Lecture 8 Notes

Genetic Mutations and Horizontal Gene Transfer

Genetic Mutation

Any change in the nucleotide (nt) sequence of the genome.
Involves:
- Switch of one base pair for another.
- Loss of base pairs.
- Addition of base pairs.
Wild-type (WT) vs. mutant organism.
- WT: Normal nucleotide sequence.
- Mutant: Bears some change in nucleotide sequence.

Causes of Mutations

Spontaneous mutation
- Due to random errors during DNA replication.
Induced mutation
- Result from exposure to physical or chemical mutagens.

Chemical Mutagens

Can insert between adjacent bases.
Can "mimic" a base - nitrogenous base analogs.

Physical Mutagens

UV rays
- Cause abnormal bonds between adjacent nucleotides, often causing thymine dimers.
X rays and gamma rays
- Introduce small breaks in DNA strands.
Horizontal gene transfer (i.e., DNA recombination)
- When genes are shared between microbes.

Selective Pressure and Mutations

Most mutations are not beneficial to the microbe.
Some mutations contribute to the success of an individual microbe.
Selective agents:
- Antimicrobial drugs.
- Disinfectants.
Protective mutations
- Aid in survival in the face of selective agents.
Microbes rely on and keep protective mutations as long as the selective agent is present.
- Ex: Antibiotic resistance.
Selective agents kill susceptible microbes but leave resistant ones behind.

Horizontal Gene Transfer (HGT) / Genetic Recombination

When a microbe receives DNA from another microbe.
- Most common in bacteria, some yeast.
- Usually plasmids or chromosomal fragments.
Recombinant
- The microbe that contains and expresses genes that originated in another microbe.
Types of HGT:
- Conjugation
- Transformation
- Transduction
Gene transfer occurs between bacteria - Bacterial gene transfer mediated by bacteriophages.

Conjugation

A mode of DNA transfer that requires contact between bacteria.
- Plasmids are usually transferred.
Gram-negative and Gram-positive bacteria perform conjugation.
- Most common in Gram-negative.
- Gram-negative bacteria conjugate via sex pilus.
- Gram-positive bacteria conjugate via mating bridge.
Usually occurs between closely related bacterial species.

Conjugation in Gram-Negative Bacteria

Conjugative Gram-negatives contain a special plasmid called the fertility (F) factor.
F+ bacteria can form sex pili – donors.
F- cannot form sex pili – recipients.
Recipient bacteria contain recognition site on their surface that enables the pilus to connect.
Once F+ and F- bacteria are connected, the pilus retracts and DNA is transferred.
Sometimes foreign DNA is kept/integrated, but sometimes it’s degraded.

F Factor Transfer via Conjugation

Sometimes only the F factor is transferred from one bacterium to the next.

High-Frequency Recombination (Hfr) via Conjugation

Occurs when bacteria have F factors integrated into chromosome.
Entire chromosome is copied and transferred.
Sex pilus breakage often terminates transfer, resulting in recipient cell receiving chromosomal fragment.
Chromosomal genes (and sometimes F factor) integrate into recipient cell.

Conjugation in Gram-Positive Bacteria

Gram-positives use pheromones to conjugate
Recipient bacteria secrete pheromones.
Pheromones bind to receptors on donor bacteria
- Binding triggers expression of transfer (tra) genes.
Cells join, cell walls and membranes fuse to form mating bridge.
Plasmid is transferred from donor to recipient.

Transformation

When bacteria capture DNA from the surrounding environment.
- DNA may be released from dead cells and degraded into small pieces.
- Degraded to small pieces that can be taken in by recipient cells.
DNA binds to proteins on recipient cell membrane and transported across.

Transduction

Delivery of bacterial DNA from a bacteriophage.
- Viruses serve as vectors - entity that delivers foreign DNA into a cell.
Generalized transduction
- Random pieces of host DNA picked up by the virus during assembly and delivered to the next host.
Specialized transduction
- Specific regions of host DNA picked up and delivered to the next host.

