Plasmid

• Plasmids are the extrachromosomal genetic elements found in bacteria.
• They are circular pieces of DNA that are extra genes.
• About 1-20 copies of plasmids are present in one bacterial cell.
• Episomes are the type of plasmid that can be inserted into the bacterial chromosome and can replicate with it.
• For normal life and functioning, a plasmid is not required in the bacteria. But their presence confers new properties in the bacteria. Example: Drug resistance, toxigenicity

Properties/Characteristics of bacterial plasmids:

1. Physical properties:
   • Plasmid is a double-stranded circular and supercoiled DNA.
   • Within a cell, it can exist autonomously. It can replicate independently of the bacterial chromosome.
   • It has a molecular weight of 10⁶-10⁸ which may encode from 40-50 genes.
   • It has about 1-3% of the weight of the bacterial chromosome consisting of 1500-400,000 base pairs.
   • Plasmid as large as 2 million base pairs can occur in some bacteria.

2. Replication:
   • It contains genes for self-replication.

3. Curing:
   • It can be lost spontaneously or by curing agents.

4. Incompatibility:
   • In the same cell, two members of the same group cannot co-exist.

5. Transferability:
   • Some plasmids are self-transferable.

6. Recombinations:
   • Episome can integrate with host chromosome.

7. Mobilisation:
   • By the process of integration, the self-transferable plasmid can mobilize the chromosomal gene or other plasmids.

Types of plasmid:

• Based on their function, plasmids are of five types:
• Resistance ( R ) plasmid
• Fertility (F) plasmid
• Bacteriocinogen or Col plasmid
• Degradative plasmid
• Virulence plasmid

 1. R-plasmid (R-factor):

• They are circular with double-stranded plasmid.
• R factor occurs in two sizes:
  • large plasmids ( mol. wt. 60 million)
  • small plasmids ( mol. Wt. 10 million)
• Large plasmids are conjugative ‘R’ factors. To code for the conjugation process, it contains extra DNA.
• Small plasmids contain only the ‘r’ genes. They are not conjugative.
• It consists of two components.
  • Resistance transfer factor (RTF): carries the genes that govern the process of intercellular transfer.
  • Resistant determinant ( R-determinant): carries resistant genes for each of the several drugs.
• The drug resistance is not transferrable in the case when RTF dissociates from the R-determinant.
• For the spread of the multiple drug resistance in the bacteria, R factor plays a vital role.
• Antibiotics can be destroyed and the membrane transport system can be modified.
• R-factor may carry the resistance genes either one, two, or more than these.
• They may also carry the gene resistance for the metal ions.
• They also carry resistance to certain bacteriophages by coding for the enzymes.

2. F-plasmids:

• It is a transfer factor or F-factor.
• It contains genetic information, which controls the mating process of the bacteria during the conjugation.
• It contains the basic genetic information necessary for:
  • Extra-chromosomal existence
  • Self-transfer
  • Synthesis of sex-pilus.
• F-plasmid carries some fourteen genes which include the structural gene for the pilin.
• Pilin is the pilus protein that functions in sex pilus formation.
• Strains of bacteria having the F plasmid are called F₊ and function as donors.
• Strains of bacteria lacking the F plasmid are called F- and function as recipients.
• It is also called the conjugative plasmid.
• The conjugative function is determined by the cluster of at least 25 transfer (tra) genes.
• These genes determine:
  • Expression of pili
  • Synthesis and transfer of DNA during mating
  • Interference with the ability of F₊ bacteria to serve as recipients.

3. Bacteriocinogen or Col plasmid:

• Coliforms produce extracellular colicins.
• In the several species of coliform, the colicinogenic (col) factors are present.
• These bacterial factors are the lethal toxins for the closely related species or even for the different strains of the same species.
• Some bacterial substances are produced not only by the coliforms but also by the other bacteria.
• This group of substances is called bacteriocins.
• Colicins are produced by coli
• Pyocin are produced by Pseudomonas aeruginosa.
• Marscesins are produced by Serratia marcescens.
• Diphthericin is produced by Corynebacterium diphtheria.
• Bacteriocin produced by the different bacterial strains helps in the interspecies typing of organisms.

4. Degradative plasmids:

• From the dead plants and animals, degradative plasmid helps in the degradation and digestion of the dead organic matter.
• It is then used in the biosynthesis process.
• It will make energy and will recycle further.

