Bacterial conjugation process

The review article focuses on the plasmid-mediated conjugation process in E. coli bacteria. A unidirectional transfer of the genetic material through a contact between the two bacterial cells is known as conjugation. A physical bridge between the two cells mediates the DNA transfer. In prokaryotes, such as bacteria, the transfer of the genetic material mostly involves a one-way process. Thus, the process of conjugation helps to transfer the genetic material from one cell to another, enabling the process of copying the genetic material. Among the bacteria, most widely used ones for genetic analysis involve Escherichia coli bacteria. 
Lederberg and Tatum first conducted a conjugation experiment on E. coli cells. William Hayes demonstrated the theory of the unidirectional transfer of the genetic material in E. coli. Bernard Davis independently conducted a U-tube experiment. With the help of the above studies, various researchers came up with different findings in bacterial genetics. Conjugation studies also help to map the genes. In the late 1950’s, Francis Jacob and Elie Wollman studied the transfer of genetic material from Hfr strains to F^- strains. Most of the conjugation occurs through plasmids in the bacterial cells.

A plasmid is an extrachromosomal genetic material present in the bacterial cell. These plasmids exhibit a property of transferring the genetic material. Hence they are used as vectors in the process of cloning and recombinant DNA technology. The contact between the two cells involves a physical bridge between the two cells. Thus, a segment of the chromosome from one cell transfers to another cell thereby undergoing genetic recombination. Hence, the cells receiving the DNA are known as trans-conjugants. The essential genetic element for a bacterial conjugation is known as a conjugon. Unlike prokaryotes, the process of conjugation in protozoa involves a two-way process.

Image 1: Conjugation in bacteria

Lederberg and Tatum experiment:

Two E. coli strains to differ in their nutritious environments were studied. Note that even bacteria require nutrients for carrying out various cellular activities.  The two bacterial strains were labeled as strain A and strain B respectively. The amino acid synthesizing bacteria do not require a supplemented medium. Such type of bacteria labeled as “+” strains, synthesize the required nutrients. The strain A had a genotype known as met bio thr⁺ leu⁺thi⁺. The strain A bacteria grew on a medium supplemented with methionine and biotin. Without these two functional molecules, the strain A would not have grown. The strain B had met⁺bio⁺thr leu thi genotype. It required threonine, leucine, and thiamine to grow. Both the strains were mixed and plated on a minimal medium. The mixed culture gave rise to the prototrophic colonies. No colonies were visible on the minimal medium after plating the strains individually. It is due to the auxotrophic cells.

A mutant organism capable of growing only on a minimal medium with the growth factor supplementation not required by the wild-type strains is known as an auxotroph. A strain of microorganisms not requiring any additional nutrient to grow is known as a prototroph. The prototrophic colonies occurred at a frequency of 1 in 10 million cells. These colonies were recombinants arising due to the exchange of the genetic material between the two cells.

Davis U-tube experiment:

Bernard Davis showed physical contact between the two bacterial cells using a U-tube apparatus. He placed both the bacterial strains in a liquid medium poured into either side of the tube separated by a filter. The medium moved between the compartments. It was later on, plated on a minimal medium. None of the colonies grew. Hence, through this experiment, Davis demonstrated the cell to cell contact of the bacteria mediated gene transfer.

William Hayes experiment:

The genetic exchange in the E. coli occurred in one direction. One cell acted as a donor and the other like a recipient. Sex factor or the F factor-mediated the transfer of the genetic material. There are two types of bacterial cells such as the donor and the recipient cells. The donor cells are the one giving the genetic material. The recipient cells accept the genetic material from the donor cells. F-factor is a plasmid capable of replicating independently. Hence, the donor bacteria are known as F+ strains. The recipient bacteria are known as F- strains. Two same types of bacteria do not undergo conjugation. The F+ and F- strains only undergo conjugation.

F+ and F- matings:

The process of conjugation involves mating between F+ and F- strains. The F factor of the donor bacteria has a nick at one of the strands extending through the sex pilli or a physical bridge. The nicked strand gets transferred to the recipient where the remaining strand gets copied. Hence, the transfer and the synthesis of the DNA gets completed. Once the transfer of the genetic material from F+ to F- strains gets completed, the F- strain with the genetic material now becomes a donor or an F+ strain. It becomes a donor with a very high frequency.

