BACTERIAL GENETICS

BACTERIAL GENETICS

13.   BACTERIAL GENETICS

Bacteria can be grown in liquid media and on solid (agar) media

Minimal media are those that contain the minimum nutrients possible for colony growth. Minimal media is generally lacking amino acids and allows only  "wild type" microorganisms to grow. Minimal media contain carbon source (e.g. glucose), salts and water.  Wild type bacteria are usually prototrophic: i.e. they can synthesize all necessary compounds. Prototrophy can be lost by mutations leading to auxotrophy.  Auxotrophy is the inability of an organism to synthesize a particular compound required for its growth. Auxotrophs cannot grow on minimal media. 
Heredity in bacteria is fundamentally similar to heredity in more-complex organisms, but the bacterial haploid genome is microscopic in size which makes the observation of their phenotypes difficult and requires different approaches and methods.

Bacterial Genetics :
The study of inheritance of information in bacteria is known as bacterial genetics. Bacteria also reproduce sexually. The sexual reproduction in bacteria is of three types. i.e. Conjugation, transformation and transduction.

Plasmids
Most plasmids are circular and several thousand base pairs in length, although plasmids consisting of several hundred thousand base pairs also have been found. Each plasmid possesses an origin of replication (Ori) I.e., a specific DNA sequence where DNA replication is initiated.
13.1.    Bacteria Exchange Genes Through Conjugation, Transformation, and Transduction
Bacteria exchange genetic material by three different mechanisms, all containing some type of DNA transfer and recombination between the transferred DNA and the bacterial chromosome.
1)    Conjugation
It takes place when genetic material passes directly from one bacterium to another. In conjugation, two bacteria lie close together and a link forms between them. A plasmid or a part of the bacterial chromosome passes from one cell (the donor) to the other (the recipient). Subsequent to conjugation, crossing over may take place between homologous sequences in the transferred DNA and the chromosome of the recipient cell. In conjugation, DNA is transferred only from donor to recipient, with no reciprocal exchange of genetic material.

2)    Transformation 
It takes place when a bacterium takes up DNA from the medium in which it is growing. After transformation, recombination takes place between the introduced genes and those of the bacterial main genome.

3)    Transduction
It takes place when bacterial viruses (bacteriophages) carry DNA from one bacterium to another. Inside the bacterium, the newly introduced DNA may undergo recombination with the bacterial chromosome.

Not all bacterial species exhibit all three types of genetic transfer. Conjugation takes place more frequently in some species than in others. Transformation takes place to a limited extent in many species of bacteria, but laboratory techniques increase the rate of DNA uptake. Most bacteriophages have a limited host range, so transduction is normally between bacteria of the same or closely related species only.
These processes of genetic exchange in bacteria differ from diploid eukaryotic sexual reproduction in two important ways. First, DNA exchange and reproduction are not coupled in bacteria. Second, the donated genetic material that is not recombined into the host DNA is usually degraded, and so the recipient cell remains haploid. Each type of genetic transfer can be used to map genes.
13.1.1.    Conjugation
In 1946, Joshua Lederberg and Edward Tatum reported that bacteria can transfer and recombine genetic information.
They use two auxotrophic strains of E. coli.
The Y10 strain which required the amino acids threonine, leucine and the vitamin thiamine for growth but did not require the vitamin biotin (bio+) or the amino acids phenylalanine (phe+) and cysteine (cys+); the genotype of this strain can be written as thr− leu− thi− bio+ phe+ cys+. 
The Y24 strain had the opposite set of alleles: it required biotin, phenylalanine, and cysteine in its medium, but it did not require threonine, leucine, or thiamine; its genotype was thr+ leu+ thi+ bio− phe− cys−. When they mixed Y10 and Y24 bacteria together and plated them on minimal medium. Neither Y10 nor Y24 grew on minimal medium when they are plated separately.
Strain Y10 was unable to grow in minimal media because it required threonine, leucine, and thiamine for growth. These were absent in the minimal medium. Strain Y24 was unable to grow in minimal media because it required biotin, phenylalanine, and cysteine for growth. These were absent from the minimal medium. 
However when  Lederberg and Tatum mixed the two strains in the liquid culture media and plated them on minimal medium a few colonies did appear in the minimal medium that means after mixing both strains, the auxotrophic strain becomes prototropic.and  the genotype of these prototropic can be given as thr+ leu+ thi+ bio+ phe+ cys+.
Lederberg and Tatum said this conversion can be because of these two reasons
(1)    Mutation 
Multiple simultaneous mutations (thr− to thr+, leu− to leu+, and thi− to thi+ in strain Y10 or bio− to bio+, phe− to phe+, and cys− to cys+ in strain Y24) would convert the auxotrophic into prototropic. However, the frequency of mutation is very low. So later they ruled out the chances of mutation.
(2)    Genetic transfer and recombination
13.1.1.1. Conjugation between F+ and F− cells 
In most bacteria, conjugation depends on a fertility (F) factor that is present in the donor cell and absent in the recipient cell. Cells that contain F factor are referred to as F+, and cells lacking F factor are F−. 
The F plasmid contains an origin of replication and a number of genes required for conjugation. These genes are called as Tra genes and Mob genes. These are must for conjugation.  For example, some of these genes product encode sex pili, i.e. slender extensions of the cell membrane some help in replication of plasmid, some help in transfer to replicating plasmid DNA from donor to recipient, some maintaining the shape and size of conjugation tube, some maintaining the durability of conjugation tube.
A cell containing F plasmid produces numerous sex pili, one of which makes contact with a receptor on an F− cell and pulls the two cells together. DNA is then transferred from the F+ cell to the F− cell.


