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Posted by : Hendra Yulisman 19 Jan 2012

Sometimes when two pieces of DNA come into contact with each other, sections of each DNA strand will be exchanged. This is usually done through a process called crossing over in which the DNA breaks and is attached on the other DNA strand leading to the transfer of genes and possibly the formation of new genes. Genetic recombination is the transfer of DNA from one organism to another. The transferred donor DNA may then be integrated into the recipient's nucleoid by various mechanisms. In the case of homologous recombination, homologous DNA sequences having nearly the same nucleotide sequences are exchanged by means of breakage and reunion of paired DNA segments. Genetic information can be transferred from organism to organism through vertical transfer (from a parent to offspring) or through horizontal transfer methods such as conjugation, transformation or transduction. Bacterial genes are usually transferred to members of the same species but occasionally transfer to other species can also occur.

Transfer of genetic material

General or homologous recombination requires extensive homology and is mediated by an enzyme, RecA protein.

TRANSFORMATION:

Transformation involves the uptake of free or naked DNA released by donor by a recipient. It was the first example of genetic exchange in bacteria to have been discovered. This was first demonstrated in an experiment conducted by Griffith in 1928. The presence of a capsule around some strains of pneumococci gives the colonies a glistening, smooth (S) appearance while pneumococci lacking capsules have produce rough (R) colonies. Strains of pneumococci with a capsule (type I) are virulent and can kill a mouse whereas strains lacking it (type II) are harmless. Griffith found that mice died when they were injected with a mixture of live non capsulated (R, type II) strains and heat killed capsulated (S, type I) strains. Neither of these two when injected alone could kill the mice, only the mixture of two proved fatal. Live S strains with capsule were isolated from the blood of the animal suggesting that some factor from the dead S cells converted the R strains into S type. The factor that transformed the other strain was found to be DNA by Avery, McLeod and McCarty in 1944.

Transformation is gene transfer resulting from the uptake by a recipient cell of naked DNA from a donor cell. Certain bacteria (e.g. Bacillus, Haemophilus, Neisseria, Pneumococcus) can take up DNA from the environment and the DNA that is taken up can be incorporated into the recipient's chromosome.

rough coniessmooth colonies

Rough colonies                      Smooth colonies

 

The steps involved in transformation are:

  1. A donor bacterium dies and is degraded.
  2. A fragment of DNA (usually about 20 genes long) from the dead donor bacterium binds to DNA binding proteins on the cell wall of a competent, living recipient bacterium.
  3. Nuclease enzymes then cut the bound DNA into fragments.
  4. One strand is destroyed and the other penetrates the recipient bacterium.
  5. The Rec A protein promotes genetic exchange (recombination) between a fragment of the donor's DNA and the recipient's DNA.

Some bacteria are able to take up DNA naturally. However, these bacteria only take up DNA a particular time in their growth cycle (log phase) when they produce a specific protein called a competence factor. Uptake of DNA by Gram positive and Gram negative bacteria differs. In Gram positive bacteria the DNA is taken up as a single stranded molecule and the complementary strand is made in the recipient. In contrast, Gram negative bacteria take up double stranded DNA.

Transfer of genetic material2

Significance: Transformation occurs in nature and it can lead to increased virulence. In addition transformation is widely used in recombinant DNA technology.

 

CONJUGATION:

In 1946 Joshua Lederberg and Tatum discovered that some bacteria can transfer genetic information to other bacteria through a process known as conjugation. Bacterial conjugation is the transfer of DNA from a living donor bacterium to a recipient bacterium.

Plasmids are small autonomously replicating circular pieces of double-stranded circular DNA. Conjugation involves the transfer of plasmids from donor bacterium to recipient bacterium. Plasmid transfer in Gram-negative bacteria occurs only between strains of the same species or closely related species. Some plasmids are designated as F factor (F plasmid, fertility factor or sex factor) because they carry genes that mediate their own transfer. The F factor can replicate autonomously in the cell. These genes code for the production of the sex pilus and enzymes necessary for conjugation. Cells possessing F plasmids are F+ (male) and act as donors. Those cells lacking this plasmid are F- (female) and act as recipient. All those plasmids, which confer on their host cells to act as donors in conjugation are called transfer factor.

