Traditional and Molecular Methods for Classifying Bacteria

 Higher organisms are easily studied according to their observable characters. Micro- organisms, especially, bacteria are difficult to separate according to size and shape. The reason being, diverse micro-organisms share common morphology.

  Likewise, study of endospore, flagella and capsules are also less helpful. So, study of biochemical, physiological and genetic characters can yield useful Information for classification. The characters that are used to study, classily and identify bacteria can be broadly divided into two categories -

  1. Classical (traditional) method,
  2. Genomic (molecular) method.

1. Traditional methods :

  Traditional or classical methods involve study different characters of organisms. They are being used for routine identification for many years. They may provide phylogenetic details too.

a). Morphological characters : 

Microbiologists have often used morphological characters in microbial taxonomy. Some of the morphological features that are used in classification and identification are -

  1. Shape and size of cells
  2. Arrangement of cells
  3. Ultra structural features
  4. Staining characteristics
  5. Occurrence of cilia or flagella
  6. Mode of motility
  7. Endospore formation,shape and location
  8. Cellular Inclusion, etc.
  These characters have been studied by various techniques of light microscopy and electron microscopy.

b). Physiological and metabolic Characteristics :

Physiological and metabolic characteristics are very useful in classification. It is because they are directly related to the nature and activity of microbial enzymes as well as transport proteins. These include :

  • Mode of nutrition - an organism can be autotrophic, heterotrophic and fastidious. It can be a phototroph or chemotroph with respect to energy source. 
  • Growth characteristics in liquid media and solid media are characteristic of each species . Colony morphology and pigmentation are useful characters.
  • Physiological characters of each species are unique. Thus, they show differences with respect to growth temperature, pH, oxygen requirement. osmotic tolerance, antibiotic sensitivity and tolerance etc. 
  • Biochemical characteristics include activity of microbial enzymes and transport proteins. Enzymes confer ability to catalyze a specific biochemical reaction. Various biochemical tests used in microbiology laboratory are helpful in identification of bacteria.

  Some of the biochemical tests that can be used are -

  • Sugar fermentation
  • Hydrolysis of polymeric substances
  • Gelatin liquefaction
  • Production of catalase and peroxidase
  • Indole production
  • Hydrogen sulfide ( H₂S ) production
  • Mixed acid fermentation
  • Diacetyl formation
  • Nitrate reduction
  • Urease, phenylalanine deaminase, etc. production
  •  Utilization or specific nutrients.

c). Ecological characteristics : 

   Ecology is the study of organisms with reference to their specific environment. Some micro-organisms that resemble in many respects but they inhabit different environments. It means that these organisms may be closely related.
  The taxonomically important properties include.
  1. Pattern of life cycle
  2. Nature of symbiotic relationship
  3. Ability to cause disease in a given host
  4. Ability to participate in biogeochemical transformations
  5. Habitat preferences such as requirements for temperature, pH, oxygen and osmotic concentration.

d). Antigenic characteristics :

  Bacterial cell is clustered with several antigens. Each set of antigen is characteristic of the bacterial cell. Therefore , antigenic characters of bacteria can be useful for classification and identification of bacteria.
  When different strains of bacteria are identified by serological reaction the method is called serotyping.

e). Phage typing :

   Phage typing is concerned with Identification of strains or species of bacteria on the basis of their susceptibility to bacteriophage or phage. Phage typing gives various phagovars

f). Genetic analysis :

   Genetic analysis aims at determining genetic seminaries or differences in the organism, and there by use it for identification or classification of organisms.
The genetic tools used for the purpose include .....

2. Molecular methods :

Micro-organisms have more or less no record of fossils. However their phylogenetic and evolutionary relationship can be established by determining their genetic similarities. Organism showing a close genetic similarity, suggest their evolutionary closeness.

Following methods are available for comparison of microbial genomes. 

1] Nucleic acid Base Composition or GC content of the DNA 

2] Nucleic acid hybridization :

  • This involves hybridization between single stranded DNA obtained from two different organisms.
  •  The extent of hybridization between the DNA obtained from different organisms, show degree of their genetic similarity.

3] Nucleic acid sequencing :

  • The determination of G + C content as well as DNA homology studies have yielded useful informations. 
  • However, studies of sequence of nucleotides in small subunit rRNA ( 16S and 18S rRNA from prokaryotes and eukaryotes respectively ) ( SSU rRNAs ) have proved more useful in inferring microbial phylogeny and taxonomic assignments. 

4] Genomic Fingerprinting :

  • Genomic fingerprinting is a technique useful for classification and determination of phylogenetic relationship.
  • It does not depend on determination of nucleotide sequence. 
  • The assays include the study of restriction fragments generated by specific restriction endonucleases and conserved repetitive sequences.

5] Amino acid Sequencing :

  • The amino acid sequences of proteins directly reflect mRNA sequences and in turn that of the genes. 
  • Different varieties of the proteins can be compared. 
  • The organisms having similar amino acid sequence and functions may be closely related.