The F plasmid replicates through rolling circle mode of DNA replication. In rolling circle mode of DNA replication, the replication process is initiated when one of the DNA strands on the F factor is nicked at an origin (oriT). The 5’ end of the nicked DNA is transferred to F– bacteria in single strand form. In F– bacteria the F plasmid become circular and become double-stranded. Now F– bacteria is converted into F+. 

13.1.1.2. Conjugation by Hfr cells 
Origin of replication (Ori T) allows a plasmid to replicate independently of the bacterial chromosome. F plasmid also contains an insertion sequence. Insertion Sequence allows integration of plasmid DNA into the bacterial chromosome. Insertion Sequence containing plasmid is known as an episome. 
The F (fertility) factor of E. coli is an episome that controls mating and gene exchange between E. coli cells. The integrated form of the chromosome is called as Hfr (high-frequency recombinant). After integration, both plasmid and bacterial genome replicate as a single unit by rolling circle mechanism. After integration, both bacterial genome and plasmid can be transferred to a recipient cell. Hfr cells are called so because they are able to transfer bacterial genes to recipient cells with high frequency. 
The DNA is nicked at the origin of transfer and is replicated by rolling circle mechanism. One DNA strand( the 5’ end) begins to passes through a conjugation tube to the F- cell. The process is just like conjugation between F+ and F- bacteria. However, in Hfr- F– conjugation bacterial genes are transferred to F- bacteria. After conjugation, the Hfr cell remains Hfr but the F- cell does not become F+ and continues to remain F–. This is because the entire F+ gene present in the Hfr is not transferred.  Only a fragment of the bacterial genome is transferred between Hfr and F-. 


In conjugation between Hfr and F– cells, the integrated F factor is nicked, and the end of the nicked strand moves into the F– cell, just as it does in conjugation between F+ and F– cells. Because, in an Hfr cell, the F factor has been integrated into the bacterial chromosome, the chromosome follows it into the recipient cell. During conjugation, the amount of the genome received by F– cell depends on the length of time that the two cells remain in conjugation.
This gene transfer between Hfr and F– cells is the reason behind the formation of recombinant prototrophic bacteria observed by Lederberg and Tatum. After the transfer of bacterial genome in the recipient cell,  the DNA circularize itself and become double-stranded. A crossing over takes place between two circular DNA. And the transferred  DNA becomes part of the genome of recipient bacteria. And the recombinant recipient genome can replicate and pass on to later generations by binary fission.

