LLecture ecture 19-19-ConjugationConjugation

By: HATİCE NUR HALİPÇİBy: HATİCE NUR HALİPÇİ

Bacterial conjugation:Bacterial conjugation:is the is the transfer of genetic material transfer of genetic material between bacteria through between bacteria through

direct cell-to-cell contact, or direct cell-to-cell contact, or through a bridge-like through a bridge-like

connection between the two connection between the two cellscells

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BACTERIAL CONJUGATIONBACTERIAL CONJUGATION

Discovered in 1946Discovered in 1946 by by Joshua LederbergJoshua Lederberg and and Edward TatumEdward Tatum,,

JOSHUA LEDERBERG JOSHUA LEDERBERG EDWARD L. TATUMEDWARD L. TATUM

(1925-2008) (1925-2008)

(1909-1975) (1909-1975)

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Discovery of conjugation in Discovery of conjugation in E. E. colicoli by Lederberg and Tatum, by Lederberg and Tatum,

1946 1946 ► a. Strain A, which is met- and bio-, produces no a. Strain A, which is met- and bio-, produces no

colonies when plated onto minimal medium.colonies when plated onto minimal medium.

► b. Strain B, which is thr-, leu-, and thi-, produces no b. Strain B, which is thr-, leu-, and thi-, produces no colonies when plated onto minimal mediumcolonies when plated onto minimal medium

► c. A mixture of strains A and B is allowed to grow c. A mixture of strains A and B is allowed to grow for a few cell divisions in complete medium and for a few cell divisions in complete medium and then plated on minimal medium, 1/10,000,000 cells then plated on minimal medium, 1/10,000,000 cells grow into colonies; therefore,a recombinational grow into colonies; therefore,a recombinational process is taking placeprocess is taking place

► d. Cells in the colonies which grew were d. Cells in the colonies which grew were prototrophs (wild-type)prototrophs (wild-type)

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Davis U tube experiment1950

Tatum and Lederberg 1947

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Davis' U-tube experiment Davis' U-tube experiment (1950)(1950)

        1. Cells of strain A were placed on one side of a U-1. Cells of strain A were placed on one side of a U-tube and cells of strain B were placed in the other tube and cells of strain B were placed in the other side.  A filter with pore size that allowed liquid but not side.  A filter with pore size that allowed liquid but not bacterial cells to pass separated the two sides of the bacterial cells to pass separated the two sides of the tubetube

        2. The medium was pumped back and forth by air 2. The medium was pumped back and forth by air pressure/vacuumpressure/vacuum

        3. Cells from both sides were plated on minimal 3. Cells from both sides were plated on minimal medium and none grewmedium and none grew

        4. He concluded that cell-to-cell contact is necessary 4. He concluded that cell-to-cell contact is necessary for genetic recombination to occurfor genetic recombination to occur

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BACTERIAL CONJUGATIONBACTERIAL CONJUGATION

►Bacterial conjugation is often incorrectly Bacterial conjugation is often incorrectly regarded as the regarded as the bacterialbacterial equivalent of equivalent of sexual reproductionsexual reproduction or or matingmating. It is not . It is not actually sexual, as it does not involve actually sexual, as it does not involve the fusing of the fusing of gametesgametes and the creation and the creation of a of a zygotezygote, nor is there equal exchange , nor is there equal exchange of genetic material. It is merely the of genetic material. It is merely the transfer of genetic information from a transfer of genetic information from a donor cell to a recipientdonor cell to a recipient

The F plasmid is transferred by The F plasmid is transferred by conjugation between bacteriaconjugation between bacteriass

1. 1. Bacterial conjugationBacterial conjugation: a : a plasmid genome plasmid genome or or host host chromosome chromosome is transferred from one bacterium to another is transferred from one bacterium to another in a in a matingmating process mediated by process mediated by F plasmidF plasmid..

2. 2. F-plasmidF-plasmid: an example of an episome in : an example of an episome in E. coliE. coli..

