MICROBIAL GENETICS

 

Microbes are preferably suited for biochemical and genetics studies and have made huge contributions to these fields of science such as the demonstration that DNA is the genetic material that the gene has a simple linear structure. Following are the most important functions of microbial genetics.

1) To understand the gene function of microorganisms

2) Microbes provide relatively simple system for studying genetic phenomenon and thus useful to other higher organisms.

3) Microorganisms are used for isolation and multiplication of specific genes of higher organisms which is referred as gene cloning.

4) Microbes provide many value added products like antibiotics, growth hormones etc. Microbial genetics will be helpful to increase these products productivity by microbial technology

5) Understanding the genetics of disease causing microorganisms especially virus, will be useful to control diseases.

6) Gene transfer among the prokaryotes play major role in the spread of the genes in a particular environment. Microbial genetics will be useful to study the gene transfer from one organism to another. Gene transfer methods are briefly explained below.

CONJUGATION

In 1946, Joshua Lederberg and Edward Tatum demonstrated that bacteria can transfer and recombine genetic information. Conjugation takes place when genetic material passes directly from one bacterium to another. In conjugation, two bacteria lie close together and a connection mediated by a conjugation pilus or sex pilus. A plasmid or a part of the bacterial chromosome passes from one cell (the donor F⁺ male) to the other (the recipient F^- female). Consequent 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. Conjugation is the transfer of a plasmid or other self-transmissible DNA element and sometimes chromosomal DNA from a donor cell to a recipient cell. Recipients of the DNA transferred by conjugation are called transconjugants. The process of conjugation can transfer DNA regions of hundreds to thousands of kilo bases and has the broadest host range for DNA transfer among the methods for bacterial exchange. Conjugation occurs in and between many species of bacteria, including Gram-negative as well as Gram-positive bacteria, and even occurs between bacteria and plants. Broad-host-range conjugative plasmids have been used in molecular biology to introduce recombinant genes into bacterial species that are refractory to routine transformation or transduction methods. Although numerous examples of conjugative plasmids exist, conjugation involving the F plasmid is the most common.

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 are referred to as F⁺ and cells lacking F are F⁻.

·         The F factor contains an origin of replication and a number of genes required for conjugation.

·          For example, some of these genes encode sex pili, small extensions of the cell membrane. A cell containing F produces the sex pili, one of which makes contact with a receptor on F⁻ cell and pulls the two cells together.

·         DNA is then transferred from the F⁺ cell to the F⁻ cell. Conjugation can take place only between a cell that possesses F and a cell that lacks F.

·         In most cases, the only genes transferred during conjugation between an F⁺ and F⁻ cell are those on the F factor Transfer is initiated when one of the DNA strands on the F factor is nicked at an origin (oriT).

·         One end of the nicked DNA separates from the circle and passes into the recipient cell. Replication takes place on the nicked strand, proceeding around the circular plasmid in the F+ cell and replacing the transferred strand.

·          Because the plasmid in the F+ cell is always nicked at the oriT site, this site always enters the recipient cell first, followed by the rest of the plasmid. Thus, the transfer of genetic material has a defined direction. Inside the recipient cell, the single strand replicates, producing a circular, double-stranded copy of the F plasmid. If the entire F factor is transferred to the recipient F− cell, that cell becomes an F+ cell.

Hfr CELLS

·         When F plasmid (sex factor) integrated with chromosomal DNA then such bacteria is known as High frequency recombination (Hfr) bacteria. Here F factor or F plasmid of the donor is not free from plasmid but it is integrated to the donor bacterial chromosomal DNA as an episome.

·         Thus F plasmid together with bacterial chromosomal DNA forms a recombinant DNA called as High frequency recombination DNA or Hfr DNA. Hfr strains can effect high rate of recombination as some portion of the donor bacterial DNA may also be get transferred. Hence they called Hfr strains.

·         In the cross (conjugation) between Hfr cell and F- cell, frequency of recombination is very high but frequency of transfer of whole F-factor is very low.

·         Hfr cell acts as donor while F- cell acts as recipient.

·         Hfr cell produces hair like appendages called sex pili which facilitates cell to cell contact with F^- strain by forming a conjugation tube. The formation of sex pili is governed by genes of F factor

·         Hfr DNA of donor undergoes replication by rolling circle mechanism

·         Now the 5’ end of this strand enters into recipient cell through conjugation tube. Since, replication origin lies somewhere in the middle of F- factor, portion of F-factor that lies at 5’ end enters first into recipient cell but the portion situated at 3’ end enters only when whole chromosomal DNA enters into the recipient cell.

