Conjugation (2)

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Bacterial conjugationBacterial conjugationis the transfer of genetic material betweenbacterial cells by direct cell-to-cell contact or by a bridge-like

connection between two cells.

Discovered in 1946 by Joshua Lederberg and Edward Tatum,

conjugation is a mechanism of horizontal gene transfer as are

transformation and transduction although these two other

mechanisms do not involve cell-to-cell contact.

In 1958 they received Nobel Prize.

Bacterial conjugation is often regarded as the bacterial equivalent

of sexual reproduction or mating since it involves the exchange ofgenetic material.


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During conjugation the donorcell provides a conjugative or

mobilizable genetic element that is most often a plasmid or

transposon.

Most conjugative plasmids have systems ensuring that the recipient

cell does not already contain a similar element.

The genetic information transferred is often beneficial to the

recipient.

Benefits may include antibiotic resistance, xenobiotic tolerance or

the ability to use new metabolites.

Such beneficial plasmids may be considered bacterial

endosymbionts.Bacterial conjugation is often incorrectly regarded as the bacterial

equivalent of sexual reproduction or mating.

Bacterial conjugation is merely a transfer of gene from one

bacterium to other unlike the fusion of gametes in sexual

reproduction in organisms.

Lederberg and Tatum studied two strains of Escherichia coliwithdifferent nutritional requirements.

Strain A would grow on a minimal medium only if the medium were

supplemented with methionine and biotin; strain B would grow on a

minimal medium only if it were supplemented with threonine,

leucine, and thiamine.

Thus, we can designate strain A as metbiothr+leu+thi+and strain


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B as met+bio+thrleuthi.

Strains A and B were mixed

together, and some of the progeny

were now wild type, having

regained the ability to grow

without added nutrients.

They plated bacteria into dishes

containing only unsupplemented

minimal medium.

On control dishes, plated either

with strain A or strain B no colonygrew but on mixed plate colonies

grew at a frequency of 1x10-7.

It could be suggested that the

cells of the two strains do not

really exchange genes but instead

leak substances that the othercells can absorb and use for

growing.

This possibility of cross feeding

was ruled out by Bernard Davis. He

constructed a U-tube in which the

two arms were separated by a fine


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filter.

The pores of the filter were too small to allow bacteria to pass

through but large enough to allow easy passage of the fluid medium

and any dissolved substances.

Strain A was put in one arm; strain B in the other.

After the strains had been incubated for a while, Davis tested the

content of each arm to see if cells had become able to grow on aminimal medium, and none were found.

In other words,physical contactbetween the two strains was

needed for wild-type cells to form.

It looked as though some kind of gene transfer had taken place, and

genetic recombinants were indeed produced.


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In 1953, William Hayes determined that genetic transfer occurred in

one direction in the above types of crosses.

Therefore, the transfer of genetic material in E. coliis not

reciprocal.

One cell acts as donor, and the other cell acts as the recipient.

This kind of unidirectional transfer of genes was originally

compared to a sexual difference, with the donor being termed

male and the recipient female.

However, this type of gene transfer is not true sexual reproduction.

In bacterial gene transfer,one organism receives geneticinformation from a donor;the recipientis changed by thatinformation.

By accident, Hayes also discovered a variant of his original donor

strain that would not produce recombinants on crossing with the

recipient strain.

Apparently, the donor-type strains had lost the ability to transfer

genetic material and had changed into recipient-type strains.In his analysis of this sterile donor variant, Hayes realized that the

fertility (ability to donate) of E. colicould be lost and regained

rather easily.

Hayes suggested that donor ability is itself a hereditary state

imposed by a fertility factor F).Strains that carry F can donate, and are designated F+.


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Strains that lack F cannot donate and are recipients.

These strains are designated F.The E. coli fertility plasmid are most extensively studied plasmid.

The general features of this plasmid are,

1. Large circular plasmid (100 kb)

2. Only 60% genes has been mapped.

3. 32 kb is organized as a unit to transfer its genome to another

bacteria (transfer region or tra genes)

4. Two methods of replication:

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

b. uses bacterial chromosomal origin when integrated (oriC);oriV is suppressed.