3. 3. EpisomeEpisome: an element that may exist as a free : an element that may exist as a free circularcircular plasmidplasmid, or that may become , or that may become integratedintegrated into the bacterial into the bacterial chromosome as a liner sequence.chromosome as a liner sequence.

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F-plasmidF-plasmid

► E.coli cells totally lacking the presence of the F E.coli cells totally lacking the presence of the F factor in any form are called factor in any form are called F – cellsF – cells

► F factor can, however, exist in a cell in three F factor can, however, exist in a cell in three different forms:different forms:

1- Cell containing an autonomously replicating F 1- Cell containing an autonomously replicating F plasmid are called plasmid are called F + cellsF + cells

2-The F factor is able to integrate in to the donor cell 2-The F factor is able to integrate in to the donor cell chromosome to give rise to an chromosome to give rise to an Hfr cellHfr cell that can that can efficiently transfer donor cell chromosal DNA to a efficiently transfer donor cell chromosal DNA to a recipient cell bye conjugation recipient cell bye conjugation

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F-plasmidF-plasmid3- 3- Prime Factor Plasmids that “leave” the

genome carrying chromosomal DNA are known as prime factors.

They “leave” the chromosome by homologous recombination, resulting in a deletion in the chromosome

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The F' state and merozygotes The F' state and merozygotes ► a. The F factor can excise from the chromosome a. The F factor can excise from the chromosome

and include a part of the bacterial chromosome to and include a part of the bacterial chromosome to become a F‘become a F‘

► b. When a F' is mated with a F-, both resultant cells b. When a F' is mated with a F-, both resultant cells become F' and both cells also contain the bacterial become F' and both cells also contain the bacterial chromosomal segment that was present in the F'chromosomal segment that was present in the F'

► c.Merozygotec.Merozygote is a state when a bacterial cell, is is a state when a bacterial cell, is temporarily temporarily diploiddiploid as result of DNA transfer as result of DNA transfer processes like conjugation processes like conjugation

4 - Conjugation-Transfer of Chromosomal DNA.swf

F-plasmidF-plasmid

1. large circular plasmid (1. large circular plasmid (100 kb100 kb))

2. only 60% (ca. 2. only 60% (ca. 60 genes60 genes) has been mapped.) has been mapped.

3. 3. 32 kb 32 kb is organized as a unit to transfer its is organized as a unit to transfer its genome to another bacteria (genome to another bacteria (transfer region transfer region or or tratra genes) genes)

4. 4. twotwo methods of replication: methods of replication:

a. a. oriVoriV as free plasmid (one copy/ bacterial as free plasmid (one copy/ bacterial chromosome)chromosome)

b. uses b. uses E. coli E. coli chromosomal origin when chromosomal origin when integrated (integrated (oriCoriC); ); oriVoriV is suppressed. is suppressed.

F-plasmidF-plasmidoriT

oriV

tra genes32 kb

tra genes32 kb

100 kb100 kb

used to initiate replication for transfer

used to initiate plasmid replication

IS elements (insertion sequences used in transposition)

Discrete region that has transfer genes:

tra & trb loci (~40 genes)(Origin of transfer)

F-F-plasmidplasmid

IS element

F-plasmid

E. coli chromosome

integrated F-plasmid

oriC

oriV

After integration, F-plasmid After integration, F-plasmid replicates as part of host replicon. replicates as part of host replicon. oriVoriV is suppressed. is suppressed.

After integration, F-plasmid After integration, F-plasmid replicates as part of host replicon. replicates as part of host replicon. oriVoriV is suppressed. is suppressed.

Hfr cell contains integrated F plasmid.

