Plasmids- Definition, Properties, Structure, Types, Functions, Examples

Plasmids are small circular DNA fragments that are found in many microorganisms, such as bacteria, archaea, and some eukaryotes. They are not part of the main chromosome of the cell, but they can replicate independently and carry genes that may confer some advantages to the host organism. Plasmids are also known as extra-chromosomal elements or genetic tools.

The term plasmid was coined by Joshua Lederberg in 1952, who discovered that some bacteria can exchange genetic material through a process called conjugation. He observed that some strains of Escherichia coli could transfer antibiotic resistance to other strains through a physical contact mediated by a hair-like structure called pilus. He proposed that this transfer was due to a circular DNA molecule that could move from one cell to another. He named this molecule plasmid, from the Greek word plasmos, meaning molded or formed.

Plasmids have been widely used in biotechnology and genetic engineering, as they can be manipulated to introduce, modify, or delete specific genes in the host cell. For example, plasmids can be used to produce recombinant proteins, such as insulin or human growth hormone, in bacteria or yeast cells. Plasmids can also be used to study gene expression and regulation, gene function and interaction, and gene therapy.

• They are extra chromosomal DNA fragments present in the cell. This means that they are not part of the main genetic material of the cell and can exist independently of it.
• They are double stranded structures. Exceptions are the linear plasmids in bacteria Streptomyces spp and Borrelia spp. Double stranded DNA consists of two complementary strands that are held together by hydrogen bonds between the nitrogenous bases.
• They can replicate independently. This means that they do not depend on the replication machinery of the host cell and can make copies of themselves using their own origin of replication (OR) and enzymes.
• The absence of a plasmid in the cell does not affect cell functioning, but the presence of a plasmid in the cell is usually beneficial. Plasmids often carry genes that confer some advantage to the host cell, such as antibiotic resistance, virulence factors, or metabolic capabilities.
• Plasmids are also known as sex factors, conjugants, extra chromosomal replicons, or transfer factors. These names reflect their ability to transfer from one cell to another through a process called conjugation, which involves the formation of a pilus (a thin tube-like structure) between two cells and the transfer of a copy of the plasmid.
• Copy number – the copy number refers to the number of copies of plasmid present in the bacterial cell. Usually, small plasmids are present in high numbers and large plasmids are present in few numbers. The copy number is regulated by various factors, such as the size of the plasmid, the origin of replication, and the availability of nutrients and energy.
• Compatibility of plasmids – this refers to the ability of two different plasmids to coexist in the same bacterial cell. Some plasmids are compatible with each other and can be maintained together in a stable state, while others are incompatible and compete for resources or interfere with each other`s replication or expression. Plasmids that belong to the same compatibility group (or incompatibility group) are usually incompatible with each other.

Plasmids are small, circular, double-stranded DNA molecules that are distinct from the chromosomal DNA of the host cell. They are extrachromosomal and not essential for the survival of the cell, but they may confer some advantages such as antibiotic resistance or virulence. Plasmids can vary in size from 1 kb to 200 kb, and they can exist in different species and get transferred from one cell to another.

Plasmids have certain essential elements that enable them to replicate and maintain themselves in the host cell. These are:

• Origin of replication (OR): This is a specific sequence of DNA where the replication of the plasmid begins. The OR is usually rich in A=T base pairs, which makes it easier to separate the strands during replication.
• Selectable marker (SM): This is a gene that confers resistance to a certain antibiotic or toxin, which helps in identifying and selecting the cells that contain the plasmid.
• Multiple cloning site (MCS): This is a region that contains several restriction enzyme recognition sites, which allow the insertion of foreign DNA fragments into the plasmid. The MCS is usually located within a lacZ gene, which encodes for β-galactosidase enzyme. This enzyme can cleave a substrate called X-gal and produce a blue color. If a foreign DNA is inserted into the MCS, it disrupts the lacZ gene and prevents the production of β-galactosidase enzyme. This results in a white color when X-gal is added. This is called blue-white screening method.
• Promoter (P): This is a sequence of DNA that initiates the transcription of a gene or a group of genes. The promoter can be constitutive (always active) or inducible (activated by certain signals). The promoter determines the expression level and regulation of the genes carried by the plasmid.
• Primer binding site (PBS): This is a short sequence of single-stranded DNA that anneals to a complementary primer during PCR amplification or DNA sequencing. The primer provides a free 3` end for the DNA polymerase to extend the DNA strand.