Each Gram negative F+ bacterium has 1 to 3 sex pili that bind to a specific outer membrane protein on recipient bacteria to initiate mating. The sex pilus then retracts, bringing the two bacteria in contact and the two cells become bound together at a point of direct envelope-to-envelope contact. In Gram-positive bacteria sticky surface molecules are produced which bring the two bacteria into contact. Gram-positive donor bacteria produce adhesins that cause them to aggregate with recipient cells, but sex pili are not involved. DNA is then transferred from the donor to the recipient. Plasmid-mediated conjugation occurs in Bacillus subtilis, Streptococcus lactis, and Enterococcus faecalis but is not found as commonly in the Gram-positive bacteria as compared to the Gram-negative bacteria.

1. F+ conjugation:

This results in the transfer of an F+ plasmid (coding only for a sex pilus) but not chromosomal DNA from a male donor bacterium to a female recipient bacterium. The two strands of the plasmid separate. One strand enters the recipient bacterium progressing in the 5' to 3' direction while one strand remains in the donor. The complementary strands are synthesized in both donor and recipient cells. The recipient then becomes an F+ male and can make a sex pilus. During conjugation, no cytoplasm or cell material except DNA passes from donor to recipient. The mating pairs can be separated by shear forces and conjugation can be interrupted. Consequently, the mating pairs remain associated for only a short time. After conjugation, the cells break apart. Following successful conjugation the recipient becomes F+ and the donor remains F+.

Transfer of genetic material3

 

2. Resistance plasmid conjugation:

Some Gram-negative bacteria harbor plasmids that contain antibiotic resistance genes, such plasmids are called R factors. The R factor has two components, one that codes for self transfer (like F factor) called RTF (resistance transfer factor) and the other R determinant that contains genes coding for antibiotic resistance. R plasmids may confer resistance to as many as five different antibiotics at once upon the cell and by conjugation; they can be rapidly disseminated through the bacterial population. The difference between F factor and R factor is that the latter has additional genes coding for drug resistance. During conjugation there is transfer of resistance plasmid (R- plasmid) from a donor bacterium to a recipient. One plasmid strand enters the recipient bacterium while one strand remains in the donor. Each strand then makes a complementary copy. R-plasmid has genes coding for multiple antibiotic resistance as well as sex pilus formation. The recipient becomes multiple antibiotic resistant and male, and is now able to transfer R-plasmids to other bacteria. When the recipient cells acquire entire R factor, it too expresses antibiotic resistance. Sometimes RTF may disassociate from the R determinant and the two components may exist as separate entities. In such cases though the host cell remains resistant to antibiotics, it can not transfer this resistance to other cells. Sometimes RTF can have other genes (such as those coding for hemolysin, enterotoxin) apart from R determinants attached to it.

3. Hfr (high frequency recombinant) conjugation:

Plasmids may integrate into the bacterial chromosome by a recombination event depending upon the extent of DNA

homology between the two. After integration, both plasmid and chromosome will replicate as a single unit. A plasmid that is capable of integrating into the chromosome is called an episome. If the F plasmid is integrated into the chromosome it is called an Hfr cell. After integration, both chromosome and plasmid can be conjugally transferred to a recipient cell. Hfr cells are called so because they are able to transfer chromosomal genes to recipient cells with high frequency.

The DNA is nicked at the origin of transfer and is replicated. One DNA strand begins to passes through a cytoplasmic bridge to the F- cell, where its complementary strand is synthesized. Along with the portion of integrated plasmid, the chromosome is also transmitted to the F- cell. The bacterial connection usually breaks before the transfer of the entire chromosome is completed so the remainder of the F+ plasmid rarely enters the recipient. Usually only a part of the Hfr chromosome as well as the plasmid is transferred during conjugation and the recipient cell does not receive complete F factor. After conjugation the Hfr cell remains Hfr but the F- cell does not become F+ and continues to remain F-. However the transferred chromosome fragment recombines with the chromosome of F- cell thereby transferring some new property to the recipient cell.

Transfer of genetic material4

Transfer of genetic material5

The integration of episome into the chromosome is not stable and the episomes are known to revert back to free state. While doing so, the episomes sometimes carry fragments of chromosomal genes along with it. Such an F factor that incorporates some chromosomal genes is called F prime (F') factor. When such a F' cell mates with F- recipient cell, it not only transfers the F factor but also the host genes that it carried with it. This process of transfer of chromosomal genes along with F factor is known is sexduction.