Lysogenic Cycle of bacteriophage

Lysogeny or lysogenic cycle is one of type of viral reproduction or life cycle.
Lysogenic cycle is characterized by
(1) integration of the bacteriophage nucleic acid into host bacterial genome by site specific recommendation and replication with bacterial genome e.g. λ phage in E.coli.
(2) integration and formation of circular replicon in bacterial cytoplasm that replicates as extra-chromosomal plasmid e.g. P1 in E.coli.
  Lysogenic virus (temperate phage) can remain in this stage for various replications of host cell DNA until it excises it self from cell DNA and undergoes a lytic life cycle.

Genetic material of temperate phages that is inserted into the DNA of host is called pro-phase.
A cell that contain a Prophase is known as lysogen.

Phage conversion :

  When a cell become lysogenised, occassionally extra genes carried by the phage get expressed in the cell and change the properties of the bacterial cell. This process is called lysogenic or phage conversion.
- Examples :
1) lysogenic conversion has shown to enable biofilm formation in Bacillus anthacis.

2) Lysogenic conversion of Bacillus subtilis and Bacillus cereus has shown an enhanced rate or extent of sporulation that produces endospore that are resistant to temperature, ionising radiation, desiccation, antibiotics and disinfectants. 

3) Virulence genes carried within Prophase as autonomous genetic elements morons that confers advantage to bacteria through enhanced lysogen survival.

4) non-virulent bacteria are transformed to virulent pathogen.
e.g. Cornebacterium diphtheriae produces diptheria toxin only when infected by Phage-B.

Stages(steps) of lysogenic cycle :

1. Attachment
2. Penetration
3. Integration of phage genetic material to bacterium
4. Replication of genetic material
5. Cell division.

1). Attachment -

The cycle begins with the attached of bacteriophage to specific host cell receptor on bacterial cell wall by adsorption.

2). Penetration -

a) injection of linear phage DNA into bacterium.
b) circularisation of phage DNA
c) The virus genetic material is known as Prophase, while it is in the dormant stage.
Dormant stage :- if the virus does not start multiplying or replicating after infecting the cell it is said to be dormant.

3). Integration of phage/ lysogenization -

The integration of the circular λ genome involves the presence of attachment sites in the genome of both bacteriophage and bacterium.
The bacterial attachment site, called attB Consists of a specific DNA sequence composed of 3 domain (B,O,B').
The attachment site of bacteriophage called attp, consist of P,O,P' domains.
The O domain has a sequence common to both attB and attP.
The O domain is also known as core sequence.

4). Replication of genetic material-

a) lysogenised cell will follow its regular metabolic activities and eventually prepare for cell division.
b) The nucleic acid replicates and nucleus divides into two parts.

5) cell division -

Division of cell body results in two daughter cells each having viruses genetic information incorporated to their genetic material.
The cells (host bacterial cell) remain normal until virus is triggered or induced.

Lysogeny is maintained by a repressor protein encoded. The DNA of λ Prophase called λ repressor. It bloks the expression of other genes responsible for phage replication and synthesis of phage proteins.

Immunity : The lysogens are immune to superinfecting phage. The resistance of a λ lysogen to superinfection by λ phage is called immunity.
  λ repressor encoded by CI gene. These protein inhibitor of transcription from PL and PR.
In lysogen CI encoded λ repressor is continuously when a phage infects a lysogen excess repressor present in cytoplasm bind to two promoters PL and PR before RNA polymerase bind.

Induction - Under some condition, the Prophase initiates synthesis of phage proteins, that leads to lysis of cells and release of new phages. This process is called induction.
Induction occur because cro protein express and inhibits Transcription of CI gene.

Induction is defined into mainly two ways.
1. UV Induction - if UV light damage host DNA ensuring pro-phase induction.
2. Zygotic induction - when Hfr lysogen mates with f- recipient (non lysogen) transcription of pro-phase begins and a non lysogenic recipient receiving a pro-phase will lyse ensuring lytic cycle.

Synchronous Growth Curve - Methods of Synchronous Growth

    It is a normal tendency of bacterial culture to grow in a heterogeneous manner. Usually the cells In a population occur in different state of physiology as well as cell division even though they are inoculated at a time in the medium. 

  Therefore, study of organisms from such heterogeneously growing population does not provide reliable information on the behavior of individual cells. This can be however, obtained by study of populations from synchronous cultures.

Synchronous growth is the one where all cells in the culture are in the same state of physiology and cell division. Therefore, they will divide at a time.

Nature of synchronous growth - 

   Since all cells in the synchronously growing culture divide at a time, they yield a zig zag pattern of growth curve.

  For some time, there will be no increase in cell number and then suddenly the cell number doubles. This is because all cells divide at a time.
  The synchrony in the growth hardly remains for 2 - 3 generation. Then the culture looses synchrony and cells start dividing In heterogeneous manner.
  All cells do not divide at same time. The rate, at which cell division takes place, depends on the age of the cell. Young cells are smaller in size as compared to old cells.
  Cells with intermediate size grow more rapidly. Again very large cells grow slowly.

synchronous growth of bacteria. The step like growth pattern indicates that all the cells of the population divide at about the same time.