F+ cell contains episomal F plasmid

Chromosome Transfer: formation of Hfr strains(high frequency recombination)

There are two mechanisms of integration:

1. Homologous recombination

2. Transposition

Depending on the site of F-plasmid integration there are different Hfr strains

7-1BacConjugation.swf

The Sex Pilus

-Some E. coli strains contain Fertility plasmids (F+)

- Carries the information required for its own transfer

- DNA is NOT transferred through the pilus

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F-Pilus

1. Extracellular filament that extends from surface (2-3 m); hair-like

-main structural component is a single subunit (pilin; coded by traA gene)

-tip protein

2. Pilus recognizes various receptors on the host cell.

-mating pair formation may occur differently on solid or liquid media

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recipientrecipient

F donorF donor

piluspilus

F pili are essential for initiating pairing but are NOT channels for DNA transport

Single stranded genomes are generated for bacterial conjugation

1. F-plasmid is ~100kb & takes about 5 min to transfer.

2. Sequences required for transfer are located within a 33.3-kb transfer region (IncF1)

3. –Pilus synthesis and assembly -Nicking & initiation of transfer -mating pair (pore) formation -transfer of DNA -mating-aggregate stabilization -surface exclusion -regulation

oriT traM J YALEKBPVRC WU N trbCDE traF trbB traH G ST D I/Z

finP

Transfer genes

traJtraJactivatoractivator

traY/ItraY/ITranscription unitTranscription unit

tratra region of the F plasmid region of the F plasmid

Direction Direction of transferof transfer

regulationregulation

tratra & & trbtrb loci; loci;~40 genes~40 genes

Expressed coordinately as a part of Expressed coordinately as a part of single transcription unitsingle transcription unit traY/traI traY/traI

F-plasmidF-plasmid

5. 5. F-positiveF-positive ( (FF++)) bacteria are able to bacteria are able to conjugateconjugate (mate) with (mate) with F-minusF-minus ( (FF--) bacteria.) bacteria.

6. In its 6. In its integratedintegrated form the F-plasmid may form the F-plasmid may cause some, or all, of the cause some, or all, of the bacterial bacterial chromosome to transfer chromosome to transfer to the F-minus to the F-minus recipient.recipient.

7. F-positive bacteria possess 7. F-positive bacteria possess pilipili formed from formed from the the pilinpilin protein. protein.

ConjugationConjugation1.1. tip of the F-pilus makes contact with recipient tip of the F-pilus makes contact with recipient

cell.cell.

a. pilus is composed of pilin subunits which form a. pilus is composed of pilin subunits which form a hollow cylinder of 8 nm with 2 nm inner a hollow cylinder of 8 nm with 2 nm inner diameter. diameter.

b. If potential recipient is F-positive, no b. If potential recipient is F-positive, no connection is formed due to connection is formed due to surface exclusion surface exclusion proteins proteins coded by coded by traStraS and and traT traT of F-plasmid.of F-plasmid.

ConjugationConjugation2. 2. Pilus retracts Pilus retracts bringing recipient closer for transfer.bringing recipient closer for transfer.

3. 3. DNA transferred DNA transferred through through channelchannel formed by protein coded by formed by protein coded by traDtraD gene. TraN and TraG may also participate in pore formation. gene. TraN and TraG may also participate in pore formation.

4. Transfer begins from 4. Transfer begins from oriToriT which is nicked by which is nicked by TraY/TraI TraY/TraI complex complex at a at a nicnic site. (TraI actually nicks) site. (TraI actually nicks)

5. 5. TraY/TraI multimeric TraY/TraI multimeric complex migrates around circle and complex migrates around circle and unwinds DNA unwinds DNA from 5’ end . from 5’ end . 1200bp/sec1200bp/sec..

6. Only 6. Only one unitone unit length is transferred. length is transferred.

Overview of Conjugation

1. Plasmids that are both conjugative and mobilizable are termed self-transmissible. Plasmids that are mobilizable but nonconjugative ,are called mob plasmid .They are often efficiently transferred to recipient cells when other plasmids present in the donor cell provide the necessary cell contact functions.

2. The transfer systems are encoded by the tra genes (contained on the plasmids themselves).

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Overview of Conjugation

3. Tra systems are linked to their incompatibility (Inc) group. F-type IncF, RP4 plasmids IncP

4. Plasmids that have transfer systems that allow transfer of DNA to unrelated species are known as promiscuous plasmids. IncW plasmids, IncP plasmids, & IncN.

