GENET 270- lec 7- transduction
What is transduction in bacteria?
It’s the transfer of bacterial DNA from one cell to another by a bacteriophage (transducing particle).
🧠 Think: “phage-mediated DNA delivery.”
What are the two types of transduction, and how do they differ?
Generalized transduction → any region of the bacterial chromosome can be transferred.
Specialized transduction → only specific genes near the prophage attachment site are transferred.
💡 Generalized = random genes; Specialized = specific neighbors.
What defines a generalized transducing phage?
A phage that accidentally packages host (bacterial) DNA instead of its own.
When this defective phage infects another bacterium, it injects donor bacterial DNA, which can recombine with the recipient’s chromosome to form a transductant.
What is a transductant?
A recipient cell that has incorporated donor bacterial DNA delivered by a transducing phage — effectively a recombinant bacterium.
🧠 Transductant = recombined recipient.
What are pac sites, and what role do they play in phage packaging?
Pac sites are specific DNA sequences recognized by phage enzymes where DNA is cut to begin packaging into phage heads during replication.
🧠 Pac = “packaging start.”
Why do generalized transducing phages have non-specific pac sites?
Because their packaging enzymes can mistakenly recognize similar sequences in the bacterial chromosome, leading to bacterial DNA being packaged instead of phage DNA.
💡 This error enables generalized transduction.
What happens after the phage enzyme cuts at a pac site in the DNA concatemer?
The enzyme begins headful packaging, cutting DNA into unit lengths that fill a phage head.
If the enzyme recognizes a bacterial site, bacterial DNA gets packaged and transferred to another cell.
🧠 Like cutting sausages — one “headful” per phage.
What is the headful packaging mechanism used by generalized transducing phages?
Once DNA packaging starts at a pac-like site, the phage enzyme cuts and fills each phage head with one headful of DNA (a little more than one genome length).
If bacterial DNA is mistakenly packaged, it also gets cut into these unit lengths and enclosed in phage heads.
🧠 Headful = “fill the head, then cut.”
How does the headful mechanism contribute to generalized transduction?
It allows random fragments of bacterial DNA to be packaged if the phage enzyme starts at a bacterial pac-like site — enabling transfer of any gene during infection of a new host.
💡 Mechanistic link: pac error → headful packaging → bacterial gene transfer.
Why is a broad host range important for transducing phages?
Because transducing phages only need to attach and inject DNA into the recipient — they don’t need to replicate there.
A broad host range increases the number of possible recipient species for transduction.
🧠 Broad host range = wider gene-sharing network.
What are the two best-known generalized transducing phages?
Phage P1 → infects E. coli
Phage P22 → infects Salmonella typhimurium
🧠 P1 = E. coli; P22 = Salmonella.
What are the two main uses of generalized transduction in bacterial genetics?
1️⃣ Mapping genes — determining gene order and distances.
2️⃣ Strain construction — transferring mutations or markers between strains to build specific genotypes.
💡 Transduction = lab tool for gene maps and mutant swaps.
Why is generalized transduction valuable for genetic mapping?
Each transducing particle carries about 50–100 genes.
Only genes close together on the chromosome can be co-transduced, so co-transduction frequency reflects genetic distance.
🧠 Closer genes = higher co-transduction rate.
What determines whether two genes can be co-transduced?
Genes will be co-transduced only if they’re close enough on the chromosome to be carried in the same transducing particle (≈50–100 genes per fragment).
The closer two genes are, the higher the co-transduction frequency.
🧠 “Close together = travel together.”
Why is generalized transduction considered a rare event?
Because multiple rare steps must occur:
1️⃣ The phage must accidentally package bacterial DNA,
2️⃣ Inject it into a recipient, and
3️⃣ That DNA must recombine successfully into the recipient chromosome.
💡 Each step is low-probability → overall rare process.
How do researchers detect or select for transduction events?
They plate the infection mixture on selective media where only recombinant transductants can grow (e.g., minimal medium lacking a nutrient restored by the donor gene).
🧠 Selection “filters out” only successful recombinants.
What does it mean if two markers are co-transduced?
It means the two genes are linked closely enough on the chromosome to be packaged together in the same DNA fragment during transduction.
🧠 Co-transduction = physical proximity.
What does it mean if two markers are not co-transduced?
It means they’re too far apart to be carried in the same fragment — indicating that the genes are unlinked on the chromosome.
No co-transduction = distant or separate genes.
What is the relationship between co-transduction frequency and gene distance?
They are inversely proportional — the closer the genes, the higher the co-transduction frequency.
Co-transduction frequency
= # of cotransductants/ total transductants
× 100
🧠 Closer = higher frequency; farther = lower frequency.
What is the purpose of the example cross (Slides 17–18)?
To show how to calculate co-transduction frequencies and use them to infer gene order on the bacterial chromosome.
🧠 Real data example: Arg, Met, Rif markers.
What is the donor genotype and recipient genotype in this example?
Donor: Arg⁺ Met⁻ Rifˢ
Recipient: Arg⁻ Met⁺ Rifᴿ
💡 Arg⁺ (can synthesize arginine), Met⁻ (cannot synthesize methionine), Rifˢ (rifampicin-sensitive)
and
Arg⁻ (requires arginine), Met⁺ (can synthesize methionine), Rifᴿ (resistant to rifampicin).