 Methods of synchronization of growth 

Various practical manipulations are applied to obtain synchronous growth from a heterogeneous population.
1. Physical selection method
2. Blological selection method

1. Physical selection method -

   These methods involve separation of the cells on the basis of their size. Selection of cells with uniform size and then their synchronous culture.
  This is based on the fact that cells with same size occur in same state of cell division and hence their use provides synchronized growth.

Cells with identical size are selected by various use provides methods:

  • By use of membrane filter.
  • By use of density gradient centrifugation.

a. Selection of cells by membrane filter -

  Use of membrane filter with specific pore size will help in separating cells with uniform diameter. Such cells are then used for obtaining synchronous growth.
  Another method for obtaining cells with uniform size by use of membrane filter is Helmstetter Cummings technique.

Helmstetter Cummings method for obtaining synchronous growth.

  When heterogeneous culture is fltered through cellulose nitrate membrane filter, some cells get adhered to the filter.
The filter is then inverted and fresh broth is allowed to low through.
Initial flow removes loosely adhered bacteria from the filter. Then, the later flow will contain only these cells which have just separated after division from adhered cells.

Therefore these cells will be in the same state of division. Hence, culture arising from these cells will be growing as synchronized culture.

b. Use of density gradient centrifugation -

  Another method of separation of cells having identical size is the use of centrifugation technique. Thus, centrifugation of a culture allows one to separate cells with identical size. They can yield synchronous growth upon inoculation to a fresh medium.

2. Blological selection methods -

  Characteristic physiological properties of organisms can also be useful in obtaining synchronised growth. Two methods based on these parameters are widely used.
a). Cyclic temperature shifft method
b). Use of limiting growth substance.

a). Use of cyclle temperature shift method -

The method is commonly used for obtaining synchronized growth of mesophilic bacteria, where they are incubated for some time at 37°C and 20°C alternately.
  The fundamental property of the bacteria is that they can initiate cycle of new chromosome replication and cell division only at a speciflc temperature ( at 37°C). and not at lower temperature, i.e. at 20°C.
  But., once the cycle has initiated, it can continue till it is completed. Therefore, cells which have been transferred to 20°C and kept for some time, all cells will continue to complete their division cycle, but will not start new cycle of cell division.
  Thus, all cells are brought to same state of cell division. These organisms will therefore give synchronized growth on further incubation.

b) Use of limiting growth substances -

  Supply of limniting growth substances in the medium controls the growth rate of fastidious organisms.
The organisms can be made to grow synchronously by supplying them with fresh supply of this limiting growth substance in the nutrient medium after they have entered stationary phase of growth. b.

In stationary growth phase, bacteria stop growth because of non availability of limiting growth substance. Hence, all cells may occur in same state of physiology in this growth phase. The transfer of these cells to a fresh medium containing limiting growth substance will allow them to divide at a time and hence will give synchronous growth.

In stationary growth phase, bacteria stop growth because of non availability of limiting growth substance. Hence, all cells may occur in same state of physiology in this growth phase. The transfer of these cells to a fresh medium containing limiting growth substance will allow them to divide at a time and hence will give synchronous growth.

Diauxic Growth of Bacteria

  Diauxic Growth is observed much often that when bacteria are allowed to grow in the medium contalning two different types of carbon/energy sources, they show a characteristic diauxic growth.

The diauxic growth is characterized by the occurrence of two lag phases which separate the log phase in two parts.

Diauxic growth of bacteria, when grown on medium, containing two different carbon sources.

Biochemical reasoning of diauxy

The phenomenon was first observed by Monod during his studies of E.coli grown on medium containing glucose and lactose as the sources of carbon and energy.
  The first lag phase was observed due to the adaptation of organisms to the new medium conditions. This was followed by utilization of glucose.
  Second lag phase was observed when glucose got exhausted from the medium and organisms become prepared to utilize lactose.

  Initially organisms start utilizing glucose, a most readily utiltzable source of carbon and energy and show first lag and log phase. But when, all glucose is consumed by the organisms, the culture again enters a second lag phase during which it induces formation of enzymes required for lactose utilization, after which It again enters second log phase.

  The ability of organisms to use only glucose and not lactose initially is due to the phenomenon of catabolite repression.
  Catabolite repression is the phenomenon where the catabolite of a pathway represses synthesis of an enzyme.

E.coli possesses ability to degrade both glucose and lactose and obtain ATP from them. However, enzymes for glucose utilization are constitutive, whereas those for lactose degradation (β galactosidase) are inducible and are produced only in presence of lactose.
  Synthesis of enzymes for lactose degradation is controlled by phenomenon of catabolite repression.