·         To transfer whole chromosomal DNA, it takes 100 minutes in E. coli. In most of the cases, sex pilus (conjugation tube) breaks before transfer of whole chromosomal DNA takes place. So, frequency of transfer of whole F-factor is very low. After the cross between Hfr cell and F- cell, recipient cell remains recipient.

 

F’  AND F^- CELLS

·         The bacterium possessing a plasmid containing the F factor and a part of the bacterial genome.

·         Existence of Hfr donor cells is not absolute. The F-factor integrated into the bacterial DNA of Hfr donor cells may dissociate and become free in the cytoplasm.

·         The dissociation may be occasionally inconsistent during which the dissociated F-factor may bring with it some genes of the bacterial chromosome.

·         Adelberg and Burns (1958) first identified such a modified F- factor and called it F (F-prime) factor; the donor cell possessing this factor is called F (F-prime) male.

·         When an F male conjugates with F^- (recipient) cell, the F-factor is transferred from donor to the recipient cell, and such a recipient bacterial cell becomes heterozygous (merozygous) for that part of the bacterial chromosome, which the F-factor had obtained during its anomalous dissociation.

·         Transfer of F-factor to recipient cell apparently occurs by the same mechanism as F-factor, transfers during in F⁺ and F^- mating and chromosome transfer in Hfr and F^- cell mating.

·         Genetic recombination of this type, mediated by F-factor, is called sexduction or F-duction.

TRANSFORMATION

·         Bacterial transformation is a process of horizontal gene transfer by which some bacteria take up foreign genetic material (naked DNA) from the environment. It was first reported in Streptococcus Pneumoniae by Griffith in 1928. DNA as the transforming principle was demonstrated by Avery et al in 1944.

·         The process of gene transfer by transformation does not require a living donor cell but only requires the presence of persistent DNA in the environment. The prerequisite for bacteria to undergo transformation is its ability to take up free, extracellular genetic material. Such bacteria are termed as competent cells.

·         The factors that regulate natural competence vary between various genera. Once the transforming factor (DNA) enters the cytoplasm, it may be degraded by nucleases if it is different from the bacterial DNA. If the exogenous genetic material is similar to bacterial DNA, it may integrate into the chromosome. Sometimes the exogenous genetic material may co-exist as a plasmid with chromosomal DNA.

·         The recipient that successfully propagates the new DNA is called the transformant.

·         During extreme environmental conditions, some bacterial genera spontaneously release DNA from the cells into the environment free to be taken up by the competent cells. The competent cells also respond to the changes in the environment and control the level of gene acquisition through a natural transformation process.

·         Transformation is adopted as the most common method of gene transfer as it is the best way for the transfer of artificially altered DNA into recipient cells.

·         The process of transformation can transfer DNA regions of one to tens of kilobases.

TYPES OF TRANSFORMATION

There are two forms of transformation

Natural Transformation

• In natural transformation, bacteria naturally have the ability to incorporate DNA from the environment directly.

Artificial Transformation

• In the case of artificial transformation, the competence of the host cell has to be developed artificially through different techniques.

PRINCIPLE OF BACTERIAL TRANSFORMATION

• Bacterial transformation is based on the natural ability of bacteria to release DNA which is then taken up by another competent bacterium.
• The success of transformation depends on the competence of the host cell. Competence is the ability of a cell to incorporate naked DNA in the process of transformation
• Organisms that are naturally transformable spontaneously release their DNA in the late stationary phase via autolysis.
• Several bacteria, including Escherichia coli, can be artificially treated in the laboratory to increase their transformability by chemicals, such as calcium, or by applying a strong electric field (electroporation) or by using a heat shock.
• Electroporation or heat shock increases the competence by increasing the permeability of the cell wall, which allows the entry of the donor DNA.
• Similarly, transformants can be selected if the transformed DNA contains a selectable marker, such as antimicrobial resistance, or if the DNA encodes for utilization of a growth factor, such as an amino acid.
• In most of the naturally competent bacteria, the free DNA binds to the bacteria, and the DNA is integrated into the chromosomal DNA.
• Sometimes, the free DNA is inserted into a plasmid which is capable of replicating autonomously from the chromosome, and thus, the insert doesn’t have to be integrated into the chromosome.
• Plasmid encodes some enzymes and antibiotic-resistant markers which are later expressed in the transformant after transformation.
• In this process of transformation, the donor DNA is first inserted into the plasmid. The plasmid containing the donor DNA is then inserted into the competent host bacteria.
• After the transformation is completed, the bacteria containing the plasmid can be detected either by using a growth media supplemented with a particular antibiotic.

