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This lecture discribes
- What is bacterial conjugation?
- How F plasmid mediates the process?
- How conjugation helped for recombination?
Conjugation
Conjugation
Bacterial conjugation (mating) is a mechanism of genetic transfer that involves cell-to-cell contact. It is a plasmid encoded mechanism. Conjugative plasmids use this mechanism to transfer copies of themselves to new host cells. Thus the process of conjugation involves a donor cell, which contains the conjugative plasmid, and a recipient cell, which does not. During the conjugation, some of the plasmids which could not transfer by themselves (non-transmissible) also can be mobilized by this plasmid. Laderberg & Tatum first demonstrated the process of conjugation through auxotrophic mutants.
Laderberg and Tatum’s “U” tube experiment
F Plasmid
F Plasmid
The F plasmid (F stands for “fertility”) is a circular DNA molecule of about 100 kbp sized. The tra region, containing genes that encode transfer functions. Many genes in the tra region are involved in mating pair formation, and most of these have to do with the synthesis of a surface structure, the sex pilus. Some of the identified genes and their functions in tra operon are as follows:
- Pili Assembly and Production - traA, traB, traE, traC, traF, traG, traH, traK, traL, traQ, traU, traV, traW
- Inner Membrane Proteins - 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
- The oriV region responsible for the origin of vegetative replication, refers the F plasmid replication in the donor itself.
- The oriT of F plasmid is responsible for origin of replication during conjugation (T refers transfer here). During conjugation, oriT initiates the replication from which rolling circular DNA replication enables to form a copy of F plasmid (single strand, linear) which will move to recipient.
- Apart from these important genes, insertion sequences (IS3 and IS2) and transposon (Tn1000) are also present. These may recombine with identical elements on the bacterial chromosome, which leads to integration and the formation of different strains.
Molecular mechanism of F plasmid transfer
Molecular mechanism of F plasmid transfer
Step I: Effective Contact
The tra operon of F plasmid produces specialized pilus called sex pilus and only the donor cells produce these pili because they alone have F plasmid. These Pili allow specific pairing to take place between the donor and recipient cells. All conjugation in gram-negative Bacteria is thought to depend on cell pairing brought about by pili. The pilus makes specific contact with a receptor on the recipient cell and then is retracted by disassembling its subunits. This pulls the two cells together. Following this process, donor and recipient cells remain in contact by binding proteins located in the outer membrane of each cell. DNA is then transferred from donor to recipient cell through this conjugation junction.
Step II. F plasmid replication
DNA synthesis is necessary for DNA transfer by conjugation. This DNA is synthesized by rolling circle replication, a mechanism also used by some viruses. DNA transfer is triggered by cell-to-cell contact, at which time one strand of the circular plasmid DNA is nicked and is transferred to the recipient. The nicking enzyme required to initiate the process, TraI, is encoded by the traoperon of the F plasmid. This protein also has helicase activity and thus also unwinds the strand to be transferred.
Step III. Transfer of newly synthesizing copy of F plasmid
As this transfer occurs, DNA synthesis by the rolling circle mechanism replaces the transferred strand in the donor, while a complementary DNA strand is being made in the recipient. Therefore, at the end of the process, both donor and recipient possess complete plasmids. For transfer of the F plasmid, if an F-containing donor cell (F+), mates with a recipient cell lacking the plasmid (F-), the result is two F+ cells. Transfer of the F plasmid, comprising approximately 100 kbp of DNA, takes about 5 minutes.
Animation of conjugation (Youtube)
Episome and High frequency of Recombination
Episome and High frequency of Recombination
The F plasmid, what we have discussed so fat is an episome, which refers a plasmid that can integrate into the host chromosome. When the F plasmid is integrated, chromosomal genes can be transferred along with the plasmid. Following genetic recombination between donor and recipient DNA, horizontal transfer of chromosomal genes by this mechanism can be very extensive.
F+ Cells refers the E. coli possessing a non-integrated F plasmid and those with an F plasmid integrated into the chromosome (as episome) are called Hfr (for high frequency of recombination) cells. This term refers to the high rates of genetic recombination between genes on the donor and recipient chromosomes. Both F+ and Hfr cells are donors, but unlike conjugation between an F+ and an F-, conjugation between an Hfr donor and an F- leads to transfer of genes from the host chromosome. This is because the chromosome and plasmid now form a single molecule of DNA. Consequently, when rolling circle replication is initiated by the F plasmid, replication continues on into the chromosome. Thus, the bacterial chromosome as a whole is also replicated and transferred. The integration of F plasmid provides the mobilizing property to the genome of an organism
How episomes are formed
The F plasmid and the chromosome of E. coli both carry several copies of mobile elements called insertion sequences (See the physical map of the F plasmid). These provide regions of sequence homology between chromosomal and F plasmid DNA within the donor. Consequently, homologous recombination occurs between an IS on the F plasmid and a corresponding IS on the chromosomal DNA, results in integration of the F plasmid into the host chromosome. Once integrated, the plasmid no longer replicates independently, but the traoperon still functions normally and the strain synthesizes pili and can facilitate the conjugal tube between donor and recipient.
Mobilization between hfr and F- cells
When a recipient is encountered, conjugation is triggered just as in an F+ cell, and DNA transfer is initiated at oriT (origin of transfer) site. However, because the plasmid is now in the part of the chromosome as episome, during the transfer of plasmid DNA by rolling circular replication, chromosomal genes begin to be transferred. As in the case of conjugation with just the F plasmid itself, chromosomal DNA transfer also involves replication.