Generalized Transduction step by step:

The bacteriophage infects 1st host cell
During assembly of new viral particles, the virus picks up random pieces of host cell DNA
Host cell lyses, viruses released
Viruses deliver host DNA (and viral nucleic acid) to a new host cell
The foreign DNA can integrate into new host chromosome

Specialized Transduction step by step:

Donor cell infected with temperate phage
Prophage enters host chromosome at specific sites.
During activation, prophage DNA pops out of chromosome along with flanking host DNA
Host and viral DNA are incorporated into new viruses
Viruses deliver host and viral DNA to next host

Transposons

Movable genetic element – “Jumping genes”.
Contains gene for transposase
- Enzyme that excises transposon from chromosome/plasmid and integrates it into a new chromosome/plasmid.
Jumps from chromosome to plasmid, chromosome to chromosome, plasmid to chromosome, plasmid to plasmid.
If foreign genes are contained within transposons, they are more likely to integrate into chromosome of recipient cell.
Example:
- Emergence of vancomycin-resistant MRSA via conjugation and transposons.
  - $VanR$
  - $MethR$ (MRSA)
  - $VanRMethR$ (VMRSA)
  - Transposon-mediated jumping
  - E. faecalis harbored a plasmid with a transposon containing the vancomycin-resistance gene.

Antibiotic Resistance

Antibiotics are selective agents
- Kill susceptible bacteria, select for resistant mutants.
Antibiotic-resistant bacteria are a threat worldwide.
In the US, high rates are attributed to overuse of antibiotics.
- Human medicine.
- Agriculture.
Emerging antibiotic-resistant bacterial pathogens:
- MRSA, VMRSA.
- Carbapenem-resistant Klebsiella pneumoniae.
- Multidrug-resistant Uropathogenic E. coli (UPEC).
- NDM1-positive bacteria.

Antibiotic Resistance in the US - CDC View

2013 – CDC director Dr. Tom Frieden warned, “if we’re not careful, we will soon be in a post-antibiotic era”.
2017 – CDC notes that we’ve arrived – “Pan-resistant bacteria are now here in the US”.
Case of carbapenem-resistant Enterobacteriaceae (CRE) infection in Reno, Nevada
- Resistant to all 26 antibiotics on the market.
- Patient died.

Emerging Drug-Resistant Pathogens of Concern

Carbapenem-resistant Klebsiella pneumoniae (aka KPC)
- K. pneumoniae is an encapsulated Gram-negative bacterium.
  - Lives in the gut of humans and animals.
  - Opportunistic pathogen.
- If it gains access to extra-intestinal sites (bloodstream, lungs, etc), infections can become deadly.
  - Major nosocomial pathogen.
- Carbapenems are antibiotics that break down bacterial cell walls.
- Genes for antibiotic resistance acquired via transposons, delivered by bacteriophages.
A “new” type of drug resistance – NDM1
- New Delhi metallo-β-lactamase.
- Mobile genetic element encoding multiple drug resistance genes.
- Some NDM1-positive bacteria are resistant to most/all antibiotics.
- Major problem in Gram-negative pathogens.
Carbapenem-resistant, NDM1-positive Klebsiella pneumoniae may be the next big superbug.
- Carbapenem-resistant NDM1-positive Klebsiella is also resistant to most disinfectants.
- Emerging nosocomial pathogen.
- Very contagious
  - Transmitted patient-to-patient, medical worker-to-patient, from fomites, indwelling devices.
- Recent outbreaks
  - NIH hospital in 2011 – 18 infected, 11 died.
  - Detroit hospital in Sept/Oct. 2012 – 5 infected, no deaths.
Multidrug-resistant Uropathogenic E. coli (UPEC)
- UPEC is the leading cause of urinary tract infections:
  - Cystitis
  - Pyelonephritis
  - Renal damage
  - Potential systemic infection
- 8-65% of UPEC strains are resistant to drugs commonly used to treat these infections.
- Most concerning drug-resistant strain: UPEC ST131
  - Received multiple antibiotic-resistance genes via conjugation and delivery of transposon.
  - Also received “extra” virulence genes - binds better to urinary tract.
- Drug-resistant strain may be nosocomial or community-acquired.

TED Talk

TED Talk (Maryn McKenna) – What do we do when antibiotics don’t work anymore?