Some plasmids may also have additional elements such as:

• Transfer (TRA) gene: This is a gene that encodes for the proteins involved in the conjugation process, which is one of the ways that plasmids can be transferred from one cell to another. The TRA gene also produces a sex pilus, which is a long protein structure that connects two cells during conjugation. Plasmids that have TRA gene are called conjugative plasmids, while those that lack it are called non-conjugative plasmids.
• Incompatibility (INC) group: This is a sequence of DNA that determines whether two plasmids can coexist in the same cell or not. Plasmids that belong to different INC groups are compatible and can coexist, while plasmids that belong to the same INC group are incompatible and cannot coexist. Incompatibility occurs because plasmids use similar mechanisms to regulate their copy number and partitioning during cell division, which leads to competition and interference between them.
• Virulence (VIR) gene: This is a gene that encodes for factors that enhance the pathogenicity of the host cell. For example, some plasmids carry genes that produce toxins, enzymes, adhesins, or other molecules that help the bacteria to infect and damage other cells or tissues. Plasmids that have VIR gene are called virulence plasmids.
• Degradative (DEG) gene: This is a gene that encodes for enzymes that can break down unusual or complex substances such as hydrocarbons, pesticides, or aromatic compounds. Plasmids that have DEG gene are called degradative plasmids, and they enable the bacteria to utilize these substances as sources of energy or carbon.

The structure of a typical plasmid can be represented as follows:

Figure 1: The structure of a typical plasmid.

Plasmids are transferred from one bacterial cell to another by different mechanisms. The most common and well-studied method is conjugation. Conjugation is a process in which two bacterial cells come into direct contact and exchange genetic material through a specialized structure called a pilus. The pilus is formed by the donor cell that contains a conjugative plasmid, such as an F plasmid or an R plasmid. The pilus attaches to the recipient cell that lacks the plasmid and initiates the transfer of a single-stranded copy of the plasmid DNA. The recipient cell then synthesizes the complementary strand and becomes a new donor cell.

Conjugation can occur between bacteria of the same or different species, and even between bacteria and eukaryotic cells. Conjugation can also transfer chromosomal genes along with plasmid genes, resulting in recombination and genetic diversity. Conjugation is regulated by various factors, such as environmental conditions, cell density, and plasmid compatibility.

Another method of plasmid transfer is transduction. Transduction is a process in which a bacteriophage (a virus that infects bacteria) carries plasmid DNA from one bacterial cell to another. The bacteriophage infects a donor cell that contains a plasmid and incorporates some of the plasmid DNA into its own genome. The bacteriophage then lyses the donor cell and releases new viral particles that contain the plasmid DNA. These viral particles infect a recipient cell that lacks the plasmid and injects the plasmid DNA into it. The recipient cell then replicates the plasmid DNA and becomes a new donor cell.

Transduction can occur by two mechanisms: generalized transduction and specialized transduction. Generalized transduction occurs when any fragment of the donor cell`s DNA, including plasmid DNA, is randomly packaged into the bacteriophage genome. Specialized transduction occurs when only specific regions of the donor cell`s DNA, such as those adjacent to the bacteriophage integration site, are packaged into the bacteriophage genome.

A third method of plasmid transfer is transformation. Transformation is a process in which a bacterial cell takes up free plasmid DNA from the environment and incorporates it into its own genome. The free plasmid DNA can be released from dead or lysed cells or artificially introduced by laboratory techniques. The bacterial cell that takes up the plasmid DNA becomes a new donor cell.