Three types of plasmids based on their mobility include conjugative, mobilizable, and non-mobilizable plasmids. A protein gets involved in the conjugative machinery. It is known as relaxase. It is an important protein capable of recognizing the origin of transfer (OriT). The OriT is a short DNA sequence required in the cis position. Relaxase catalyzes the initial and final stages of conjugation. It resembles rolling circle replication proteins. The mobilizable plasmids thus carry OriT, relaxase gene, and nicking auxiliary proteins. Though conjugative and mobilizable plasmids appear similar in their properties, still they exhibit a difference in the machinery required for gene transfer.

Hfr strain:

The high-frequency recombination strains (Hfr) originate by rare crossovers. The Hfr strain arises due to the integration of the F factor into the bacterial chromosome. Such type of F factors is known as episomes. Hence, it replicates as a part of the bacterial chromosome. The Hfr cells conjugate with the F- strains. The nicked strands in the integrated factor F get transferred to the recipient F- strain, thereby transferring the bacterial genes. The transferred strand gets copied along with the genes. Recombination occurs in the recipient. Though the genes get copied, an F- strain never acquires Hfr phenotype because a complete copy of the F factor of the Hfr strain does not retain. Only a part of the F factor gets transferred.

Occasionally, the Hfr cell may not be efficient in excision of the F factor. The host chromosome adjacent to the F factor sometimes gets integrated into it due to an aberrant excision. Not only one but many segments get aberrantly inserted into it. During this excision, the F factor plus bacterial genes loop out of the chromosome. It leads to the formation of F’ factor. This type of conjugation is known as F-duction or sexduction.  

Image 2: Hfr strain

Bacterial gene mapping using conjugation:

The interrupted mating experiment helped in mapping the bacterial genes. It involved a cross between the F – and Hfr strains.

1.     Hfr strain had genes such as Hfr H thr⁺ leu⁺ azi^R ton^R lac⁺ gal⁺str^s

2.     The recipient had genes such as F^- thr leu azi^S ton^S lac gal str^R

“S” indicates sensitive and the “R” indicates resistant. The generation of the recombinants results from a double crossover. At various time intervals, the conjugating pairs broke apart and the transconjugants plated on a selective agar medium. It helped in studying the gene transfer. A single F factor gets integrated into Hfr strain. The interrupted mating experiment revealed the circular structure of the E. coli linkage map.

What is an inter-kingdom conjugation?

Nitrogen-fixing bacteria undergo an inter-kingdom conjugation. Agrobacterium tumefaciens and Agrobacterium rhizobium undergo inter-kingdom conjugation. A few pieces of evidence also report the inter-kingdom conjugation between the bacteria and the yeast. Hence, it is not necessary for the bacteria to undergo conjugation between their species. Inter-kingdom gene transfer is an example of horizontal gene transfer between two species, or different organisms.

Applications in genetic engineering:

The transfer of the genetic material through the process of conjugation involves convenience. It is possible to transfer genes from one bacterium to another, from bacteria to the yeast, plants or other cells. With conjugation, it is possible to use or synthesize a metabolite. Conjugative bacteria show the ability to pick up new plasmids from the environment. Recombinant DNA technology uses plasmids as cloning vectors. 

References:
[1] Int Std Ed-General Biology, Peter Russel
[2] Genetics of Bacteria, Sheela Srivastava
[3] Introduction to Genetics: A Molecular Approach, Terry Brown

© Copyright, 2018 All Rights Reserved.

DNA Replication in Prokaryotes

Replication is a process of DNA synthesis or DNA copying. It follows the semiconservative type of replication. A semiconservative replication results into two double-stranded DNA molecules. Each DNA has one strand retained from the parent and one newly synthesized strand. Various experiments tried revealing the mechanisms of replication. The process of replication in prokaryotes is clear in E. coli bacteria.

The process of DNA replication involves three steps such as initiation, elongation, and termination. The DNA replication requires replication enzymes and certain proteins. Arthur Kornberg and his colleagues worked on bacterial DNA replication and identified the DNA replication enzymes. They studied replication in E. coli.