Basis of catabolite repression

  ATP is a catabolite, produced as a result of degradation of sugars. High concentration of ATP represses synthesis of enzymes required for lactose degradation. Thus, when glucose is already present in the medium, organisms will start degrading It and produce ATP.
  This ATP in turn will repress formation of enzymes for lactose degradation. Hence organisms will not be able to degrade lactose Initially.
  However, when all glucose is consumed by organisms again ATP level in the cell will drop. This will make condition favorable for   β galactosidase synthesis and organisms will become capable of utilizing lactose.
   Thus, during second lag phase, organisms will become prepared to utilize lactose and hence will be able to enter another log phase. 

Continuous Culture - Definition, Types of method, instrumentation and applications

The culture in which organisms can grow continuously for indefinite period of time is called continuous culture

  Generally in batch culture, the organisms cannot grow for long. This is due to the development of unfavourably conditions In the growth medium, as a result of depletion of nutrients, accumulation of toxic metabolites, Increased cell density etc.

  However if the environment conditions are maintained favorable continuous culture is possible. Based on this principle, continuous culture is obtained. Continuous culture system is also referred to as pen culture system.

Principle of obtaining continuous culture

  If the environment conditions, optimal for growth can be maintained in the culture medium, continuous growth is possible. In the growing culture, this can be achieved by
  1. Continuous removal of toxic waste metabolites from medium produced during the growth.
  2. Continuous feeding of the growth medium with fresh nutrients at a rate, they are being utilized.
  3. Removal of excess of cells such that the desirable cell density is maintained in the growth medium.
The methods used for obtaining continuous culture are based on these principles.

Methods of obtaining continuous culture

Two methods are used for obtaining continuous culture.    
  1). Turbidostat
  2). Chemostat

Turbidostat :

  In turbidostat, the basic approach for obtaining continuous culture is by maintaining uniform turbidity or cell density in the growth tube.  
   The turbidity of the growth medium is measured by an optical device. The turbidity in the growth medium is maintained by replacing spent medium with fresh medium.

Method of Turbidostat

  The device consists of a growth tube, facility for removal of spent medium by an overflow device through a siphon,  reservoir of fresh medium and optical sensing device for measurement of turbidity in growth tube.

  In the growth tube, when the turbidity exceeds beyond a set limit, an electronic signal is obtained from optical sensing device which measures turbidity.

  This signal allows a flow of fresh medium into the growth tube. Simultaneously, the spent medium, along with growth, is allowed to leave the growth tube by overflow device through a siphon.

  This continues till the culture gets diluted and turbidity drops to a set low level. Thus, In the turbidostat, number of cells in the growth tube controls the flow rate. The rate of growth in the culture adjusts to this flow rate.

Apparatus for continuous cultivation of Bacteria. The system consists of (a) Fresh culture medium reservoir, (b) Flow rate regulator, (c) growth tube with over flow device to remove excess cell mass and spent medium and (d) spent culture bottle

Chemostat :

 In Chemostat, the basic approach for obtaining continuous culture is by monitoring uniform chemical composition in the growth medium. The method involves replacement of the spent medium by fresh one at a rate; the nutrients are being consumed from the growth medium.

Method of Chemostat

  The device and assembly for chemostat is almost similar to removal of spent medium by a siphon device and reservoir for the fresh medium.
  In chemostat, fresh medium is allowed to enter the growth tube at a specified flow rate. The spent medium is removed from the growth tube by an over flow device using siphon. Thus the device allows 
  1. Continuous removal of toxic waste metabolites and excess of cell mass from the growth tube as well as
  2. Supply of the fresh nutrients at a rate, they are being consumed.
  Thus, uniform chemical environment for growth of organisms is maintained continuously in the growth tube, which allows continuous cultivation. The fresh medium contains some nutrients ingrowth limiting concentration (usually vitamin or growth factor).
  Their concentration decides the growth rate of the culture in chemostat and the growth rate adjusts to the flow rate of fresh medium.

Growth rate and flow rate of fresh medium in the chemostat

  In continuous culture, the growth rate of the organisms adjusts to the flow rate of the fresh medium. For example, In chemostat, rate of entry of fresh medium is changed, growth rate adjusts to match the new rate of loss of cells through overflow and maintain new level of cell density. This can be explained as under.

  1. The rate of growth of organisms in continuous culture is always limited by the concentration of nutrients. Initially the organisms grow at a maximum growth rate.
  2. As the culture density increases, the rate of utilization of nutrients will increase. This continues till level of one of the nutrient depleted. The depleting of this nutrient will limit the growth rate.
  3. As long as the growth rate exceeds the rate of loss ol cells by siphon, cell density will continue to increase In the growth tube and concentration of nutrient in the growth tube will decrease.
  4. As a consequence to this, the growth rate decreases till the rate of Increase in cells is equal to the rate of loss of cells and a balance between the growth rate and loss of cells is achieved. Thus. the continuous culture system is a self regulatory system and it can be decided as - (Rate of production of cells  through growth) = (Rate of loss of cell through overflow)

However, if the flow rate for addition of fresh nutrients exceeds far beyond the rate of new cell formation. It will result Into washing out of the culture from the growth tube.