Plasmid replication requires a "mating bridge" between the donor

and recipient cells.

Before the mating bridge can form:

donor must recognize recipient cell

donor must make contact with recipient cellThe conjugative functions of the F plasmid are specified by a cluster

of at least 25 transfer (tra) genes.

They determine:

expression of F pili.

synthesis and transfer of DNA during mating

interference with the ability of F+bacteria to serve as recipients.


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Each F+bacterium (donor) contains F pili.

Binding of F pili to specific outer membrane protein on recipient

cells takes place.

An intercellular cytoplasmic bridge formation is formed.

Transfer of single strand of DNA from donor to recipient takes place.

Transferred strand is converted into double stranded circle in

recipient.

The copy is retained in donor.

The newly synthesized double stranded DNA is called exogenote and

native DNA as endogenote.

The tra locus includes the pilin gene and regulatory genes, whichtogether form pili on the cell surface, polymeric proteins that can

attach themselves to the surface of F-bacteria and initiate the

conjugation.

The tra genes encode proteins which are useful for the propagation

of the plasmid from the host cell to a compatible donor cell or

maintenance of the plasmid.DNA transfer genes traY and traI product make a single stranded

nick at OriT.

The 5 end separates progressively and transferred to F -cell.

Separation takes place under the influence of traY and traI

multimers.

Separation is very rapid at the rate of 1200bp/sec.


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The roles of some tra-gene encoded proteins

Pili Assembly and

Production

traA, traB, traE, traC,

traF, traG, traH, traK,

traL, traQ, traU, traV,

traW,Inner Membrane traB, traE, traG,traL,

traP

Periplasmic Proteins traC, traF, traH,traK,

traU, traW

DNA transfer traC, traD, traI,traM,

traY

Surface Exclusion

Proteins

traS, traT

Mating Pair Stabilization traN, traG

Only single strand is transferred.

DNA replication does not take place.In the recipient cell it becomes double stranded.

Conjugation is governed by ~33 kb transfer region (Tr).

It contains around 40 genes, arranged in two loci (Tra and Trb).

There arranged in three transcriptional units.

TraJ regulates other two units: Tra M, and TraY-I, a 32 kb

transcription unit.


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On the other strand finP regulator of antisense RNA is present that

turns of TraJ.

FinP needs finO product to function as regulator.

TraA gene codes for pilin protein that form F pili,

Twelve other genes govern pili formation.

F pili is 2-3 m long, ~8 nm in diameter with a 2 nm internal

diameter.

Tra S and Tra T products have role in surface exclusion.

Once contact with pili is made. It disassembles and conjugation

bridge is made by tra D gene product (other gene products may be

involved).Tra M recognizes the mating pair.

Then Tra Y binds near the Ori T.

Ori T is nicked by Tra I at nic site and bind at 5 of the nick.

This binding causes unwinding of ~200 bases.3 OH end is used for

repair or other strand synthesis.

The separated single strand is transferred into recipient cell.Recently it has been shown that the F pili are involved only in

establishing cell contact, not in DNA transfer.

After the F pili bring a donor cell and a recipient cell together, a

conjugation channel forms between the cells, and DNA is transferred

from the donor cell to the recipient cell through this channel.

When an F+cell conjugates (or mates) with an F-recipient cell,


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only the F factor is transferred.

Both cells (donor and recipient) become F+cells because the F

factor is replicated during transfer, and each cell receives a copy.

The F factor can also integrate at any of the number of sites

present in recipient cell chromosome by site specific recombination.The recombination is believed to be mediated by short DNA

sequences called IS elements.

Cells carrying the integrated form of the F factor are called Hfr

(high frequency recombination).

In the integrated state the F factor mediates the transfer of a

chromosome of the Hfr cell into recipient cell.In its integrated state, the F factor mediates the transfer of the

chromosome from the Hfr cell to a recipient (F-) cell during

conjugation.

Usually, the cells separate before chromosome transfer is complete;

thus, only rarely will an entire chromosome be transferred from an

Hfr cell to a recipient cell.