Initiation of replication:

A DNA sequence known as the replicator directs the initiation of replication. This replicator includes the origin of replication. The origin of replication is known as Ori C. It is a specific region where the DNA denaturation occurs. Thus, it is a point at which the loss of native conformation of DNA results into the single-stranded structure, making the process of replication easier. The local DNA denaturation results in DNA replication bubble, leading to segments of single strands known as the template strands. The Y-shaped replication fork arises due to the exposure of two template strands. At this point, the two DNA strands get separated. A sequentially replicating segment of DNA is known as a replicon. The bacterial chromosomes contain a single replicator. The replication fork moves in the direction of untwisting of the DNA. There arise two replication forks while untwisting. Hence, the process of DNA replication is bidirectional. The Ori C is an AT-rich region. It gets denatured very easily.

An initiator DNA-a protein binds to the replicator. Thus, the DNA replication gets initiated due to stimulation of the denaturation process. The DNA helicase starts untwisting the DNA bi-directionally. It leads to the release of energy by hydrolysis of ATP. The energy gets utilized for untwisting. Then comes the role of DNA primases. The enzyme DNA primase forms a primosome complex. This enzyme synthesizes a short RNA primer. New nucleotides get added to the primer. At each replication fork, 10 to 100 nucleotides or pairs get added per second. The single strands created by the unwinding of DNA get stabilized by single-strand binding protein (SSB). Since there are two strands, one at the top and one at the bottom, the synthesis of RNA primer occurs on both the templates. The RNA primers get further lengthened by DNA polymerase III.

Okazaki fragments:

The DNA strands get synthesized from 5’ to 3’ direction at the Y-shaped replication fork. Two types of strands get synthesized. Leading strand is known as a continuous strand. A discontinuous strand is known as a lagging strand. The lagging strand gets synthesized in the form of discontinuous fragments known as Okazaki fragments.

Since the leading strand gets synthesized continuously, and the lagging strand is discontinuous, the process of replication is known as a semi-discontinuous type of replication. The two Okazaki strands get joined by DNA ligase enzyme. 

Image: DNA Replication steps: The image describes five main steps of DNA replication in prokaryotes. (1)The unwinding of the DNA occurs due to helicase activity. The replication fork proceeds further. Two types of strands form including the leading strand and the lagging strand. The SSBPs, primase, and the DNA polymerase-III get recruited. These components allow new DNA synthesis. (2) In this step, the DNA polymerase-III get dissociated. Discontinuous strand synthesis starts on the lagging strands. These strands occur in the form of fragments. Thus, they are known as Okazaki fragments. (3) The replication fork gets extended little more. Thus, the discontinuous strand synthesis proceeds further. (4) The DNA polymerase-I replaces the RNA primer. (5) DNA ligase seals the gaps.

The gist of events occurring at the replication fork:

1.     DNA primase synthesizes an RNA primer.

2.     Replication of the lagging strand occurs.

3.     Untwisting and elongation of new DNA strands.

4.     Primer removal by DNA polymerase I.

5.     Ligation of the adjacent fragments.

Circular DNA replicates bidirectionally:

The E. coli parental strand remains circular. These circular forms exhibit theta shaped structures. These structures arise as replication bubbles. As the two DNA strands untwist, the positive supercoils arise. Hence, the rotation helps in moving the molecule ahead. Topoisomerase solves the problem of supercoiling by introducing the negative supercoils. It keeps both the parent strands intact during replication. Hence, the unreplicated part undergoes a repeated negative supercoiling.

Rolling circle model:

It efficiently synthesizes multiple circular genome copies. Lambda bacteriophage uses this type of replication. A nick gets initiated at one of the parent strands. Then the strand rolls of a linear copy of the circular genome. The synthesis of the second strand converts the linear single-stranded genome into a double-stranded DNA.

References:

[1] IGenetics, Peter Russell, second edition

[2] DNA Replication, Arthur Kornberg, ‎Tania A. Baker, 2005
[3] The Cell Cycle: Principles of Control, Page 61, David Morgan, ‎David Owen Morgan, 2007

© Copyright, 2018  All Rights Reserved