Transformation can occur naturally or artificially. Natural transformation occurs when certain bacteria have the ability to take up extracellular DNA under specific conditions, such as nutrient limitation or stress. Artificial transformation occurs when bacteria are treated with chemicals or electric pulses that make them more permeable to DNA uptake.

Plasmid transfer by conjugation, transduction, and transformation can have significant impacts on bacterial evolution, adaptation, and pathogenesis. Plasmids can confer new traits to bacteria, such as antibiotic resistance, virulence factors, metabolic capabilities, or bioluminescence. Plasmids can also facilitate horizontal gene transfer between different bacteria or between bacteria and other organisms, increasing genetic diversity and creating novel combinations of genes.

Plasmids can be classified into different types based on various criteria, such as the presence or absence of the transfer gene (TRA), the function or role of the plasmid in the host cell, the size and copy number of the plasmid, and the compatibility or incompatibility of the plasmid with other plasmids. Here are some of the common types of plasmids:

• Conjugative plasmids: These are plasmids that contain the TRA gene, which encodes for the proteins involved in the formation of a pilus or a conjugation bridge between two bacterial cells. Conjugative plasmids can be transferred from one cell to another through this bridge, and they can also mobilize other non-conjugative plasmids by providing them with the TRA gene. Conjugative plasmids are usually large and low-copy plasmids. An example of a conjugative plasmid is the F plasmid or fertility plasmid, which also carries genes for sex determination and incompatibility in bacteria.
• Non-conjugative plasmids: These are plasmids that lack the TRA gene and cannot be transferred by conjugation. They can only be transferred by other methods, such as transformation, transduction, or mobilization by conjugative plasmids. Non-conjugative plasmids are usually small and high-copy plasmids. An example of a non-conjugative plasmid is the ColE1 plasmid, which is widely used as a cloning vector.
• Resistance plasmids (R plasmids): These are plasmids that carry genes for resistance to various antibiotics or other toxic substances. Resistance genes can be located on conjugative or non-conjugative plasmids, and they can confer protection to the host cell against harmful agents. Resistance plasmids are often associated with multidrug resistance and horizontal gene transfer in bacteria. An example of a resistance plasmid is the R100 plasmid, which confers resistance to several antibiotics, such as ampicillin, chloramphenicol, kanamycin, streptomycin, sulfonamide, and tetracycline.
• Colicinogenic plasmids (Col plasmids): These are plasmids that produce colicins, which are bacteriocins or toxins that kill other closely related bacteria that lack the same Col plasmid. Colicins can act by disrupting the cell membrane, inhibiting protein synthesis, or degrading DNA or RNA of the target cells. Colicinogenic plasmids provide a competitive advantage to the host cell in a mixed bacterial population. An example of a colicinogenic plasmid is the ColE1 plasmid, which produces colicin E1 that inhibits protein synthesis in sensitive cells.
• Degradative plasmids: These are plasmids that encode enzymes for the degradation of unusual or complex organic compounds, such as aromatic hydrocarbons, camphor, naphthalene, salicylic acid, toluene, etc. Degradative plasmids enable the host cell to utilize these compounds as carbon and energy sources and to detoxify them from the environment. Degradative plasmids are often found in soil bacteria and can be transferred horizontally among different species. An example of a degradative plasmid is the TOL plasmid, which allows Pseudomonas putida to degrade toluene and xylene.
• Virulence plasmids: These are plasmids that carry genes for virulence factors, which are molecules that enhance the ability of the host cell to infect and cause disease in other organisms. Virulence factors can include adhesins, toxins, invasins, siderophores, capsules, etc. Virulence plasmids are often found in pathogenic bacteria and can be transferred horizontally among different strains or species. An example of a virulence plasmid is the Ti (tumor-inducing) plasmid, which is carried by Agrobacterium tumefaciens and causes crown gall disease in plants by transferring a segment of its DNA (T-DNA) into the plant genome.