Significance: Among the Gram negative bacteria this is the major way that bacterial genes are transferred. Transfer can occur between different species of bacteria. Transfer of multiple antibiotic resistance by conjugation has become a major problem in the treatment of certain bacterial diseases. Since the recipient cell becomes a donor after transfer of a plasmid, an antibiotic resistance gene carried on a plasmid can quickly convert a sensitive.

 

TRANSDUCTION:

Bacteriophage are viruses that parasitize bacteria and use their machinery for their own replication. During the process of replication inside the host bacteria the bacterial chromosome or plasmid is erroneously packaged into the bacteriophage capsid. Thus newer progeny of phages may contain fragments of host chromosome along with their own DNA or entirely host chromosome. When such phage infects another bacterium, the bacterial chromosome in the phage also gets transferred to the new bacterium. This fragment may undergo recombination with the host chromosome and confer new property to the bacterium.

Life cycle of bacteriophage may either by lytic or lysogenic. In the former, the parasitized bacterial cell is killed with the release of mature phages while in the latter the phage DNA gets incorporated into the bacterial chromosome as prophage.

Following are the stages of transduction involving a lytic phage:

  1. A lytic bacteriophage adsorbs to a susceptible bacterium.
  2. The bacteriophage genome enters the bacterium. The phage DNA directs the bacterium's metabolic machinery to manufacture bacteriophage components and enzymes.
  3. Occasionally during maturation, a bacteriophage capsid incorporates a fragment of donor bacterium's chromosome or a plasmid instead of a phage genome by mistake.
  4. The bacteriophages are released with the lysis of bacterium.
  5. The bacteriophage carrying the donor bacterium's DNA adsorbs to another recipient bacterium.
  6. The bacteriophage inserts the donor bacterium's DNA it is carrying into the recipient bacterium.
  7. The donor bacterium's DNA is exchanged by recombination for some of the recipient's DNA.

Transfer of genetic material6

In case of temperate phages that undergo lysogenic cycle, the phage DNA gets incorporated into the bacterium chromosome. This is called a prophage and it behaves as if it were a part of bacterial chromosome. This process is known as lysogenic conversion and the bacteria are called lysogenic bacteria. The genes present in the phage DNA also get expressed in the bacterium. Only those strains of Corynebacterium diphtheriae that have been lysogenised with beta prophage produce the diphtheria toxin. The prophage sometimes disassociates itself from the host chromosome during multiplication of lysogenic bacteria, and in doing so; it sometimes carries along with itself fragments of bacterial chromosome. The separated prophage then initiates lytic cycle and the subsequent phage progeny may have a piece of chromosomal DNA. When such phage infects another bacterium, newer characteristics coded by that chromosomal gene are conferred.

Two types of transduction are known; restricted transduction and generalized transduction. Generalized transduction can transfer any bacterial gene to the recipient. This process may occur with phages (lytic phages) that degrade their host DNA into pieces the size of viral genomes. If these pieces are erroneously packaged into phage particles, they can be delivered to another bacterium in the next phage infection cycle. Phages P22 of Salmonella typhimurium and P1 and ยต of E. coli carry out generalized transduction. In restricted transduction only those chromosomal genes that lie adjacent to the prophage are transmitted. The lambda phage that infects E.coli always transfers gal+ gene (responsible for galactose fermentation). Specialized transduction is only effective in transducing a few special bacterial genes while generalized transduction can transduce any bacterial gene.

 

PLASMIDS:

Plasmids are extrachromosomal elements found inside a bacterium. These are not essential for the survival of the bacterium but they confer certain extra advantages to the cell.

Number and size: A bacterium can have no plasmids at all or have many plasmids (20-30) or multiple copies of a plasmid. Usually they are closed circular molecules; however they occur as linear molecule in Borrelia burgdorferi. Their size can vary from 1 Kb to 400 Kb.

Multiplication: Plasmids multiply independently of the chromosome and are inherited regularly by the daughter cells.

Types of plasmids: R factor, Col factor, RTF and F factor.

F factor: This is also known as fertility factor or sex factor. Most plasmids are unable to mediate their own transfer to other cells. Vertical (inheritance) or horizontal (transfer) transmissions maintain plasmids. F factor is a plasmid that codes for sex pili and its transfer to other cells. Those bacteria that possess transfer factor are called F+, such bacteria have sex pili on their surface. Those cells lacking this factor are designated F-. The F factor plasmid is

transferred to other cells through conjugation. An F- cell will become F+ when it receives the fertility factor from another F+ cell.

