Bacterial Genetics and Recombination Comprehensive Notes

Bacterial Genetics Notes

Figure 8-2: Auxotrophic Strains Experiment

• Strains A and B have reciprocal genotypes. This means that where one strain has a gene, the other lacks it.

• Neither strain can grow on minimal medium individually because each lacks a nutrient-synthesizing capability.

• Strain A: (met- bio- thr- leu+ thi+), meaning it can't synthesize methionine, biotin, or threonine, but can synthesize leucine and thiamine.

• Strain B: (met+ bio+ thr+ leu- thi-), meaning it can synthesize methionine, biotin, and threonine, but cannot synthesize leucine and thiamine.

• Auxotrophic strains: cannot grow on minimal media because they're missing something they can't synthesize. They can grow on complete media because it provides the missing nutrients.

Experiment Steps:

1. Grow strains A and B separately in complete medium.

2. Mix A and B in complete medium and incubate overnight.

3. Plate the mixture on minimal medium.

4. Controls:

   • Strain A plated on minimal medium: No growth (auxotrophic).

   • Strain B plated on minimal medium: No growth (auxotrophic).

   • Strains A + B plated on minimal medium: Colonies of prototrophs appear at a low frequency of 1/10^7 of total cells.
     *Something about putting them together overnight, letting them mix and sit together allowed you to get the prototrophs

• Conclusion: Something happened when you mixed strain A and B together that allowed them to exchange information somehow to make a prototroph.

• The goal of this experiment was to determine what happens when 2 strains of reciprocal genotypes combine them together.

Bacterial Recombination

• Bacterial recombination is not reciprocal.

• It is unidirectional exchange.

Prototrophs

• A prototroph can grow on minimal media.

• In the tube where strains were mixed, prototrophs were found, indicating genetic exchange.

Davis U-tube Experiment

• Goal: To determine if direct contact between the two strains is required for genetic exchange and prototroph formation.

Experiment Setup:

• Strain A (F+) stays on one side of the filter, Strain B (F-) stays on the other side of the filter.

• A U-shaped tube with a filter in between separates strain A from strain B.

• The filter pore size is small enough to prevent bacteria from passing through but allows liquid and media to pass.

• Medium is passed back and forth across the filter using alternating pressure and suction.

Results:

• When plated on minimal media, neither side shows growth.

Conclusion:

• Contact is required between the strains for recombination to occur. Bacteria can exchange information, but at a very low frequency and contact IS required.

F-plasmid Mediated Transfer (Conjugation)

• F-plasmid: An extra-chromosomal element (plasmid) separate from the bacterial chromosome.
  *Catalyses the ability of the bacterium to take the DNA of one bacterium and put it in another bacterium, uni directionally.

• F+ cell: A cell with the F factor (plasmid).

• F- cell: A cell without the F factor.

• Process:

  1. Conjugation occurs between F+ and F- cell. The F+ cell extends a pilus (sex pilus) to connect to the F- cell.

  2. One strand of the F factor is nicked by an endonuclease and moves across the conjugation tube.

  3. The DNA complement is synthesized on both single strands.

  4. Movement across the conjugation tube is completed; DNA synthesis is completed.

  5. Ligase closes circles; conjugants separate.

• When this unidirectional recombination happens, everything on the F-plasmid has moved into a new cell.

• Exconjugants: Cells formed by conjugation. 2 daughter cells are created (F+ cell and F+ cell)

Hfr: High-Frequency Recombination

• A different type of transfer still involving the sex pilus.

• Experiment: Hfr strain is put in contact with an F- strain.

  • Hfr H (thr+ leu+ aziS tonS lac- gal-, StrS) x F- (thr- leu- aziR tonR lac+ gal+, StrR)

• Conjugation is interrupted by shaking the mating cells to break them apart at different time intervals.

• The longer the strains stay together, the more genetic information is transferred from one bacterium to another.

• The time of transfer is gene-dependent, indicating a specific order of transfer.

Interrupted Mating

• The longer you keep them together, the more things transferred from one bacterium to another… more genetic information is sent from one bacteria to another.

• There is a time-dependent relationship.

• Genes are transferred in a specific order, e.g., thr and leu early, azi, ton, and lac later.

Mapping Genes by Interrupted Mating

• The order of transfer is different for each Hfr strain.

• Origin of transfer changes, and direction of transfer can change.

• The relative position of genes can be mapped based on the time it takes for them to be transferred during conjugation.

F-Plasmid Integration

• In Hfr strains, the F-plasmid has been integrated into the bacterial genome.