5. Virulence plasmids:

• With the help of this plasmid, bacteria will be transformed into a pathogen.
• It carries the genes which are responsible for causing disease.

Based on the role in conjugation, plasmids are of two types:

• Conjugative plasmid
• Non-conjugative plasmid

i. Conjugative plasmids:

• These large plasmids (F plasmids) carry genes that are responsible for transferring themselves to other cells.
• It includes the genes that direct the synthesis of sex pilli.

ii. Non-conjugative plasmids:

• These plasmids are present in Gram-positive bacteria, especially in the Gram-positive cocci.
• It is also present in the Gram-negative organism. Example: Haemophilus influenza, Neisseria gonorrhoeae.
• They are usually small, 1-10 dal.
• In each bacterium, multiple copies (more than 30 ) may be present.
• When the same bacterium carries both the conjugative and non-conjugative plasmids, they can be mobilized for transfer to another cell.
• When the conjugation is established then the donor can transfer non-conjugative plasmids.

Functions/applications of plasmid:

• The main function of the plasmid is the spread of antibiotic-resistant genes. These resistant genes are carried within the plasmid and are transferred from one cell to another.
• Plasmid is used in recombinant DNA technology.
• To deliver the desired drug into the body, a plasmid is used.
• For the insertion of the human insulin on the body
• Insertion of human growth hormone in mammalian cells of animals.
• Plasmids are used in Gene Therapy:
  • For the insertion of the therapeutic genes in the human body. It helps to fight against diseases.
  • Easy manipulation and can be replicated in bacterial cells easily.
  • Targeting the defected cells easily and triggering the therapeutic genes in them.
• Plasmids carry the genes involved in metabolic activities. They aid in the digestion of pollutants from the environment.
• Plasmids can produce antibacterial proteins.
• Plasmid can carry genes that increase the pathogenicity of the bacteria.
• When the nutrients are scarce, the plasmid can help bacteria by:
  • Fix the nitrogen
  • Degrade organic compounds

Host Range of plasmid

• The host range of a plasmid means the types of bacteria in which the plasmid can replicate.
• It is usually determined by the ori region from where the replication starts.
• Plasmid having the narrow host range includes:
  • ColE1 plasmid type; Example: pBR322, pET, and pUC.
  • Replication of these plasmids occurs only in coli.
  • It may occur in Salmonella and Klebsiella also which are closely related bacteria.
• Plasmids having the broad host range includes:
  • RK2
  • RSF1010 plasmids
  • RC plasmids; Example: pBBR1MCS .
  • Plasmids with the ori region of RK2 can replicate in most types of Gram-negative proteobacteria.
  • Plasmids with the RSF1010-derived plasmids can replicate in Gram-positive bacteria too. Example: Firmicutes.
  • Replication of the same plasmid can occur even in the distantly related bacteria.
  • Broad-host-range plasmids do not depend on the host cell because they encode their proteins. These proteins are essential for the initiation of replication.
  • Broad host-range plasmids should be for gene expression in many types of bacteria.

Determining the Host Range

• It is sometimes difficult to ensure the particular plasmid will replicate on the other host or not. So, The actual host ranges of most plasmids are unknown.
• Initially, plasmids need to be introduced to the other bacteria.
• So, for this process, a system has been developed which is known as transformation.
• By this method, the plasmid can be introduced into the bacteria to see if it could replicate or not.
• But it has limitations as it can’t be applied to all types of bacteria.
• Similarly, to introduce DNA into cells, electroporation can be used.
• Plasmids can be introduced into other types of bacteria by the conjugation process.
• It is also found that the expression of the gene present in one plasmid does not function well or doesn’t get expressed in the other bacterium.
• Sometimes the selected gene can be introduced into the different bacteria.
• A bacterium might possess resistance to any antibiotic due to the presence of a particular gene. Such resistance property can be transferred to other bacteria too when they will uptake those resistant genes.
• For example, the kanamycin resistance gene, which is first found in the Tn5 It can be expressed in most Gram-negative bacteria. Then, it will make resistant to kanamycin antibiotic.
• By this property, a marker gene can be cloned in the plasmid. i.e making numerous copies.
• A transposon carrying a selectable marker into the plasmid can also be introduced by this method.
• Care must also be taken to ensure that the plasmid has not recombined into the host chromosome.
• Determining the host range of a plasmid is laborious too. Many barriers hinder the transfer of plasmid into the host. The same method can’t be approached to all sorts of plasmids and bacteria.