STEPS IN BACTERIAL TRANSFORMATION

·         Development of competence

·         Binding of DNA to the cell surface

·         Processing and uptake of free DNA

·         Integration of the DNA into the chromosome by recombination

·         The artificial development of competence can be achieved either through electroporation or through heat shock treatment. The choice depends on the transformation efficiency required, experimental goals, and available resources.

·         For heat shock, the cell-DNA mixture is kept on ice (0°C) and then exposed to 42°C.

·         For electroporation, the mixture is transferred to an electroporator and is exposed to a brief pulse of a high-voltage electric field.

·         The double-stranded DNA released from lysed cells binds noncovalently to cell surface receptors. There is no DNA sequence-specific recognition; thus, these organisms can potentially incorporate DNA from outside their species.

·         The bound double-stranded DNA is nicked and cleaved into smaller fragments by membrane-bound endonucleases, allowing the single strand to enter the cell through a membrane-spanning DNA translocation channel.

·          The transformed DNA integrates into the chromosome and replaces the chromosomal DNA fragment by recombination. This integration, however, requires significant nucleotide sequence homology between the donating DNA fragment and the fragment in the chromosome.

 

TRANSDUCTION

·         Transduction is a mode of genetic transfer from one bacterium to another through a virus. A virus is a simple replicating structure made up of nucleic acid surrounded by a protein coat.

·         Viruses come in a great variety of shapes and sizes. Some have DNA as their genetic material, whereas others have RNA; the nucleic acid may be double stranded or single stranded, linear or circular.

·         Viruses that infect bacteria (bacteriophages) have played a central role in genetic research since the late 1940s. They are ideal for many types of genetic research because they have small and easily manageable genomes, reproduce rapidly, and produce large numbers of progeny.

·         Bacteriophages have two alternative life cycles: the Lytic and the Lysogenic cycles.

·          In the Lytic cycle, a phage attaches to a receptor on the bacterial cell wall and injects its DNA into the cell. Inside the host cell, the phage DNA is replicated, transcribed, and translated, producing more phage DNA and phage proteins. New phage particles are assembled from these components. The phages then produce an enzyme that breaks open the host cell, releasing the new phages. Virulent phages reproduce strictly through the lytic cycle and always kill their host cells.

·         Temperate phages can undergo either the lytic or the lysogenic cycle. The lysogenic cycle begins like the lytic cycle but, inside the cell, the phage DNA integrates into the bacterial chromosome, where it remains as an inactive prophage. The prophage is replicated along with the bacterial DNA and is passed on when the bacterium divides. Certain stimuli can cause the prophage to dissociate from the bacterial chromosome and enter into the lytic cycle, producing new phage particles and lysing the cell.

 

TYPES OF TRANSDUCTION

Transduction is common in both virulent and temperate phages, specifically. By lytic or lysogenic cycle. Transduction is of two types:

• Generalized Transduction – In this, the phage can carry any part of DNA.
• Specialized Transduction – In this, the phage carries only the specific part of DNA.

GENERALIZED TRANSDUCTION

• Generalized transduction can occur by either lytic or lysogenic cycle.
• In generalized transduction, phage mistakenly packages bacterial DNA instead of their own phage DNA during phage assembly.
• This results in an infectious virus particle containing bacterial DNA, but one that can no longer replicate in the bacterium due to the loss of all of the phage DNA.
• The phage particle then attaches to a bacterial cell surface receptor and injects the packaged DNA into the cytoplasm of the bacterium.
• If the bacterial DNA in the phage is from the bacterial chromosome, the DNA recombines with the homologous DNA of the bacterial recipient to generate stable transductants. This process requires a host recombinase, recA.
• However, studies have indicated that the majority of transduced DNA is not stably integrated into the bacterial genome but rather remains extrachromosomal.
• Generalized transduction is used for mapping genes, mutagenesis, transferring plasmids and transposons, and determining whether different genera of bacteria have homologous genes.
• Example of generalized transduction includes E.coli transduction by P1 phage

·        SPECIALIZED TRANSDUCTION

·         Specialized transduction can occur only through the lysogenic cycle specifically by temperate phage.

·         Here, only the specific part of the bacterial DNA is packed into the virus. It occurs when the prophage, i.e. viral DNA, which gets inserted into the bacterial genome in the lysogenic cycle excises.

·         When prophage excises from bacterial DNA, some parts of bacterial DNA, which are flanked on both sides of the prophage are also excised. Here, the newly packed phage genome consists of both bacterial and viral genome.

Later, when the virus with the recombinant genome infects a new bacterial cell, the bacterial gene also gets inserted into the host genome with the viral genome through lysogeny. The recipient cell now shows the newly acquired characteristics

·         Specialized transduction is commonly used for isolation and insertion of genes of choice.

·         Example of specialized transduction includes E.coli transduction by 𝝀 phage.