What is being selected and what is being screened in this experiment?
Selection: Plate on minimal medium lacking arginine — only cells that gained Arg⁺ from the donor can grow (these are transductants).
Screening: Among those Arg⁺ colonies, test which also gained Met⁻ or Rifˢ to calculate co-transduction frequencies.
🧠 Select for Arg⁺ → then screen for other donor markers.
What is the biological logic behind this setup?
Phage grown on the Arg⁺ Met⁻ Rifˢ donor picks up fragments of DNA randomly.
If a fragment carrying Arg⁺ also contains Met or Rif genes, those can be co-transduced.
By measuring how often Arg⁺ is transferred with each marker, you can infer how close the genes are on the chromosome.
💡 Co-transfer frequency = genetic distance indicator.
What is the goal of the Arg–Met–Rif transduction experiment?
To use co-transduction frequencies to determine how close these genes are on the chromosome and to infer their order.
🧠 We measure how often genes “ride together” inside one transducing fragment.
How are co-transduction frequencies calculated?
Arg + Met⁻ pair: 13.5 % co-transduction
Arg + Rifˢ pair: 34 % co-transduction
🧠 Higher % = genes closer together.
What can we conclude from these frequencies?
Because Arg and Rif show a higher co-transduction frequency (34 %) than Arg and *Met (13.5 %)**,
Arg and Rif are closer on the chromosome than Met.
Possible gene orders:
1️⃣ rif — arg — met or 2️⃣ arg — rif — met
(orientation can’t be told yet because flipping the map doesn’t change order).
💡 Co-transduction tells distance, not direction.
What if two distances are very similar?
When co-transduction frequencies are close, there may be two equally plausible gene orders (e.g., rif – arg – met or arg – rif – met).
To resolve this, you perform a 3-factor cross, analyzing which recombinant type is rarest to reveal the correct order.
🧠 3-factor cross = tie-breaker for close gene distances.
Why do we perform a 3-factor cross in transduction mapping?
When two gene pairs have similar co-transduction frequencies, a 3-factor cross helps determine the correct order by analyzing the rarity of recombinant classes (i.e., how many recombination events are needed).
🧠 It’s the “tie-breaker” when distances are too close.
What is the basic principle behind a 3-factor transduction cross?
Incoming donor DNA (linear fragment) must undergo an even number of recombination events to integrate stably into the recipient chromosome.
Odd numbers of crossovers break the chromosome (non-viable).
Rarer recombinant types require more recombination events, revealing gene order.
💡 Common = 2 crossovers; Rare = 4 crossovers.
How do we use recombination frequency to infer gene order?
1️⃣ List all possible gene orders (e.g., rif–arg–met vs arg–rif–met).
2️⃣ Identify the rarest class of recombinants from the data (in this case: Arg⁺ Met⁻ Rifᴿ).
3️⃣ Draw both possible gene orders and mark crossovers needed to create that rare class.
4️⃣ The order that requires more crossovers matches the rarest class → this is the correct gene order.
🧠 More crossovers = rarer class = correct order.
What was the result of the 3-factor cross in the Arg–Met–Rif example?
The rarest class (Arg⁺ Met⁻ Rifᴿ) fit with the gene order:
Arg — Rif — Met
This order requires four crossovers in the diagram, confirming it as the true gene order.
💡 Rare class = Arg⁺ Met⁻ Rifᴿ → Arg–Rif–Met is correct.
What data identifies the rare class in this experiment?
The Arg⁺ Met⁻ Rifᴿ recombinant occurred least often → it’s the rarest class.
This means producing it requires more recombination events than other classes.
💡 Rarest = most crossovers = defines gene order.
How do we determine which order matches the rare class?
1️⃣ Draw both possible orders:
• Order I: Rif – Arg – Met • Order II: Arg – Rif – Met
2️⃣ Keep donor and recipient alleles consistent (+/–).
3️⃣ Map crossovers needed to yield Arg⁺ Met⁻ Rifᴿ.
4️⃣ The order requiring four crossovers (Arg–Rif–Met) fits the rare class data.
🧠 “Four-crossover fit” = true order.
What is the final confirmed gene order from this 3-factor cross?
✅ Arg – Rif – Met
Requires 4 crossovers for the rarest class.
Supported by the observed recombinant frequencies.
Confirms that Arg lies *between Rif and Met on the chromosome.
🧠 Remember: rare class → Arg⁺ Met⁻ Rifᴿ → Arg-Rif-Met.
What is the goal of strain construction using transduction?
To create isogenic strains that are genetically identical except for one mutation or gene of interest — allowing precise study of gene function.
“Isogenic = same genome except one target difference.”
How can transduction be used to construct isogenic strains?
By moving a desired mutation (or a gene insertion) from one strain into another using a generalized transducing phage, often selecting for a linked marker (like antibiotic resistance).
💡 Example: use an antibiotic marker close to the gene to “drag” it into the recipient.
Describe the example from the slides for strain construction.
