Conjugation - Lecture notes 5 PDF

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Conjugation Horizontal gene transfer:

Conjugation  Occurs in both archea and bacteria  Conjugative element can be plasmids or integrative elements (may bring part of the host chromosomal DNA)  Transfer require direct cell-cell contact between a Donor (the organism to transfer genetic material) and Recipient (the organism receive)  It is done by a specialized secretion system (Type IV secretion system, T4SS -> not the same as Type IV pili)  Different conjugative systems have different range of host o F: narrow host range (only E. coli and a couple of closely related species) o RP4: broad host range (most if not all Gram-negative bacteria) o Ti: can be conjugated to Eukaryotic cell (plants) Note that: Genes for conjugative systems that transfer between bacteria are usually tra/trb; genes for conjugative system that transfer between bacteria and eukaryotes are usually called vir (virulence)

Steps of Conjugation 1. Pili formation  Pro-pilin inserts into inner membrane  Pro-pilin is manipulated and cleaved to form pilin  Pilin then attaches to a phospholipid  Pilin-phospholipid complex is then assembled into the pilus The F pilus formation can be characterized by use of fluorescent bacteriophage MS-2 as it only binds F+ E. coli. F pilus will only be formed in bacteria having F plasmid. Pili extends and attaches to a recipient cell, then retract to pull the recipient into close contact. On the other hand, RP4 pili is called P-like pili or P-pili. It is shorter than F pili and more vulnerable. Thus, P-pili is not used to capture and pull a recipient for close contact. It enables the donor to adhere to the recipient. When coupling protein is inserted in the inner membrane (which is outside the T4SS structure), it allows the pilin subunit to insert into the pilus (which is the formation of pilus). 2. Mating pair formation As the Recipient and Donor have close contacts, there will be a smooth association of membrane from both the donor and recipient. (However, it may be an artefact created by electron microscope which makes them like smooth association)

3. Relaxosome assembly  Relaxosome = Relaxase + oriT (origin of transfer) + accessory proteins  Relaxase is always essential but other accessory proteins vary from system to system  Accessory protein is used for stabilization of DNA complex or bend the gene for better contact to relaxase 1) Relaxase binds to the oriT 2) Nicks the DNA (cuts one strand and starts unwinding it) 3) Accessory proteins bind to stabilize this complex 4) Coupling protein then bridges the relaxosome with the T4SS Note that Relaxosome and coupling protein is not required for mating pair formation but the transfer of genetic material to the recipient. Relaxosome structure: Note that: TraJ is the only accessory protein involved in the RP4 system, which is encoded by the plasmid.

4. DNA Transfer  Coupling protein associates with the relaxase, it then inserts itself into T4SS structure and allows the relaxosome to pass through the structure (transfer of DNA material) 5. Complementary strand synthesis  Rolling circle DNA replication 1. Circular dsDNA is nicked by relaxase and one strand is brought to the recipient through the pilus 2. The 3’ end is elongated using the unnicked DNA as a template, 5’end is displaced 3. Displaced DNA is a lagging strand and is made ds by series of Okazaki Fragments 4. Another relaxase binds to the joint connecting the original strand and the newly synthesized strand to stop transferring of new strand to recipient 5. The displacement DNA then replace the original strand DNA (which is transferred to the recipient)

Integrative conjugative elements (ICE)  Lacks ori so can only be replicated within a chromosome  It will excise from the chromosome to forma a piece of circular DNA in order to conjugate  However, it may not excise from chromosomal DNA everytime. When relaxase bind to it, it may rip off some of the chromosomal DNA from the donor and transferred to the recipient Hfr strains of E. coli  High frequency of recombination (F plasmid is integrated to the chromosome) – See bacteriophage integration of gene  Conjugated DNA undergoes the same process and transfer some of the donor DNA to the recipient cell.  The conjugated DNA then undergoes homologus recombinant to insert itself to recipient DNA or be degraded by recipient.  It is used for chromosome mapping by calculating the frequency of the gene conjugated along with F. The closer a gene to F, more frequently it will be transferred. 6. Donor and Recipient separate Fertility inhibition  Plasmid inhibits the transfer of other conjugative elements by producing proteins to do the followings: 1. Interfere with conjugation 2. Prevent pilus formation 3. Block coding protein 4. Other mechanism  Donor bacteria with multiple conjugative plasmids often only transfer a subset of these plasmid (Plasmid incompatibility) Exclusion  Some of the bacteria may already have the conjugative element. Duplicated conjugative element is a metabolic burden to the recipient  Conjugative elements have exclusion factors 1. Surface exclusion (preventing formation of mating pairs) 2. Entry exclusion (prevents DNA transfer)  RP4 only prevents DNA transfer as it is not used for formation of mating pairs.

Application of Conjugation  Genetic Engineering – Delivering DNA into plants/bacterial cells  It is very efficient on transferring genetic material (when compared to transformation)  Broad host range (unlike virus)  Easy to remove the delivery vehicle (use of antibiotics) RP4 and plasmid mobilization    

RP4 integrated to the chromosome Plasmids that carry oriT can be mobilized (insertion sequence) Useful for bacteria that are not naturally competent Necessary for rare event such as allele exchange by homologus recombination and transposition)

Ti Plasmid  Virulence genes that transfer T-DNA into plant cells to produce opine for metabolism  It is used to make genetically modified plants by separating the virulence gene and the shuttle vector (have 2 ori, one for bacteria and one for plants) ....