Plasmids are not only important for the survival and adaptation of the host organisms, but also useful for various biotechnological and ecological applications. Some of the functions and applications of plasmids are:

• Genetic engineering: Plasmids are widely used as vectors to introduce foreign DNA into bacteria or other cells. The foreign DNA can be a gene of interest, a reporter gene, a selectable marker, or a combination of these. Plasmids can also be used to manipulate or delete certain genes from the host cell. Plasmids can be modified by inserting or deleting DNA sequences using restriction enzymes and ligases. Plasmids can also be amplified by bacterial replication or by polymerase chain reaction (PCR).
• Antibiotic resistance: Plasmids often carry genes that confer resistance to one or more antibiotics. These genes can protect the bacteria from the effects of antibiotics in human medicines or in the natural environment. Antibiotic resistance plasmids can be transferred from one bacterium to another by conjugation, transduction, or transformation. This can lead to the spread of antibiotic resistance among bacterial populations and pose a challenge for public health.
• Degradation of pollutants: Plasmids can also carry genes that enable the bacteria to degrade unusual substances such as hydrocarbons, pesticides, herbicides, or heavy metals. These genes can help the bacteria to survive in contaminated environments and also reduce the environmental pollution. Degradative plasmids can also be transferred among bacteria by horizontal gene transfer.
• Virulence and pathogenesis: Plasmids can also carry genes that produce virulence factors, such as toxins, adhesins, invasins, or siderophores. These factors can help the bacteria to infect and colonize the host cells, such as plants, animals, or humans. Virulence plasmids can also be transferred among bacteria by horizontal gene transfer. Some examples of virulence plasmids are Ti plasmid in Agrobacterium tumefaciens, which causes crown gall disease in plants, and pXO1 and pXO2 in Bacillus anthracis, which cause anthrax in animals and humans.
• Bacteriocin production: Plasmids can also carry genes that produce bacteriocins, which are proteins that kill or inhibit the growth of closely related bacteria. These genes can provide a competitive advantage for the bacteria that produce them over other bacteria that lack them. Bacteriocin plasmids can also be transferred among bacteria by horizontal gene transfer. Some examples of bacteriocin plasmids are Col plasmids in Escherichia coli, which produce colicins, and pAD1 in Enterococcus faecalis, which produces enterocin A.

There are many examples of plasmids that have been isolated from different bacteria and used for various purposes. Some of the notable examples are:

• pBR322: This is one of the first plasmids that was widely used as a cloning vector. It is derived from a natural plasmid found in E. coli and contains genes for ampicillin and tetracycline resistance. It also has multiple restriction sites for inserting foreign DNA fragments.
• pUC19: This is another popular cloning vector derived from pBR322. It has a high copy number and a lacZ gene that allows blue-white screening of recombinant colonies. It also has a multiple cloning site with many unique restriction sites.
• pGLO: This is a plasmid that contains a gene for green fluorescent protein (GFP) from the jellyfish Aequorea victoria. It also has a gene for arabinose operon regulator (araC) and a gene for ampicillin resistance. This plasmid can be used to transform bacteria and make them glow green under UV light when exposed to arabinose.
• Ti plasmid: This is a large plasmid found in Agrobacterium tumefaciens, a soil bacterium that causes crown gall disease in plants. It contains genes for virulence factors that enable the bacterium to infect plant cells and transfer a part of the plasmid (T-DNA) into the plant genome. The T-DNA can be modified to carry genes of interest and used for plant genetic engineering.
• F plasmid: This is a conjugative plasmid that allows bacterial cells to undergo sexual reproduction by forming a sex pilus and transferring a copy of the plasmid to another cell. It also contains genes for fertility inhibition (fin) that prevent multiple F plasmids from coexisting in the same cell. The F plasmid can integrate into the bacterial chromosome and form an Hfr (high frequency of recombination) strain that can transfer chromosomal genes along with the plasmid.

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