damage

A break in the DNA which can be lethal if not repaired

Translesion synthesis

Nucleotide excision repair: type of repair

Single strand repair

Nucleotide excision repair: proteins involved

Uvr proteins

Nucleotide excision repair: bases repaired

CPD, radiation

Nucleotide excision repair: error free or prone?

Error free

Translesion synthesis: type of repair

Single strand repair

Translesion synthesis: proteins involved

DinB (DNA Poly IV/V) - Umu proteins

Translesion synthesis: bases repaired

CPD, ROS, nucleotide analogs

Translesion synthesis: error prone or free?

Error-prone

Why is perfect fidelity counterproductive?

It prevents evolution

True or false: the mutation rate of an organism is subject to change

True

Are water bears extremophiles?

Yes

What protects DNA during environmental stress?

Protiens

Adaptive mutations

Improve fitness of an organism, increasing its survival

Neutral mutations

Have no effect

Severely deleterious mutations

Cannot be tolerated

Evolutionary adaption in microbes can be driven by…

• Mutation

• Genetic exchange

What is the difference between transformation and transduction in bacteria?

Three methods for genetic exchange in bacteria

1. Transformation

2. Conjugation

3. Transduction

All rely on recombination to incorporate DNA

Transformation

Genetic transfer process by which naked DNA is incorporated into a recipient cell and brings about genetic change

Competent

Cells capable of taking up DNA and being transformed

In ___ _____ bacteria, competence is regulated

Naturally transformable

Transformation usually requires…

DNA binding proteins and nucleases

Com genes

Species of bacteria that are naturally competent

• Often regulated by quorum sensing

What procedures can be used to make cells competent?

• Electricity

• Heat + cations

Conjugation

Mechanism of genetic transfer that involves cell-to-cell contact

Conjugation is typically a ___-encoded mechanism

Plasmid

Donor cell

Contains conjugative plasmid

Recipient cell

Does not contain plasmid

Sex pilus

Essential for conjugation

• Only produced by donor cell

Transduction

Transfer of DNA from one cell to another by a bacteriophage

What are the two modes of transduction?

1. Generalized transduction

2. Specialized transduction

Generalized transduction

DNA from any portion of the host genome is packaged inside the virion

Specialized transduction

DNA from a specific region of the host chromosome is integrated directly into the virus genome

Generalized transduction: What is packaged inside the mature virion?

DNA derived from any portion of the host genome

Specialized transduction: What is integrated directly in the virus genome?

DNA from a specific region of the host chromosome

What has higher efficiency, generalized or specialized transduction?

Specialized

What are the four fates of foreign DNA inside cells?

1. Degraded by cellular enzymes

2. Stable maintenance outside the chromosome

3. “Random” insertion into genome

4. Site-specific recombination into genome

Degraded by cellular enzymes

• RMS systems

• CRISPR systems

Stable maintenance outside the chromosome

• Need to have replication origins recognized by/compatible with the host

• Mainly plasmids

“Random” insertion into genome

• Not truly random

• Need to have special enzymes for incorporation

• Transposons and pathogenicity islands can insert this way

Site-specific recombination into genome

• Need to have a specific recognition site and recombinases

• Foreign DNA needs to have some similarity to the host genome

• Integrates through homologous recombination

Homologous recombination results in…

Genetic exchange between similar DNA from two different sources

RecA

Mediates homologous recombination

Transposable elements

Discrete segments of DNA that move as a unit from one location to another within other DNA molecules

Are transposable elements found in all three domains of life?

Yes

Two main types of transposable elements in BACTERIA

Transposons and insertion sequences

• Both carry genes encoding transposase

• Both have inverted repeats at their ends

Insertion sequences

• Simplest transposable element

• Only gene is for the transposase

• Found in plasmids and chromosomes of BACTERIA and ARCHAEA (few viruses)

Transposons

• Larger than insertion sequences

• Has additional accessory genes between IR

• Moves any DNA between inverted repeats

• Insertion of a transposable element generates a duplicate target sequence

• May include antibiotic resistance

Does transformation work well in archaea?

Yes

Does genetic manipulation of archaea lag behind bacteria?

Yes

Signal transduction

Using intracellular and intercellular signaling to sense environments and respond

Three main mechanism for signal transduction

1. Two component systems

2. Quorum sensing

3. Heat shock

Similarities between signaling in bacteria vs eukaryotes

• Receptors on surface of cell

• Bound by ligand

• Phosphoryl transfer

• Transcriptional or functional outputs

Differences between signaling in bacteria vs eukaryotes

• Fewer interacting partners in bacteria

• Signal may need to get through more membranes

  • Organelles in eukaryotes

  • Outer membranes in bacteria

Bacteria: 2-components

• Sensor kinase (H)

• Response regulator (D)

Chemotaxis

Movement along a chemical gradient

• Regulates the direction of turning for flagella

1. Response to signal

2. Movement

3. Adaptation

What types of behavior would a bacterium want to do only when there are a lot of related bacteria around?

• Cause virulence in a host

• Bioluminescence

Quorum sensing

Asses population in a local environment

• G+ and G- do differently

Small QS molecules

Accumulate in the extracellular milieu

Signal receptors

Receive the signal and translate it into new gene expression (induced/repressed genes)

• Bioluminescence

Heat shock sensing

Largely controlled by alternative sigma factors

Heat shock proteins

Counteract damage of denatured proteins and help cell recover from temp. stress

Heat shock response occurs in…

Bacteria and archaea

T9

Microbial Genomics

What kinds of reagents/enzymes might you need to sequence DNA in a tube?

• DNA polymerase

• dNTPs

• Primers (3’ OH)

• DNA template

• Mg

• Zn

How is DNA sequenced?

Genome

• Entire complement of genetic information

• Includes genes, regulator sequences, and noncoding segments of the genome

Genomics

Discipline of mapping, sequencing, analyzing, and comparing genomes

Steps to sequence a genome

1. Sequence the DNA

2. Assemble the sequences into chromosomes or large fragments called contigs

3. Predic genes (annotation)

Sequencing

Determining the precise order of nucleotides in a DNA molecule

Sanger dideoxy method

• Dideoxy analogs of dNTPs used in conjunction with dNTPs

• Analog prevents further extension of DNA chain

• Very high accuracy

What currently is a major limitation to Sanger sequencing for large scale genome sequencing?

Time and cost to obtain data for a single 900 bp of DNA

Next gen. sequencing

More parallel sequences but shorter reads

Read

A single sequence of a piece of DNA

Run

A single sample that is sequenced by a given technology, can be pure or mixed

Second gen. sequencing: 454 sequencing

• Generates data 90x faster than Sanger method

• Massively parallel liquid handling and pyrosequencing

• Nucleotides flow through plate one at a time

• NOT RLY USED ANYMORE

Second gen. sequencing: illumina

• Similar to 454 in the beginning

• Solid phase amplification to make clusters of identical DNA pieces

• Sequencing chemistry is a bit different

MiniSeq

MiSeq