13.1.1.3. F-prime plasmid
Hfr is an integrated product of bacterial genome and F plasmid. However, when a F factor excise from Hfr, a small amount of the bacterial genome may be removed with it, and these chromosomal genes will then be carried with the F plasmid. Bacterial cells containing an F plasmid with some bacterial genes are called F prime (F′). 
For example, if an F factor integrates into a bacterial genome near the lac genes to form Hfr, during excision it may take lac genes of bacteria. 
When these lac gene containing F′ cells conjugate with F– cells. It can transfer lac genes to recipient bacteria during conjugation. The F’ cell is capable to form conjugation tube as it possesses the F plasmid with all the genetic information necessary for conjugation and gene transfer. 

13.1.1.4. Characteristics of different mating types of E. coli (cells with different types of F) are summarized below
Conjugating     Cell Types Present after Conjugation
F' X F-             Two F' cells (F- cell becomes F')
Hfr X F-           One Hfr cell and one F- (no change)*
*     Rarely the F- cell becomes F' in an Hfr X F- conjugation if the entire chromosome is transferred during conjugation.
During conjugation between an F′lac cell and an F− cell, the F plasmid is transferred to the F− cell, which means that any genes on the F plasmid, including those from the bacterial chromosome, may be transferred to F− recipient cells. The transfer of bacterial genes to F- by F’ cell is known as seduction. It produces partial diploids, or merozygotes, which are cells with two copies of some genes, one on the bacterial chromosome and one on the newly introduced F plasmid.
Now LET’S TALK About Conjugation Mapping-
Conjugation can be used in the mapping of genes in bacteria as we know the genes are transferred one by one to recipient bacteria. The amount or number of gene transfer depends upon the time. 
13.2.    Interrupted Mating Mapping 
1.    Allow conjugation to start between hfr and F- bacteria. 
2.    Genes closest to the origin of replication site (in the direction of replication) are moved through the pili first. 
3.    After a set time, interrupt the conjugation process.
4.     Genes which are close to the origin of replication manage to transfer itself in recipient bacteria. In recipient F- the transferred gene become circular and show recombination with the main genome of bacteria. This makes bacteria recombinant for that particular gene.
5.    Only those genes closest to the origin of replication site will conjugate. 
6.    The longer the time, the more genes are able to transfer from conjugation tube. 
7.    Notice which genes are recombined genes that recombine within X distance (conjugation time-distance) of the origin of replication. 
8.    Using different strains of F-plasmids and the interrupted mating technique, we can determine the order of genes present in the bacterial genome.

To study this problem, Bernard Davis constructed a U shaped tube that was divided into two compartments by a filter having fine pores. This filter allowed liquid medium to pass from one side of the tube to the other, but the pores of the filter were too small to allow the passage of bacteria. 
When two auxotrophic strains of bacteria were placed on opposite sides of the filter and a bacterial filter is placed between them, and suction was applied alternately to the ends of the U-tube, causing the medium to flow back and forth between the two compartments.
After several hours of incubation in the U-tube when bacteria are plated out on minimal medium, bacteria did not form a colony on agar media. This indicates that no conjugation takes place between donor and recipient. This U tube experiment clearly indicates that for conjugation, direct contact is required between bacterial cells.

13.3.    Transformation mapping :
Transformation mapping (T.M.) is defined as the process of mapping of the gene acquired by the bacteria from the outer environment or the given nutrient media. Only autotroph bacteria show transformation because they are unable to synthesize a particular amino acid. Thus, the number of hypotheses were given to explain the transformation in which 'Single strand, DNA fragment hypothesis' is widely accepted. Transformation mapping shows the principle sequence of gene transfer that these genes are transferred together or separately.
For eg. : X and Y genes have to be transferred. If X and Y are very far on different chromosomes then the chances of X & Y coming together in the same bacteria are less. That means two separate transformation events are required to transform the cell for having both genes X & Y (double mutant). This is called "Double transformation". If they are present on the same chromosome i.e. together then there is a possibility that they transfer as a single unit that means only a single transformation event is sufficient to transform the double mutant bacteria. This is called co-transformation."
13.4.    Transduction Mapping :
It is a process of mapping of gene accepted by the recipient through the transduction from the donor through the infection. It was discovered by Lederberg and Zinder in 1951 in Salmonella while studying on the transfer of genes by phage in bacteria.
    2 type of Transduction :
(i)    Generalized transduction: It is a type of transduction when phage contains a random fragment of gene or DNA which is transferred to the recipient bacteria (new). It occurred during the lytic growth. Generalized transduction can be abortive or complete.


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