Bacterial Conjugation Steps

Why This Matters

Bacterial conjugation is one of the three major mechanisms of horizontal gene transfer (alongside transformation and transduction), and it's the only one requiring direct cell-to-cell contact. You're being tested on your understanding of how bacteria share genetic information, why this matters for antibiotic resistance spread, and what molecular machinery makes it possible. Conjugation explains how resistance genes can jump between species in a hospital ward or how metabolic capabilities spread through soil bacterial communities.

When you encounter conjugation on an exam, you need to think beyond the sequence of events. Focus on the molecular players (relaxase, F pilus, DNA polymerase), the directionality of transfer, and the outcomes for both donor and recipient cells. Don't just memorize the steps—know what each step accomplishes and what would happen if it failed.

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Cell Recognition and Contact

The first phase of conjugation establishes physical contact between genetically distinct cells. The F pilus acts as a molecular grappling hook, identifying compatible recipients and initiating the mating process.

Formation of F Pilus

• F+ (donor) cells produce the F pilus—a retractable, hair-like appendage encoded by the F plasmid's tra genes
• Pilin protein subunits assemble into a hollow tube that extends outward to scan for potential recipients
• Pilus retraction is active and ATP-dependent, physically pulling cells together once contact is made

Contact Between Donor and Recipient Cells

• The F pilus tip binds specific receptors on F- (recipient) cell surfaces, typically outer membrane proteins like OmpA
• Species specificity of conjugation depends partly on receptor compatibility—not all bacteria can mate with each other
• Initial contact is reversible—cells can separate if stabilization doesn't occur quickly

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Mating Pair Stabilization

Once contact occurs, cells must lock together firmly enough to withstand physical disruption. Stabilization involves secondary adhesins and membrane fusion events that create a protected environment for DNA transfer.

Stabilization of Mating Pair

• Surface adhesins beyond the pilus (like TraN and TraG proteins) create multiple contact points between cell envelopes
• Membrane fusion zones form where outer membranes of both cells come into close apposition
• Stable mating pairs resist shear forces—this is why conjugation efficiency drops dramatically in agitated cultures

Formation of Conjugation Bridge

• The conjugation bridge (or mating channel) is a protein complex spanning both cell envelopes, distinct from the pilus itself
• Type IV secretion system components assemble to create a conduit approximately 10 nm in diameter
• This channel protects DNA from nucleases in the environment during transfer

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DNA Processing and Transfer

The molecular heart of conjugation involves precise enzymatic processing of the F plasmid. Relaxase nicks one strand at the origin of transfer (oriT), then pilots that strand into the recipient while rolling-circle replication replaces it in the donor.

Nicking of F Plasmid DNA

• Relaxase (TraI protein) creates a site-specific nick at the oriT sequence, remaining covalently bound to the 5' end
• Only one strand is cut—the strand that will be transferred (called the T-strand)
• Rolling-circle replication initiates in the donor, using the 3' end as a primer to replace the departing strand

Transfer of Single-Stranded DNA

• The relaxase-DNA complex threads through the conjugation bridge in a 5' to 3' direction
• Transfer is unidirectional and processive—once started, the entire plasmid transfers as a continuous strand
• The donor retains a complete F plasmid because rolling-circle replication synthesizes a replacement strand simultaneously

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Recipient Cell Processing

After DNA enters the recipient, the cell must convert the incoming single strand into a stable, functional genetic element. The recipient's own replication machinery completes the process, transforming an F- cell into an F+ cell.

Synthesis of Complementary Strand

• Recipient DNA polymerase III synthesizes the complementary strand using the transferred strand as template
• RNA primase provides the primer—standard replication machinery handles this step
• Both donor and recipient end up with complete double-stranded F plasmids by the end of successful conjugation

Circularization of Transferred DNA

• Relaxase catalyzes recircularization by joining the ends of the transferred strand before complementary synthesis completes
• Circular topology is essential for stable plasmid replication using the theta or rolling-circle mechanism
• Linear DNA would be degraded by recipient exonucleases, making circularization a survival requirement

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Post-Transfer Outcomes

The consequences of conjugation depend on the donor cell type and whether the transferred DNA integrates or remains autonomous. Understanding these outcomes explains how conjugation spreads both plasmid-borne and chromosomal genes.

Integration into Recipient Chromosome

• Hfr (High frequency recombination) donors transfer chromosomal DNA because their F plasmid is integrated into the chromosome
• Homologous recombination can incorporate transferred chromosomal sequences into the recipient's genome
• Interrupted mating experiments exploit this to map bacterial genes—genes closer to oriT transfer more frequently

Separation of Donor and Recipient Cells

• Cells separate after transfer completes (or after mating pair disruption), typically within 5-30 minutes
• The former F- recipient is now F+ and can serve as a donor in subsequent rounds
• Exponential spread of plasmids through populations occurs because every successful conjugation doubles the donor pool

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Quick Reference Table

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Self-Check Questions

1. Which two steps both require the relaxase enzyme, and what different functions does it perform in each?

2. Compare F+ × F- and Hfr × F- conjugation: why does only the first reliably convert recipients to donors?

3. If you disrupted ATP hydrolysis in the donor cell, which steps of conjugation would fail first, and why?

4. A student claims that conjugation and transformation both result in double-stranded DNA in the recipient. Explain what's similar and different about how each process achieves this.

5. FRQ-style: Design an experiment using interrupted mating to determine whether a gene for antibiotic resistance is located on the F plasmid or the bacterial chromosome. What results would you expect in each case?