Recombination by hfr cells
- The donor DNA strand typically breaks during transfer step of conjugation due to its size and only part of the donor chromosome is transferred to recipient.
- So, the recipient does not become Hfr (or F+) because incomplete transfer of the integrated F plasmid.
- However, after this transfer, the Hfr strain (donor) remains Hfr because it retains a copy of the integrated F plasmid.
- In recipient cell, as a partial chromosome has been received, it cannot be replicated and for the survive, the incoming donor DNA recombines with the recipient chromosome.
- Following recombination, the recipient cell may express a new phenotype due to incorporation of donor genes.
- As this process induce high chance of recombinants, hfr cells as donor are highly useful than F+ cells.
HFR x F- matting can be useful for determining gene arrangement and orientation
Several distinct insertion sequences (IS) are present on the chromosome, F plasmid has several choice of integration as episome and resulted a number of distinct Hfr strains are possible. Hfr strains that differ in the chromosomal integration site of the F plasmid transfer genes in different orders. The orientation of the F plasmid determines which chromosomal genes of donor should enter the recipient first. Hence different hfr cells matting with recipient and interrupting the DNA transfer at frequent intervals, it was possible to determine the arrangement and orientation of most of the genes in the E. coli chromosome long before it was sequenced.
Hfr cells can be used in genetic crosses
Unlike transformation and transduction (where donor will not survive), conjugation is typical genetic transfer mechanism in which both donor and recipient are viable. Hence it is necessary to select the recombinants only after matting using some phenotypic characters like antibiotic sensitivity, auxotrophic mutants, etc.
For example, in an experiment, an Hfr donor sensitive to streptomycin (StrS) and is wild type for synthesis of the amino acids threonine and leucine (Thr+ and Leu+) and for utilization of lactose (Lac+), is mated with a recipient cell that cannot make these amino acids or cannot use lactose, but that is resistant to streptomycin (StrR). The selective minimal medium contains streptomycin so that only recombinant cells can grow. The composition of each selective medium is varied depending on which genotypic characteristics are desired in the recombinant. The frequency of gene transfer is measured by counting the colonies grown on the selective medium. Using this principle, it is possible to mat two parents to get desire recombinants.
F’ Plasmid
Sometimes, the F plasmid integrated with donor chromosome may excised from the genome accidently. During this excision process, some portions of chromosomal DNA also incorporated with the original genes of F plasmid. The reason behind this mistake is due to identical insertion sequences present in both F plasmid as well as chromosomal DNA. Hence after liberated, if the F plasmid contains some portions of donor’s chromosomal genes, it is called as F’ (variant) plasmid. When, F’ plasmids promote conjugation, they transfer the donor’s chromosomal genes at high frequency to the recipients. The F’ plasmid mediated conjugation resembles the specialized transduction. If a F’ plasmid (with known donor’s chromosomal genes) is effectively transferred to the recipient (F-), and makes into F’ cells. At this junction, some chromosomal genes will be in two copies (one in F’ plasmid from donor and another at recipient chromosomal DNA). Such partial diploids are rare phenomenon in prokaryotic cells.
Partial diploids are used for complementation assay
A bacterial strain that carries two copies of any particular chromosomal segment is known as a partial diploid or merodiploid. In general, one copy is present on the chromosome itself and the second copy on another genetic element, such as a plasmid or a bacteriophage. Nowadays, recombinant DNA technology (otherwise Gene cloning) allows to such partial diploids of any portions of DNA. The introduced copy will be in phage or plasmid. Therefore, if a chromosomal gene copy is defective, it is possible to supply a functional gene through a plasmid or phage. For example, in lac operon, if lacZis inactivated by mutation, the mutant will be of LacZ- phenotype and cannot utilize the lactose. If a copy of functionally active lacZgene is introduced to this mutant by any of these vectors, the LacZ- mutant will restore its wild character (Positive for lactose utilization). This process is called as complementation, because the introduced wildtype gene is said to complement the mutation.
Complementation assay to identify the mutants
When two mutants strains are genetically crossed (by conjugation, transduction or transformation), homologous recombination can yield wild-type recombinants unless both mutations affect exactly the same base pairs. For example, if two different Trp- E. colimutants are crossed and Trp+ recombinants are obtained, it is obvious that the mutations in the two strains were not in the same base pairs. However, this kind of experiment cannot determine whether two mutations are in two different genes that both affect tryptophan synthesis or in different regions of the same gene. This can be determined by a complementation test. How complementation test will identify the gene’s functions and how mutation affects the biosynthesis, is described below:
If we wish to identify those two Trp- mutants have a mutation in a same gene. For this, these two mutants should be arranged in such a way that one mutation (A) should be in chromosome and another one (B) is on plasmid. Now, the mutations are said to be trans with respect to one another. Under such condition, if the mutations are in same genes, the recombinant will have two defective copies of the same gene and again the recombinant will be Trp-. On the other hand, if the two mutations are in different genes, the recombinant will have one copy of wild type (non-mutated) gene and thus will have Trp+. If one DNA molecule carries both the mutations, then the mutation is said to be cis. Here, the second DNA molecule can complement the function and thus makes the recombinant as positive to Trp. This type of complementation test is called as Cis-Trans test. Based on the cis-trans test, the gene can be defined as cistron and is equivalent to defining a structural gene as a segment of DNA that encodes a single polypeptide chain.
If two mutations occur in genes encoding different enzymes, or even different protein subunits of the same enzyme, complementation of the two mutations is possible, and the mutations are therefore not in the same cistron.
Hfr versus F- recombination (youtube)
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