Ch 6 Bacterial Genetics and Phage-Mmediated Gene Transfer Notes

Bacterial Growth and Growth Phases

• Three phases of bacterial growth: lag phase, exponential (log) phase, and stationary phase.
  • Lag phase: slow growth; bacteria are adapting to the environment.
  • Exponential growth during the lag phase as bacteria divide; described in the transcript as an exponential growth period.
  • Stationary phase: loss of nutrients; growth halts; the number of cells that divide is balanced by the number that die, so net growth is not zero but the culture is not simply flat in terms of activity.
• Experimental data representation mentioned:
  • X-axis: time in hours.
  • Y-axis: actual number of cells per milliliter on a logarithmic scale.
  • This log scale is used because bacteria numbers span large ranges quickly, so multiplicative growth appears as a straight line in log space.

Nutritional Requirements: Prototrophs vs Auxotrophs

• Prototrophs
  • Bacteria that can synthesize all essential organic compounds themselves.
  • They require only inorganic salts and sugars for energy.
  • They do not need added amino acids in the medium.
• Auxotrophs
  • Bacteria with mutations that cause loss of the ability to synthesize certain organic compounds.
  • Require the missing compound to be supplied in the medium (e.g., an amino acid).
  • Without the added compound, auxotrophs cannot grow.

Plasmids and Genes in Bacteria

• Plasmids
  • Small circular DNA molecules that are extrachromosomal and replicate independently of the bacterial chromosome.
  • Carry various genes; not part of the main chromosome.
• F factor
  • A specific plasmid important in this discussion; capable of transfer between bacteria.
• Other plasmid-borne genes
  • Antibiotic resistance genes can be carried on plasmids.
  • Some plasmids may carry toxin genes.

Replica Plating and Mutation Detection

• Replica plating concept
  • A method to determine if bacteria have lost the ability to synthesize certain organic compounds.
  • Uses a velvet/soft transfer surface to imprint colonies from one plate to another, preserving the colony pattern.
• Plates used
  • Minimal media: contains salts and sugars but no organic supplements.
  • Complete media: contains salts, sugars, and organic components.
• Interpretation of results
  • If colonies grew on complete media but not on minimal media, this indicates mutations causing auxotrophy (loss of self-synthesis).
  • If colonies can grow on complete media but not minimal media, the bacteria have mutations that prevent synthesis of certain organic compounds.
• Broader significance
  • Replica plating is a classic method to screen for mutants affecting biosynthetic capabilities.
  • Bacteria are a convenient model for studying mutations due to rapid growth and ease of culture.
  • Genetic recombination is present in bacteria as well as eukaryotes, enabling genetic exchange across domains.

Bacterial Recombination: Vertical vs Horizontal Gene Transfer

• Recombination in bacteria involves transfer of genetic information that creates new combinations of alleles.
• Vertical gene transfer
  • Transfer of genes between organisms of the same species (parent to offspring).
• Horizontal gene transfer
  • Transfer of genes between organisms of different species.
• Three major mechanisms of horizontal gene transfer (recombination) in bacteria:
  • Conjugation
  • Transformation
  • Transduction

Conjugation

• Donor and recipient
  • F+ (donor) cell contains the F factor on a plasmid and can transfer DNA to F− (recipient) cells via the sex pilus (F pilus).
  • After transfer, both cells become F+.
• Mechanism overview
  • The F factor plasmid is transferred from donor to recipient.
  • One DNA strand of the plasmid is transferred while the other strand is replicated in both donor and recipient.
  • Result: two F+ cells (donor becomes F+ and recipient gains F+).
• Integration and high-frequency recombination (HFR) cells
  • The plasmid can integrate into the host chromosome, creating an HFR (high-frequency recombination) cell.
  • When integration occurs at the origin of replication (O), transfer starts with a portion of the chromosome along with plasmid DNA.
  • The transferred chromosomal genes can recombine with the recipient's chromosome, introducing new genes into the recipient.
• Mapping the chromosome via conjugation
  • The order in which chromosomal genes are transferred reflects their proximity to the origin of transfer (origin of replication).
  • By using different donor strains with the plasmid integrated at different chromosomal locations, the transfer order can reveal the gene order on the chromosome.
  • Conceptually: genes closer to the origin are transferred earlier and at a higher rate, while more distal genes transfer later or less frequently.
• Applications
  • Conjugation can be used to map the order of genes on the bacterial chromosome.

Transformation

• Competent cells
  • Cells capable of taking up free DNA from the environment.
  • Some bacteria are naturally competent; others are made competent in the laboratory.
• Lab methods to induce competence
  • Calcium chloride treatment or electroporation to increase membrane permeability.
  • Electroporation uses electrical pulses to create temporary pores in the cell membrane.
• Process
  • Free DNA in the environment is taken up by competent cells.
  • The absorbed DNA can recombine with the host chromosome if there is a region of homology.
  • Result: transformed cells that contain new genetic material.
• Outcome
  • The donor DNA can be integrated into the host genome at homologous regions, producing a recombinant host.

