genet 270- lec 6- conjugation
What is bacterial conjugation?
It’s the process where a donor bacterium transfers plasmid DNA to a recipient cell through a direct connection called a pilus.
🧠 Think of it as bacterial “mating” — one cell physically gives DNA to another.
What structure allows a donor cell to attach to a recipient during conjugation?
The pilus, a thin protein tube that connects the cells to form a mating pair.
💡 Analogy: The pilus acts like a “grappling hook” pulling the cells together.
How is DNA transferred during conjugation?
One strand of the plasmid is cut and transferred to the recipient; both donor and recipient then synthesize complementary strands — similar to rolling-circle replication.
🧬 So both end up with a complete plasmid.
What happens to the plasmid in the donor and recipient during transfer?
Each cell replicates its own single DNA strand to form a double-stranded plasmid, making both F⁺ donors after conjugation.
what is the f plasmis
The F plasmid (fertility plasmid) carries the genes that allow a bacterium to form a pilus and transfer DNA.
What Happens During Conjugation
The F⁺ donor connects to an F⁻ recipient via the pilus.
The F plasmid is nicked, and a single strand of its DNA is transferred to the recipient.
Both cells then synthesize the complementary strand to make a complete double-stranded plasmid.
Who first discovered bacterial conjugation, and when?
Lederberg and Tatum (1947) — they observed genetic recombination in E. coli when mixing two auxotrophic strains (each missing different nutrients).
🧠 Historical anchor: 1947 = birth of bacterial “sex.”
What result led Lederberg & Tatum to conclude gene exchange occurred?
After mixing the two mutant (auxotropic) strains, they obtained progeny that could grow without any supplements — meaning they had new combinations of functional genes from both parents.
What explanations did they consider for this gene exchange?
Possible causes included cell fusion, transforming factors (free DNA in medium), or a new process later identified as bacterial sex = conjugation.
What is the F plasmid?
The “fertility” plasmid, the first conjugative plasmid discovered. It enables DNA transfer (conjugation) between bacterial cells.
How large is the F plasmid, and what’s known about it?
It’s about 100 kilobases (kb) long, and its entire DNA sequence is known — a model system for studying conjugation.
What is special about the genetic composition of the F plasmid?
It contains sequences derived from multiple genetic sources, reflecting its evolutionary history and mosaic structure.
🧠 Mnemonic: “F = Fusion” of genes from many origins.
What genes are essential for DNA transfer during conjugation?
The tra genes — they encode the proteins required to form the mating pair and transfer DNA between cells.
🧠 “tra” = transfer genes.
How many replication origins does the F plasmid have, and which are functional?
Three: RepFIA, RepFIB, RepFIC — but only RepFIA (oriV) is functional for replication.
What systems maintain the F plasmid within a bacterial population?
A partitioning system to ensure equal plasmid segregation.
Toxin–antitoxin (post-segregational killing) systems that kill cells which lose the plasmid.
🧠 Mnemonic: “Keep it or die.”
What extra “selfish” features does the F plasmid carry?
Genes that suppress the host SOS response and block T7 phage infection, helping the plasmid protect itself and its host cell.
What are the two main components encoded by tra genes?
1️⃣ Mpf (Mating Pair Formation) – builds the physical bridge between donor and recipient.
2️⃣ Dtr (DNA Transfer and Replication) – processes and replicates plasmid DNA for transfer.
What is the overall role of these two components in conjugation?
Together, Mpf forms the connection/channel for transfer, while Dtr ensures correct plasmid processing & replication as DNA moves from donor to recipient.
🧠 Memory tip: Mpf = make pair; Dtr = deliver the replicon.
What is the Mpf component in conjugation?
The Mating Pair Formation (Mpf) complex — it’s the structure that connects donor and recipient cells and provides the channel through which DNA is transferred during conjugation.
What are the two main functions of the Mpf component?
1️⃣ Holds the donor and recipient cells tightly together.
2️⃣ Forms the transfer channel for DNA movement during mating.
🧠 Think of Mpf as the “bridge and clamp” that enables DNA transfer.
What is the pilus, and what is it made of?
The pilus is a 10 nm diameter tube made of repeating pilin proteins. It’s produced by the donor cell and attaches to a specific receptor on the recipient.
🧠 Think: pilus = “protein straw” that starts the connection.
What happens during pilus retraction?
The pilus retracts, pulling the donor and recipient cells together into close contact to form a mating pair, enabling DNA transfer.