Advantages of continuous culture system:

  1. Isolation of an organism capable of growth on a simpler medium make basic of cheaper commercial process and the process should be more resistant to contamination.
  2. Use of high temperature during isolation results in the isolation of thermophilic strain minimising cooling problems in the subsequent process.

Limitation of continuous culture system:

Use of continuous enrichment process may lead to washout of the inoculum before an adapted culture is established.

Application of continuous culture system

The continuous culture system has two Important features:
  1. Cells in culture always occur in exponential phase of growth
  2. Cells are allowed to grow continuously at extremely low concentration of substrate or nutrients.
This allows studies on the regulation of metabolism of limiting growth substance.
  Continuous culture systems has also a potential application in the fermentation Industries, which allows processing of large bulk of medium by installation of relatively smaller capacities of reservoir for fresh medium, fermentation vessel and product recovery plants.

Transformation of genetic recombination in bacteria

Genetic analysis of recombination in bacteria

Defination of recombination :
Bacterial recombination is characterized by DNA Transfer from one organism called donor to another organism as recipient.

Genetic recombination process in Bacteria occurs in 3 main ways -
1) Transformation,
2) Conjugation,
3) Transduction.

Bacteria (Prokaryotes) do horizontal gene transfer at the time of recombination.
Gene transfer has 2 machanism
1) Horizontal gene transfer - in this machanism gene transfer between two independent organisms. E.g. conjugation, transduction and transformation.
2) Vertical gene transfer - in this machanism gene transfers parent to offspring. E.g. sexual reproduction by Binary Fission in Prokaryotes.

The fragment of DNA that is Transferred during horizontal gene transfer from donor to recipient is referred as Exogenote.
The recipient bacterial cell's own genetic material is called Endogenote.

A bacterial cell that has received an exogenote is initially deploid for part of its genome is called merozygote(partially diploid).

Recombination generally requires that the two DNA molecules be homologous (very similar/ not identical). If they are non homologous due to origin from different species than recombination is unlikely.

Exogenote are often degraded rapidly so that there is a race between degradation of exogenote and recombination.

Transformation :

Transformation is the process of uptake a naked DNA molecule or fragment from the medium by a cell and it's incorporation into chromosome of recipient.

Introduction :

- Transformation was first gene transfer mechanism discovered in bacteria by Fred and Grifth in 1928.
- Transformation may be natural or artificial.
Natural transformation is a rare event and occur both in gram positive and gram negative bacteria.

Competence -
- The ability of recipient bacterium to take up free DNA and become transformed is called competence. It is an inheritable characteristic.
- Competent bacteria that take up DNA from environment at high frequency encode proteins called competence factor.
- Competence factor facilitate binding of DNA fragment to cell surface and uptake of DNA in cytoplasm. e.g. competent bacteria produces competent protein which binds to foreign DNA on cell surface and then uptake by cytoplasm.

Process of Transformation :

Transduction in bacteria
1). Cell free transforming DNA in the bacterium immediate environment can be naturally released when cells die and lysis.

2). Once DNA comes in contact with competent bacteria, linear dsDNA converts to single stranded DNA and one strand is degraded.
3). Single stranded DNA (exogenote) is unstable and degraded unless they are integrated into endogenote by homologous recombination.

Genetic analysis of transformation :
- Transformation is used for gene mapping.
- Genetic analysis of Transfer together is they are near enough to be carried on same DNA fragment.
- Frequency of transformation is inversely proportional to the distance between two genes.

Transduction of Bacteria

Transduction is a Horizontal gene transfer event in which bacteriophages function as a Vactor/Vehical to transfer DNA from donor bacteria to recipient bacteria.

Transfer of bacterial genes by bacteriophagephage (Transduction) was discovered by Zinder & Lederberg in 1951 in bacterium Salmonella-  typhimurium.

Transduction is of two types :
1) Generalized Transduction
2) Specialized Transduction.

The bacteriophages containing the bacterial DNA of donor is called transducing phage.

Bacteriophage :

Bacteriophage is a virus that infect and replicate within a bacterium.
Bacteriophages has two types :
1) Virulent bacteriophage
2) Temperate bacteriophage.

• Virulent bacteriophage follow lytic cycle and they are capable of causing bacterial infection and eventually destruction and death of bacterial cell. E.g. T4 phage
Temperate bacteriophage does not cause disruptive infection, instead phage DNA incorporated into bacterial DNA and replicate lysogenic cycle and after some time become virulent cause lysis of bacterial cell. E.g. λ phage.

* Generalized Transduction :

In Generalized Transduction a DNA fragment is transferred from one bacterium to another by a lytic phage which carry donor bacterial DNA due to an error.
Virulent phage act as vehicle for transduction.

Generalized TransductionSteps

Steps of Generalized Transduction :

1). A lytic bacteriophage infect a susceptible bacterium.
2) when phage genome enters the bacterium it cause degradation of bacterial host DNA into fragments.
3). Phage genome replicate using host replication machinary and synthesize enzymes and coat protein.
4). During maturation and packaging of virus particles, few phage heads may envelope fragments of bacterial DNA by error that is double stranded. So bacterial (host) DNA is present in the transducing phage.
5). Transducing phage carrying bacterial(host) DNA or donor will infect another cell and transfer the donor DNA to the recipient bacterial cell.
6). When bacterial DNA of donor is introduced into the bacterial Chromosome of host and transfer several bacterial gene at one time forming recombinant DNA.
7). Bacteria(recipient) multiply with new genetic material.