Donor: cpxA24 zii::Tn10 (Tetᴿ)
Recipient: cpxR cpxA⁺ (wild-type)
→ After transduction and selection for Tetᴿ, colonies are screened for the presence of the cpxA24 mutation.
🧠 Transfer of Tetᴿ marker helps identify recipients that also got the nearby cpxA mutation.
What is specialized (restricted) transduction, introduced on Slide 31?
A process where only specific bacterial genes near a prophage’s integration site are transferred.
It occurs when a lysogenic phage makes a mistake during excision, carrying adjacent bacterial DNA with its own.
💡 Specialized = “site-specific DNA pickup.”
How does a specialized transducing phage form?
When a lysogenic prophage is induced to re-enter the lytic cycle, a rare mistake in excision can occur — the phage cuts imprecisely, taking adjacent bacterial genes with it.
The resulting phage genome contains both phage DNA + neighboring bacterial DNA in one continuous molecule.
🧠 Imprecise excision = hybrid phage + bacterial genes.
What type of bacterial genes can be transferred by specialized transduction?
Only genes that lie next to the prophage’s integration site (e.g., near attB in the host chromosome).
Genes farther away cannot be included because only a small fragment is mistakenly excised.
💡 Specialized = “only the neighbors get invited.”
What are key characteristics of specialized transducing particles?
Some phage genes are lost during aberrant excision (depends on where it occurs).
The transducing phage is often defective and requires a helper phage to replicate or form particles.
It can only carry bacterial genes near the attachment site.
🧠 Defective phage + helper phage = successful infection cycle.
What do terms like λ d gal or λ pbio represent?
They describe specialized transducing phages derived from phage λ that have captured specific bacterial genes:
λ d gal → carries gal genes (for galactose metabolism)
λ pbio → carries bio genes (for biotin synthesis)
💡 Prefix “λ d” or “λ p” = λ phage derivative carrying host DNA
Visual Explanation (Slides 32–36 combined)
1️⃣ Normal lysogeny: Phage DNA integrates into host chromosome at attB site → forms prophage.
2️⃣ Induction: Prophage excises to enter lytic cycle.
3️⃣ Rare error: Excision cuts beyond the normal att sites, pulling out adjacent host genes.
4️⃣ Outcome: Hybrid phage DNA = part phage + part bacterial DNA → packaged into phage head.
🧠 Normal excision = clean cut; aberrant excision = bacterial hitchhiker.
Why is specialized transduction rare?
Because it depends on a specific, rare error during prophage excision — only a tiny fraction of lysogenic cells excise incorrectly to include bacterial DNA.
🧠 Normal excision is precise → rare “mis-cut” causes specialized transduction.
What are LFT and HFT lysates in specialized transduction?
LFT (Low-Frequency Transduction) → produced after induction of a lysogen, before transducing phages multiply; contains mostly normal phages, with very few transducing particles.
HFT (High-Frequency Transduction) → produced when a defective transducing phage (e.g., λ d gal) is replicated with help from a normal λ helper phage; contains many transducing particles.
💡 Helper phage = factory; transducing phage = passenger.
What role do helper phages play in specialized transduction?
Helper phages supply the missing functions (e.g., structural proteins or replication genes) that the defective transducing phage lost during aberrant excision.
This allows the hybrid DNA to be packaged and propagated, creating an HFT lysate.
🧠 Without helper phage → no viable transducing particles.
How do HFT and LFT lysates differ experimentally?
LFT: few transductants; each event is unique and rare.
HFT: reproducible, frequent transduction of the same bacterial gene(s) near the prophage site, because many identical transducing particles are made.
💡 LFT = first rare mistake; HFT = amplification of that mistake.
Visual Summary (Slides 37–39 combined)
1️⃣ Normal prophage excision → clean λ genome (no host DNA).
2️⃣ Rare incorrect excision → λ d gal (λ DNA + host gal genes).
3️⃣ Helper phage co-infection provides missing functions → replication of defective λ d gal phage.
4️⃣ Result = HFT lysate, rich in identical transducing particles.
🧠 LFT = rare origin event; HFT = amplified batch via helper.
What is the key difference between generalized and specialized transduction?
Generalized transduction: Any bacterial gene can be transferred.
Specialized transduction: Only specific genes near the prophage integration site can be transferred.
💡 Generalized = random genes; Specialized = neighboring genes.
How do the mechanisms of DNA packaging differ between the two?
Generalized: Caused by accidental packaging of host DNA during phage assembly (headful mechanism from pac-like sites).
Specialized: Caused by aberrant prophage excision during induction of a lysogen.
🧠 Generalized = packaging error; Specialized = excision error.
Which phages perform each type?
Generalized: Virulent or temperate phages like P1 and P22.
Specialized: Only temperate phages that can integrate into the host genome (e.g., λ phage).
💡 Integration ability = requirement for specialized transduction.
🧩 Visual Recap (Slides 40–41 combined)
Generalized:
Phage cuts DNA randomly → bacterial DNA packaged → random gene transfer.
Specialized:
Phage excises with neighboring host DNA → specific gene transfer → defective phage needs helper.
💡 Different errors, same outcome: bacterial gene transfer.