💡 Analogy: like reeling in a fish — the donor pulls the recipient in.
What is the Type IV Secretion System (T4SS), and what is its role?
A multi-protein complex that builds the pilus and later serves as a conduit for DNA transfer once the cells are connected.
🧠 T4SS = “DNA delivery machine.”
Why is the Type IV Secretion System biologically significant?
It’s conserved across many species and used not only for bacterial DNA transfer but also for delivering virulence factors into eukaryotic cells (e.g., Agrobacterium transferring T-DNA into plants).
What is the Relaxosome, and what does it do?
The Relaxosome is a protein complex that binds at the origin of transfer (oriT) and prepares the plasmid for DNA transfer by nicking and processing it.
🧠 Think: Relaxosome = “prep station” for DNA export.
What is the function of the TraI relaxase protein?
TraI is a site-specific endonuclease that:
1️⃣ Nicks one strand of the plasmid DNA at oriT via transesterification.
2️⃣ Attaches to the 5′ end, guiding the strand into the recipient cell.
3️⃣ Recircularizes the transferred strand in the recipient via another transesterification reaction.
💡 Mnemonic: “TraI = Nick → Pilot → Close.”
Which accessory proteins assist the Relaxosome during DNA transfer?
TraY & host IHF → essential for nicking DNA.
TraM & TraD → coordinate DNA processing with the transfer apparatus.
TraU → part of Dtr system.
Helicase → unwinds DNA during transfer.
🧠 Support crew ensures smooth nicking, transfer, and coordination.
What is the role of coupling proteins (e.g., TraD)?
Link the Dtr (DNA processing) and Mpf (mating pair) systems.
Signal that a recipient is ready to receive DNA.
Pump DNA into the recipient through the Type IV channel (functionally similar to FtsK).
Convert the secretion system into a DNA transport machine.
💡 Coupling protein = the “bridge” between DNA prep and transfer.
Why does plasmid transfer slow down after initial high activity in new recipients?
Because conjugation is tightly regulated — once initial mating pairs form efficiently, later transfer events are reduced through control of tra gene expression.
💡 First burst → high transfer, then self-regulation prevents overuse.
Which key genes regulate conjugation in F plasmids?
FinO, FinP, and traJ — they control expression of the tra operon that drives DNA transfer.
What are the roles of FinP and FinO?
FinP = small regulatory RNA (sRNA) that binds traJ mRNA, blocking its translation and triggering degradation.
FinO = protein that stabilizes FinP and helps it form a duplex with traJ mRNA.
🧠 Mnemonic: “FinP silences, FinO protects.”
What is the function of TraJ in conjugation?
TraJ is a transcriptional activator of the tra genes. When FinP/FinO repress TraJ, conjugation gene expression shuts down, reducing plasmid transfer.
💡 FinO/FinP act like a safety brake on conjugation.
What are self-transmissible plasmids?
Plasmids that encode all the functions needed for transfer between cells — they have their own Dtr (DNA transfer) and Mpf (mating pair formation) systems.
🧠 They can travel independently — no helper needed.
What are mobilizable plasmids?
Plasmids that lack a full transfer system, but can be mobilized by a self-transmissible plasmid in the same cell.
They have mob genes (Dtr system) and an oriT site, which allows them to use another plasmid’s tra system.
💡 Mobilizable = “has luggage, needs a driver.”
What are promiscuous plasmids, and why are they important?
Plasmids that can transfer DNA between unrelated species (e.g., Cyanobacteria, Gram-positives, plants).
They are important for evolution and spreading antibiotic resistance, often moving R plasmids between species.
🧠 Promiscuous = “cross-species sharers.”
What are the two major biological roles of bacterial conjugation?
1️⃣ Spread of antibiotic resistance
2️⃣ Bacterial evolution through gene transfer
🧠 Conjugation = engine for survival & adaptation.
How does conjugation contribute to antibiotic resistance?
Conjugative plasmids carry resistance (R) genes, allowing bacteria to transfer antibiotic resistance traits rapidly between species.
💡 Antibiotic resistance spreads like a “plasmid pandemic.”
Why is conjugation such an effective mechanism for resistance spread?
Because once a plasmid enters a new host, it can replicate and transfer again, spreading resistance through an entire bacterial population — even across different species.
How does conjugation contribute to bacterial evolution?