Due to the phage gene are very small only genes that are located close together will be transduced together
Typical example of generalized transduction include P1 in E.coli and P22 in Salmonella sp.

Generalized transduction may be abortive or complete

Aborative transduction :

- The transient expression of one or more donor without formation of recombinant progeny
- The donor DNA fragment exogenate does not integrate with endogenate and also not replicate.
- Only one bacterium contain exogenate and other bacterium is like parent among the progeny.

Complete transduction :

- Production of stable recipient recombinants that inherits donor genes and retain the ability to express them.

The frequency of abortive transduction is greater than complete transduction.

* Specialised transduction :

- A DNA fragment is transferred from one bacterium to another by a temperate bacteriophage (lysogenic), Which carry donor DNA along with phage genome to an error.
- In specialised transduction phage insert donor genome at specific site and perform site specific recombination.
- Specialised transduction only occurs when lysogenic (temperate) infected donor bacteria enter into lytic cycle and release phage progeny.

Specialised Transduction

- The only bacterial genes that can be transduced are very near to the site at which pro phage is integrated.
E.g. the only site at which λ phage integrated is between genes for galactose fragmentation (gal gene) and biotin (bio)synthesis gene. So at the time of abnormal integration of prophase only gal or bio gene could be transduced.
Specialised transduction always occur by aberrant excision of λ lysogen

Steps for specialised transduction :

- A temperate phase infect a susceptible donor bacterium and it's DNA into its genome, it called pro-phage.
- When bacteriophage enters lytic cycle, occasionally during spontaneous induction a small piece of donor bacterial DNA is picked up during detachment as a part of phage genome.
- When the λ Phage dettaches  from bacterial DNA it contains some non essential parts of bacterial DNA which is called defective phage.

λ d-gel defective phage lack phage gene required to grow in lytic cycle but pro phase that still contain head/tail gene help in its packing for lysis.
  When this defective phage infects recipient cell(bacteria), due to lack of genes it could not transduced because lack of essential genes.
  So specialised transduction occur if a recipient bacteria is double infected with a wild type λ phase or λ d-gal. wild type phage supply the functions missing in defective phase and progeny will contain about numbers of both type.

1) Low frequency transducing lysate -

It forms when initial abberent excision of lysogen yield less than 1 phage per 1,000,000 wild type.

2) High frequency transducing lysate -

It forms by combination of λ d-gal and λ phase coinfect and it results into dilysogen.

Co transduction :

During transduction, only a specific size of DNA segment can be packaged by bacteriophagephage. If two genes are close along the chromosome, a phase may package a single piece of chromosome that carries both genes and transfer that piece to another bacterium. This is called co-transduction.

Frequency of Co-transduction is inversely proportional to distance between 2 genes.
Formula : C=(1-d/L)³ 
C= Frequency,
d= distance in minutes
L= size of transducing fragment in minutes.

Enzymes and Proteins Involved in the DNA Replication

Enzymes and proteins involved in DNA replication 

  A number of enzymes and proteins are associated with the replication fork to help in the initiation and continuation of DNA synthesis, Most prominently, DNA polymerase synthesizes the new strands by adding nucleotides that complement each (template) strand.
   DNA replication occurs during the S-phase of interphase. At the replication fork, many replication enzymes assemble on the DNA into a complex molecular machine called the replisome. The following is a list of major DNA replication enzymes that participate in the replisome.

1) DNA Helicase :

  • Helicase enzyme opens up the DNA double helix by breaking hydrogen bond between two strands of DNA and provide single template strand.
  • DNA-B is a primary replicative Helicase it binds and move on lagging strand in 5' to 3' direction unwinding the duplex as it goes.
  • Helicase requires ATP as energy source

2) Single Stranded Binding Proteins (SSB proteins) :

  • SSB proteins binds to both seperated single stranded DNA and prevent the DNA double helix from re-annealing after helicase unwinds.
  • SSB proteins are maintaining the strand seperation and facilitating the synthesis of the nascent strand.

3). Topoisomerase :

  • DNA Topoisomerase is a nuclease enzyme that break a phosphodiester bond in a DNA strand.
  • The function of Topoisomerase is relaxes the DNA from its super coiled nature.

4).DNA Gyrase :

  • This enzyme is used to make sure the double stranded areas out side of the replication fork do not supercoil, DNA Gyrase is one type of topoisomerase.

5). Primase :

  • Primase provides a starting point of RNA (or DNA) for DNA polymerase to begin synthesise of the new DNA strand.
  • Because DNA polymerase requires free 3'-OH group for bind to DNA for starting replication.