R factor: Those plasmids that code for the transmissible drug resistance are called R factor. These plasmids contain genes that code for resistance to many antibiotics. R factors may be transferred by conjugation and its transfer to other bacteria is independent of the F factor. Bacteria possessing such plasmids are resistant to many

antibiotics and this drug resistance is transferred to closely related species. R factors may simultaneously confer

resistance to five antibiotics. They are usually transferred to related species along with RTF.

Significance of plasmids:

  1. Codes for resistance to several antibiotics. Gram-negative bacteria carry plasmids that give resistance to antibiotics such as neomycin, kanamycin, streptomycin, chloramphenicol, tetracycline, penicillins and sulfonamides.
  2. Codes for the production of bacteriocines.
  3. Codes for the production of toxins (such as Enterotoxins by Escherichia coli, Vibrio cholerae, exfoliative toxin by Staphylococcus aureus and neurotoxin of Clostridium tetani).
  4. Codes for resistance to heavy metals (such as Hg, Ag, Cd, Pb etc.).
  5. Plasmids carry virulence determinant genes. Eg, the plasmid Col V of Escherichia coli contains genes for iron sequestering compounds.
  6. Codes resistance to uv light (DNA repair enzymes are coded in the plasmid).
  7. Codes for colonization factors that is necessary for their attachment. Eg, as produced by the plasmids of Yersinia enterocolitica, Shigella flexneri, Enteroinvasive Escherichia coli.
  8. Contains genes coding for enzymes that allow bacteria unique or unusual materials for carbon or energy sources. Some strains are used for clearing oil spillage.

 

Application of plasmids:

  1. Used in genetic engineering as vectors.
  2. Plasmid profiling is a useful genotyping method.

 

Episomes: Jacob and Wollman coined the term episome. Previously, it was considered synonymous with plasmids. F factors are those plasmids that can code for self transfer to other bacteria. Occasionally such plasmids get spontaneously integrated into chromosome. Plasmids with this capability are called episomes and such bacterial cells are called Hfr cells i.e. high frequency of recombination.

TRANSPOSABLE GENETIC ELEMENTS:

Transposable genetic elements are segments of DNA that have the capacity to move from one location to another (i.e. jumping genes).

 

Properties of Transposable Genetic Elements:

    1. Random movement: Transposable genetic elements can move from any DNA molecule to any DNA other molecule or even to another location on the same molecule. The movement is not totally random; there are preferred sites in a DNA molecule at which the transposable genetic element will insert.
    2. Not capable of self replication: The transposable genetic elements do not exist autonomously and thus, to be replicated they must be a part of some other replicon.
    3. Transposition mediated by site-specific recombination: Transposition requires little or no homology between the current location and the new site. The transposition event is mediated by an enzyme transposase that is coded by the transposable genetic element. Recombination that does not require homology between the recombining molecules is called illegitimate or nonhomologous recombination.
    4. Transposition can be accompanied by duplication: In many instances transposition of the transposable genetic element results in removal of the element from the original site and insertion at a new site. However, in some cases the transposition event is accompanied by the duplication of the transposable genetic element. One copy remains at the original site and the other is transposed to the new site.

 

Types of Transposable Genetic Elements:

A. Insertion sequences (IS): Insertion sequences are transposable genetic elements that carry no known genes except those that are required for transposition. Insertion sequences are small stretches of DNA that have at their

ends repeated sequences, which are involved in transposition. In between the terminal repeated sequences there are genes involved in transposition and sequences that can control the expression of the genes but no other

nonessential genes are present.

Importance of IS:

i) Mutation - The introduction of an insertion sequence into a bacterial gene will result in the inactivation of the gene.

ii) The sites at which plasmids insert into the bacterial chromosome are at or near insertion sequence in the chromosome.

iii) Phase Variation: In Salmonella there are two genes, which code for two antigenically different flagellar antigens. The expression of these genes is regulated by an insertion sequences.

B. Transposons: Transposons are transposable genetic elements that carry one or more other genes in addition to those, which are essential for transposition. The structure of a transposon is similar to that of an insertion sequence. The extra genes are located between the terminal repeated sequences.

Importance of transposons: Many antibiotic resistance genes are located on transposons. Since transposons can jump from one DNA molecule to another, these antibiotic resistance transposons are a major factor in the development of plasmids, which can confer multiple drug resistance on a bacterium harboring such a plasmid.

These multiple drug resistance plasmids have become a major medical problem.

Source : Sidhar Rao

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