Transduction

• Bacteriophage as the vector
  • Bacteriophages (phages) are viruses that infect bacteria and can transfer bacterial DNA between cells.
• Defective phages
  • Some phages can package bacterial DNA instead of their own DNA; these defective phages carry bacterial genes.
  • When such a phage infects a new bacterium, it can introduce bacterial DNA into the new host, enabling recombination.
• Phage life cycle (general outline)
  • Phage binds to the bacterial host.
  • Injects its DNA and uses the host machinery to replicate, transcribe, and translate phage genes.
  • Phage DNA replication and protein synthesis lead to assembly of new phage particles.
  • Lysis releases phages to infect other bacteria.
• Transduction mechanics
  • If the phage carries bacterial DNA instead of phage DNA, it introduces that DNA into a new host, enabling recombination.
• Co-transfer concepts
  • Transduction (like transformation) often results in co-transfer of linked genes: genes that are close together have a higher chance of being transferred together.
  • The likelihood of co-transformation or co-transduction depends on the distance between genes on the chromosome.
• Gene mapping via transfer frequency
  • Higher frequency of co-transfer for closely linked genes helps map gene order and distance on the chromosome.

Co-Transformation and Co-Transduction

• Co-transformation
  • When two or more genes close to each other are transferred together via transformation.
  • The percentage of recombinants with two or more specific genes decreases as the distance between genes increases.
• Co-transduction
  • Similar concept for transduction: multiple nearby genes are transferred together.
• Implication for mapping
  • The observed frequencies of recombinant strains help infer genetic distances and gene order.

Bacteriophages: Structure and Function

• Structure overview
  • A bacteriophage typically has:
  • A protein-coated head that contains phage DNA.
  • Tail fibers that recognize and attach to specific bacterial hosts.
  • The tail fibers determine host specificity; attachment precedes DNA injection.
• DNA packaging and transfer potential
  • During assembly, chromosomal DNA fragments may be unintentionally packaged into phage heads; these fragments can later be transferred to another host via transduction.

Phage Lifecycle and Plaque Assays

• Lifecycle basics
  • Phage attaches to host, injects DNA, hijacks host machinery to replicate phage DNA and produce phage proteins, assembles new phages, then lyses the cell to release progeny.
  • In some cases, phages can persist in the host without immediate lysis (lysogenic pathway not elaborated in the transcript).
• Plaque assay as a measurement tool
  • Plaque assays quantify phage density by counting plaques on a lawn of bacterial cells.
  • Procedure overview:
  • Prepare serial dilutions of phage stock.
  • Mix each dilution with a fresh culture of susceptible bacteria and plate on agar.
  • Incubate to allow phage infection and bacterial lysis, forming clear zones called plaques.
  • Interpreting plaques
  • Large numbers of phages (low dilutions) may lyse all bacteria, leaving no plaques.
  • At appropriate dilutions, discrete plaques appear; counting plaques enables calculation of phage concentration in the stock.
• Calculation of phage concentration (Pfu/mL)
  • Let N be the number of plaques observed at dilution d, and v be the volume plated (in mL).
  • Phage concentration in stock (Pfu/mL) is given by:
    $P = \frac{N}{d \, v}$
  • Example from the transcript:
  • Suppose 20 plaques observed at a 10^{-5} dilution and 0.1 mL plated.
  • Then $P = \frac{20}{(10^{-5}) imes 0.1} = \frac{20}{10^{-6}} = 2 imes 10^{7} ext{ phages/mL}.$

Phage Recombination and Wild-Type Restoration

• Recombination between two mutant phages
  • When two mutant phages infect the same host, recombination can restore wild-type genes if the mutations are distant enough on the chromosome.
  • The frequency of restoration depends on the distance between the genes that were mutated; greater distance increases the chance of recombining the wild-type alleles.

Real-World and Practical Implications

• Antibiotic resistance and horizontal gene transfer
  • Plasmids carrying antibiotic resistance genes can spread among bacterial populations via conjugation, transformation, or transduction, contributing to rapid resistance spread.
• Genetic engineering applications
  • Transformation and transduction are commonly used in laboratories to introduce new DNA into bacteria for cloning, gene expression, or genome editing.
  • Conjugation and Hfr mapping provide tools for understanding gene order and chromosomal organization.
• Phage biology and therapeutic potential
  • Phages are not only tools for genetic transfer studies but also potential therapeutic agents against bacterial infections (phage therapy).
• Ethical and biosafety considerations
  • The ability of bacteria to acquire new traits through horizontal gene transfer underscores the importance of antibiotic stewardship and biosafety in both research and clinical settings.
• Foundational connections
  • The described mechanisms underpin core concepts in genetics: mutation effects, Mendelian-like recombination in bacteria, and the modular nature of plasmids carrying accessory genes.