6) DNA Polymerase :

  • DNA dependent DNA polymerase enzyme that can synthesise a new strand on a DNA tamplate.
  • DNA polymerase has different types in Prokaryotes and Eukaryotes.

a). Prokaryotic DNA polymerase -

Prokaryotes has 3 types of DNA polymerase, these are 
  • DNA pol-I, 
  • DNA pol-II and 
  • DNA pol-III.

DNA polymerase I -

  • It is made up of one subunits. It has 3' to 5' and 5' to 3' exonuclease activity.
  • Function - DNA repair, Gap filling and synthesis of new lagging strand.

DNA polymerase II -

  • It is made up of 7 subunits. It has only 3' to 5' exonuclease activity.
  • Function - DNA repair and DNA proof reading.

DNA polymerase III -

  • It is made up of at least 10 subunits. It has 3' to 5' exonuclease activity.
  • Function - This is the main replication enzyme in Prokaryotes.

b) Eukaryotic DNA polymerase -

Eukaryotes has 5 types of DNA polymerase which are DNA polymerase α, β, γ, δ and ε.

DNA polymerase α -

  • It has no any exonuclease activity.
  • Function - DNA replication in the  nucleus.

DNA polymerase β -

  • It has no any exonuclease activity.
  • Function - DNA replication and base excision repair.

DNA polymerase γ -

  • It has 3' to 5'  exonuclease activity.
  • Function - DNA replication in Mitochondria.

DNA polymerase δ -

  • It has 3' to 5'  exonuclease activity.
  • Function - Synthesis of lagging strand during DNA replication.

DNA polymerase ε -

  • It has 3' to 5'  exonuclease activity.
  • Function - Synthesis of leading strand during DNA replication.

7) Beta Clamp Proteins :

  • Beta clamps are the protein which prevents elongating DNA polymerase from dissociating from the DNA parent strand.
  • It helps hold the DNA polymerase in place on the DNA.

8) DNA Ligase :

  • DNA Ligase Catalyse the joining of ends of two DNA chains by forming phosphodiester bond between 3'-OH group at one end of DNA strand and  and 5'-Phosphate group at the end of other DNA strand.
  • DNA ligase joins the Okazaki fragments of two lagging strand.

9) Telomerase :

  • Lengthens the telomeric DNA by adding repetitive nucleotide sequence to the ends of eukaryotic chromosomes. 
  • This allows germ cells and stem cells to avoid the Hayflick limit on cell division.

Conjugation in Bacteria

Conjugation is a process in which the genetic information (DNA) is transferred to, recipient bacteria by direct contact between them through establishment of cytoplasmic bridge.
In this process Donor bacteria posses fertility plasmids
(F plasmid )or sex plasmids that are self transmissible. They exist in both gram positive and gram negative bacteria.
F plasmids are transferred with in strains of same species.
A recipient cell that has received DNA as a result of conjugation is called trans-conjugant or ex- conjugant.

Promiscuous Plasmid
Generally plasmid transfer is intraspecific, but many plasmid transfer DNA between unrelated species, called promiscuous plasmid.

Types of Conjugation

• F+– F Conjugation
• Hfr – F Conjugation
• F' – F Conjugation

F+ –  F conjugation :

F plasmid (F-factor) in E.coli
E.coli strains with extra chromosomal F plasmid are called F+ and act as donors.
Strains that lack F plasmids are F−, act as recipient.
F− plasmid spread by infection among genetically compatible populations of bacteria.

F Plasmid :
F plasmid Consist of 25 transfer genes. That function for expression of sex pili, synthesise & transfer of DNA interfere with F+ ability to serve as recipient

These transfer gene has two type
1). (Mpf) Mating pair formation gene.
2). (Dtr) DNA transfer gene (plasmid).

Steps of F+ and F Conjugation :
1) Formation of 1 to 3 sex pili of F+ bacteria bind to specific outer membrane protein on recipient bacteria.
2) Intracellular cytoplasmic bridge is formed.
3) Relaxase Protein makes a single-strand cut at ori T(Origin of Transfer) site in Plasmid.
4) Transfer of one strand from F+ cell to F− cell, simultaneously F− plasmid replicated in F+ cell.
5) Completion of transfer - Transferred strand in converted to double strand in recipient and both the exconjugant bacteria are becomes F+.

Hfr –  F Conjugation :

Hfr : (High frequency recombination strains) Hfr strains are strains which has F plasmid attached with it's Chromosomes.
An E.coli strain with integrated F Plasmids that retain its ability to function as donor in conjugal mutation.
Integrated F plasmid transfer genes with high efficiency.

Steps of Hfr - F− conjugation :
1) Transfer or single stranded DNA from Hfr donor to F- recipient start at ori T.
2) A part of F plasmid is transferred lost after complete bacterial genome.
3) complete transfer takes 100 min and conjugation takes before this process ends.
Thus complete F plasmid is not transferred and exconjugant remain Hfr and F-.
After donor Chromosome enter recipient it may be degraded or incorporated by Homologous recombination.