• The F-plasmid integrates in different places and orientations on the genome, explaining why some genes transfer first.

• The integrated F-plasmid drives the direction of transfer. It explains time map.

F' Plasmids: Imperfect Excision

• During excision of the F factor from the chromosome, it can sometimes carry parts of the bacterial chromosome with it.

• This creates an F' plasmid, which carries bacterial genes (e.g., A and E regions).

Merozygote

• The F' cell can conjugate with an F- cell, transferring the plasmid and the bacterial genes it carries.

• The recipient cell becomes partially diploid for the genes on the plasmid, creating a merozygote (partially diploid).
  Bacteria is dependent on the plasmid.

• Imperfect excision means it grabs some other crap around it

R Plasmids: Antibiotic Resistance

• R plasmids carry genes that confer resistance to antibiotics.

• They contain:

  • r-determinants: Genes conferring antibiotic resistance (e.g., tetracycline resistance, sulfonamide resistance, ampicillin resistance).

  • RTF (Resistance Transfer Factor) segment.

Bacteriophages

• Two components:

  1. DNA

  2. Proteins

• Structure:

  • Head (containing DNA)

  • Tail

  • Tail fibers

• Once one of these infects the bacterium, it can make more of itself and assemble more of itself.
  *Self-assemble

Lytic Cycle

1. Adsorption: Phage attaches to the bacterial host cell.

2. Injection: Phage injects its DNA into the host cell; host DNA is degraded.

3. Replication and Synthesis: Phage DNA is replicated; phage protein components are synthesized.

4. Assembly: Mature phages are assembled.

5. Lysis: Host cell is lysed, releasing phages.

• The 1st collapses the host cell's machinery to shred the host's DNA then completely take over the bacterial machinery.

Serial Dilutions

• Serial dilutions can be used to determine the concentration of phage (phage titer).

• Plaques: Clear areas in the bacterial lawn where phages have lysed the bacteria.

• That's evidence for the lysis of bacteria in a small area.

Calculation

CFU/mL = Number of colonies on plate \times reciprocal of dilution of sample = number of bacteria/mL

Liederberg and Zinder Experiment

• Used reciprocal genotypes

• Strain LA-2 had a bacteria phage

• Popped some of these accidentally took their genetic information.

• If this goes on too long, the bacteria Phage will kill everything.

Transduction

• Genetic information was carried over by a bacteria phage

Phage-Mediated Recombination (Transduction)

1. Phage Infection: Phage infects a bacterial cell and injects its DNA.

2. Destruction and Replication: Host DNA is destroyed, and phage DNA is replicated.

3. Assembly: Phage protein components are assembled.

4. Release: Mature phages are assembled and released. The difference here: the phage is so dumb it couldn't distinguish between the shredded pieces of bacterial genome and its own genome.

5. Subsequent Infection: A defective phage (containing bacterial DNA) infects another cell and the Phage mediated.

6. Integration: Bacterial DNA is integrated into the recipient chromosome.

Naked DNA Transformation

• Bacteria can take up foreign genetic material (naked DNA) from the environment.

• Just another way they can recombine genetic material.

Three Ways Bacteria Share Information

1. Conjugation via the pilus

2. Phage-mediated transduction

3. Naked DNA transformation

Antibiotics

• Examples: Penicillins, Cephalosporins, Tetracyclines, Macrolides, Fluoroquinolones, Sulfonamides, Glycopeptides

• Bactrim: Sulfamethoxazole and trimethoprim are both antibiotics that treat different types of bacterial infections.

Why Bacterial Genetics Matter

• As a healthcare professional and/or an educated citizen

• Every time you take an antibiotic, you are conducting a genetic selection.

• The genetic analysis of drug resistance allows for new drugs to be developed.

  • Change cell wall components

  • Deactivate antibiotics with enzymes

  • Inhibit entry or pump out antibiotics

  • Change antibiotic target sites

  • Increased mutation rate

Staphylococcus aureus: Antibiotic Resistance

• Penicillin: Resistance by 1947 (4 years)

• Methicillin: Resistance by 1960 (2 years)

• Vancomycin: Resistance by 1997 (2 years)

• Linezolid: Resistance by 2001-2003 (2 years)

• 50% of all S. aureus infections in the US are multidrug-resistant (some are Superbugs).

Superbugs

• Klebsiella pneumoniae: A species that killed a woman resistant to all 26 American antibiotics.

• Every 15 minutes, someone in the US dies of a drug-resistant superbug.

• The Superbug crisis threatens to kill 10 million per year by 2050.