1. A site on the plasmid, known as the origin of transfer (oriT) is nicked by a specific endonuclease (TraI; TraY is also a part of the complex).

2. A pore is formed between the two cells and only ONE strand of DNA is passed through to the other cell (5’ end first).

5’

3. The single strand in each cell undergoes replication to form double stranded DNA.

Mechanism of self-transmissible transfer

Only a single unit length of F factor is transferred

F F-

FF

Free 5’ end

Mechanism of nonconjugative

plasmid mobilization1. The mob plasmid cannot transfer without another plasmid

2. The other plasmid (helper plasmid) may or may not be a self-transmissible plasmid but MUST contain tra functions (cell contact, nicking).

3. If the helper plasmid is self-transmissible it may also transfer.

tra

tra

tra

tra

tra mob

helper

Major Functions During Transfer (>40 genes)Major Functions During Transfer (>40 genes)

2. TraI has nuclease & helicase (ATP) activity. Function enhanced by TraY & IHF (integration host factor)

TraI is a transferase covalent attachment of the 5’ end of the DNA to the protein

3. TraD –active transport, binds DNA, ATP/GTP binding sites, necessary for DNA transfer

1. TraY binds near oriT and recruits traI relaxase,

Surface Exclusion

Reduces conjugation among cells carrying closely related plasmids .

1. TraT –outer membrane protein that blocks mating-pair formation

2. TraS –blocks DNA transfer

Chromosome Transfer: Hfr Recipient cell(Part 1)

1.

2.

3. Free 5’ end

Hfr cells

F+

Chromosome Transfer: Hfr Recipient cell(Part 2)

5.

3.

4.

oriT region only

1. The 1. The transfer process transfer process uses the uses the rolling circlerolling circle method of method of replication. The complement to the transferred strand is replication. The complement to the transferred strand is synthesized in the recipient. synthesized in the recipient.

It takes It takes 100 min100 min to transfer entire chromosome of to transfer entire chromosome of E. coli.E. coli.

22. F-positive strains support . F-positive strains support high levels of recombinationhigh levels of recombination and and are described as are described as Hfr Hfr strains (strains (high frequency of high frequency of recombinationrecombination).).

Chromosome transfer

33. The . The transfer of the host chromosome transfer of the host chromosome is is away away from from the the tratra region and F-plasmid, except for a small region and F-plasmid, except for a small part around part around oriToriT. .

44. . Typically only relatively short stretches of DNA are transferred & are integrated into the recipient. & are integrated into the recipient.

Chromosome transfer

5. Chromosome transfer usually does not result in conversion of recipient cell to F+.

6. In chromosome transfer, donor DNA integrates into the host genome by recombination or transposition

-In plasmid transfer, this does not occur.

Chromosome transfer

7. Bacterial contact usually broken before DNA transfer complete;

A gradient of transfer frequencies around the chromosome

Chromosome transfer

8. E. coli chromosome as a map divided into 100 minutes; the starting point for the gradient of transfer is different for each Hfr strain; determined by the F factor integration site. 0 &100

50

25 75E. coli

micro_conjugation.swf

oriT traM J YALEKBPVRC WU N trbCDE traF trbB traH G ST D I/Z

finP

Transfer genes

traJtraJactivatoractivator

pilinpilin Surface Surface exclusionexclusion

ChannelChannel

Negative Negative regulator regulator transcripttranscript

traY/traItraY/traIDNA nicking and unwindingDNA nicking and unwinding

tratra region of the F plasmid region of the F plasmid

Senses Senses that that mating mating pair pair formedformed

Direction Direction of transferof transfer

regulationregulation

traT-outer membrane protein that blocks mating pair formation.

traS-blocks DNA transfer.

traI- covalently attaches to 5’ end of DNA & unwinds it

finOHelps finPHelps finP

traY- recruits traI to 5’ end of DNA

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