Conjugative plasmids can transfer chromosomal DNA between bacteria, allowing the acquisition of new genes or mutations that lead to adaptation and evolution.
🧠 Think of conjugation as “gene mixing” for bacteria — new DNA = new abilities.
What are Hfr strains and how are they formed?
Hfr = High Frequency of Recombination strain.
Formed when an F plasmid integrates into the bacterial chromosome via homologous recombination, usually between shared insertion sequence (IS) elements.
💡 “Hfr” = F plasmid fused into the chromosome.
What happens when an Hfr cell conjugates with a recipient?
The transfer starts at the F plasmid’s oriT and continues into chromosomal genes.
Both plasmid and part of the chromosome can be transferred.
Full chromosome transfer is rare because mating pairs often break before completion.
🧠 Like copying a huge file — it often disconnects mid-transfer.
What did Lederberg & Tatum originally observe that relates to Hfr strains?
Their 1947 experiment showing recombination between E. coli strains was later explained by Hfr-mediated transfer of chromosomal DNA — the basis for understanding bacterial genetics.
What are F′ (F-prime) factors, and how do they form?
Created by imperfect excision of an integrated Hfr plasmid from the chromosome.
The excised plasmid carries extra chromosomal genes along with the F plasmid.
Formed through recombination between IS elements near the insertion site.
💡 F′ = “F plasmid + bonus chromosomal genes.”
Why are F′ factors important?
They can transfer chromosomal genes to new cells very efficiently.
Commonly used in genetic mapping and complementation experiments in labs.
🧠 In lab genetics: F′ plasmids = mobile copies of useful chromosomal genes.
Diagram summary (if shown on slides):
Arrows usually show the F plasmid integrating into the chromosome → forming Hfr.
Later, imperfect excision pulls out the F plasmid plus neighboring DNA → forming F′.
When this F′ donor conjugates, it moves those chromosomal genes into a new cell.
💡 Visual sequence:
F⁺ plasmid → integrates (Hfr) → excises imperfectly (F′) → transfers hybrid DNA.
What exactly happens when an F′ factor forms?
An Hfr plasmid excises imperfectly from the chromosome, taking a piece of chromosomal DNA with it.
This creates an F′ (F-prime) plasmid, which is larger and carries both F genes and extra bacterial genes (e.g., pro⁺, lac⁺).
🧠 Think: F′ = F plasmid “with souvenirs” from the chromosome.
What happens when an F′ donor conjugates with a recipient?
The F′ plasmid is transferred, giving the recipient two copies of the same gene (one on the chromosome, one on the plasmid).
This creates a partial diploid (merodiploid) cell useful for studying gene function and dominance.
💡 F′ transfer = duplication of genes in the recipient.
What are triparental matings, and why are they used in labs?
A lab method using three bacterial strains to efficiently transfer genes:
1️⃣ A strain with a self-transmissible plasmid (provides tra functions)
2️⃣ A strain with a mobilizable plasmid carrying the gene of interest
3️⃣ A recipient strain that receives the mobilizable plasmid
🧠 Self-transmissible = “helper plasmid”; mobilizable = “cargo plasmid.”
: Why are triparental matings useful experimentally?
They allow researchers to move engineered genes or plasmids between bacterial species with high efficiency, even when the plasmid alone can’t self-transfer.
💡 In lab terms: use conjugation as a “genetic delivery service.”
How does a triparental mating work mechanistically?
Three strains are mixed:
1️⃣ A helper strain (with a self-transmissible plasmid providing tra genes),
2️⃣ A donor strain (with a mobilizable plasmid containing the gene of interest and oriT),
3️⃣ A recipient strain.
The helper’s tra functions mobilize the donor’s plasmid into the recipient, while the helper itself doesn’t transfer its own DNA.
🧠 Helper = machinery; donor = cargo; recipient = destination.
What is a toxin–antitoxin (TA) system on plasmids?
A maintenance mechanism that ensures plasmid retention:
The toxin is stable and harmful.
The antitoxin is unstable and neutralizes the toxin only while the plasmid is present.
If a cell loses the plasmid, the antitoxin degrades first, the toxin acts, and the cell dies.
💡 “Lose me, and you die” — ensures only plasmid-containing cells survive.
What’s the evolutionary role of TA systems?
They make plasmids selfish but stable genetic elements, ensuring their survival in a population even if they don’t benefit the host.
🧠 TA systems act like plasmid “insurance policies.”