The mechanism that transfers DNA from a donor cell to a recipient

cell during conjugation appears to be the same.

Transfer is initiated at a special site called oriT (origin of transfer).

The other two sites, oriV and oriS, are used to initiate replication

during cell division, not during conjugation.


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oriV is the primary origin of replication

during cell fission; oriS is asecondary origin

that performs this function when oriV is

absent or nonfunctional.

Because transfer is initiated within theintegrated F factor, part of the F factor is

transferred prior to the transfer of

chromosomal genes in HfrxF-matings.

The rest of the F factor is transferred after

the chromosomal genes.

Thus, the recipient cell acquires a completeF factor and is converted to an Hfr cell only

in rare cases when an entire Hfr

chromosome is transferred.

Several of the details of conjugation were worked out using one

particular Hfr strain called Hfr H (for William Hayes).

In this strain, the F factor is integrated near the thr (threonine) and

leu (leucine) loci.

At varying times after the Hfr H and F-cells were mixed to initiate

mating, samples were removed and agitated vigorously in a blender

to break the conjugation bridges and separate the conjugating cells.


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Following this technique Elie Wollman and Franois Jacob (1957),

working at the Pasteur Institute in Paris, provided new insight into

the process of Conjugation.

This helped in developing map of E. coli chromosome.

There are three major

types of plasmids in E. coli:

F factors, R plasmids, and

Col plasmids.

R plasmids (resistance

plasmids) carry genes thatmake host cells resistant

to antibiotics and other

antibacterial drugs.

Col plasmids (earlier called

colicinogenic factors)

encode proteins that killsensitive E. coli cells.

There are a large number

of distinct Col plasmids.


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Some plasmids endow host cells with the ability to conjugate.

All F plasmids, many R plasmids, and some Col plasmids have this

property; or they are conjugative plasmids.

The conjugative nature of many R plasmids plays an important role

in the rapid spread of antibiotic and drug resistance genes through

populations of pathogenic bacteria.

F factor and certain other genetic elements had unique properties

and were named as episome.

An episome is a genetic element that is unessential to the host andthat can replicate either autonomously or be integrated (covalently

inserted) into the chromosome of the host bacterium.The ability of episomes to insert themselves into chromosomes

depends on the presence of short DNA sequences called insertion

sequences (or IS elements).

The IS elements are present in both episomes and bacterial

chromosomes.

These short sequences (from ~ 800 to ~ 1400 base pairs) aretransposable; that is, they can move from one chromosome to a

different chromosome.

In addition, IS elements mediate recombination between otherwise

nonhomologous genetic elements.

The role of IS elements in mediating the integration of episomes is

well documented in the case of the F factor in E. coli.


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Crossing over between IS elements in the F factor and the bacterial

chromosome produces Hfrswith different origins and directions of

transfer during conjugation.SEXDUCTIONThe F plasmid gets integrated to form Hfr cells, this process isreversible and some times rare F+ cells are seen in Hfr cultures.But generally, anomalous excision events occurproducing


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autonomous F factors carrying bacterial genes.These modified F factors, called F (F prime) factors, were first

identified by Edward Adelberg and Sarah Burns in 1959.

F factors range in size from those carrying a single bacterial gene to

those carrying up to half the bacterial chromosome.

Transfer of F factors to recipient (F-) cells is called sexduction; itoccurs by the same mechanism as F factor transfer in F+x F-matings.

There is one important difference: bacterial genes incorporated into

F factors are transferred to recipient cells at a much higherfrequency.

F factors can be used to produce partial diploids carrying two

copies of any gene or set of linked genes.

Thus, sexduction can be used to determine dominance relationships

between alleles and perform other genetic tests requiring two copies

of a gene in the same cell.


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DISTINGUISHING CHARACTERISTICS OF

CONJUGATIONDNA transfer requires cell-cell

contact.

DNA transfer occurs via a conjugal pore.DNA

transfer occurs via a conjugal pore.

DNA transfer occurs in one direction - from

donor to recipient not vice versa

DNA transfer does not require protein

synthesis in donor.

DNA transfer requires energy in donor cell -

primarily ATP.

Conjugation (2)

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