F' - F Conjugation :

F' Plasmid : Integrated F plasmids in Hfr strains can sometimes excised from bacterial Chromosomes. In rare cases excision occurs by recombinations involving insertion sequences or other genes on bacterial Chromosome to become integrated into hybrid F plasmid.
Due to error in excision of F− Plasmin from Hfr the chromosomal gene of donor is picked up.
This gene is transferred to recipient and it become partially diploid merozygote. As it has 2 sets of genes carried by plasmid and need not be incorporated in genome of bacteria.

Genetic Code : Definition, Nature & Characteristics, genetic code table and genetic bias

  Central dogma of molecular biology describes the two step process by which information in genes flow into proteins.
  DNA ➞ RNA ➞ Protein
DNA to RNA by Transcription and RNA to Protein by Translation.

As the language of nucleotide sequence on mRNA is translated to language of an amino acid sequence.
  Translation requires a genetic code through which information contained in nucleic acid is expressed in specific sequence of amino acid and this collection of codons as we known as Genetic codon.

The letters A,G,T,C correspond to nucleotides in DNA they are organised into codons.
For 20 Amino acid (standard) requires at least 20 codons.

  • If 1 nucleotide act as a codon there will be 4 combinations.
  • If 2 nucleotide act as a codon there will be (4)² = 16 combination.
  • If 3 nucleotide act as a codon there will be (4)³ = 64 combination.

George Gamow postulated that 3 letter codon must be employed to encode 20 standard amino acid used by living cells for protein synthesis.

Definition :

Genetic code is a set of rules (defined by 64 triplet codons) by which information encoded in genetic material (DNA or mRNA sequences) is translocated into protein by living cells.

  • Codon is a set of 3 letters combination of nucleotide bases(A,G,C,T).
  • Genetic code defines how codons specify which amino acid will be added next during protein synthesis.

Types of Codons :

There are two type of Codons

  1. Sense codons : the codon that codes for amino acid.
  2. Signal codons : Those codons that code for signal during protein synthesis are called signal codons.

Signal codons has two types

a). Initiation Codon :

   This codon also called start codon. AUG is the initiation codon. It is the first codon of mRNA transcript that is translated and codes for first amino acid in all proteins.
  • AUG codon - it code for methionine in eukaryotes, formyl methionine in prokaryotes and ubiqutinated lysine in protists, bacteria, mitochondria and plastids.
  • The most common start codon is AUG. In few mRNA GUG and UUG also act as initiation codon as AUG codon is unavailable (arches & bacteria).
  • In E.coli AUG and UUG is read as formyl-methionine if it is used as start codon, when used with in coding region it is read as methionine.
  •  Similarly GUG as formyl-methionine, if act as start codon. GUG act as valine if read in between coding region.
  • In eukaryotes initiation codon is located between kozak sequence.
  • In prokaryotes initiation codon is located after 3-10 nucleotides of shine-Dalgarno sequence.

b). Termination codons :

  This codon also called chain termination codon or stop codon or non sense codon.
Out of 64 codons 3 do not code for any amino acids and terminate translation. At stop codon ribosome pauses and fall off the mRNA
Termination codons are -
UAA - Orche codon
UAG - Amber codon
UGA - Opal codon

Anticodon -

  • The base sequence of tRNA which pairs with codon of mRNA during translation is called anticodon
  • Codons could be present in both DNA and RNA but anti codon is always present in tRNA.
  • Codon written in 5' ➞ 3' direction while anticodon written in 3' ➞ 5' direction.
  • Codons are arranged in sequence in nucleic acid strand while anticodons are present discretely in cells with amino acid attached or not.
  • Codon defines which anticodons to come next with an amino acid to create the protein strand.
  • Anticodons helps in bringing a particular amino acid at its proper position during translation.
  • Anticodons of some tRNA molecules have to pair with more than one codon.

Genetic code table :

Ala - Alanine 
Arg - Arginine 
Asn - Asparagine 
Asp - Aspartic acid  
Cys - Cysteine 
Gln -Glutamine  
Glu - Glutamic acid
Gly - Glycine 
His - Histidine 
Ile - Isoleucine 
Leu - Leucine 
Lys - Lysine 
Met - Methionine 
Phe - Phenylalanine 
Pro - Proline 
Ser - Serine 
Thr - Threonine 
Trp - Typtophan 
Tyr - Tyrosine 
Val - Valine

Characteristics & Nature of genetic code :

1) Triplet code -

  • Genetic code is triplet code consisting of 64 codons. Codon is a set of 3-latter combination of nucleotide.

2) comma less or continuous translation -

  • The gene is transcribed and translated from a fixed starting point to fixed stop point. Punctuations are not present between the codons.

3) Non overlapping -

  • Genetic code is non-overlapping, any single ribonucleoprotein at specific location in mRNA is part of only one triplet codon.
  • Codon is read as a continuous sequence of bases, taken 3 at a time called as reading frame.

4) Universal

  •  Means that the same code is used for all life forms. This is not strictly true, there is few context dependent codons also. in this context the genetic code is nearly universal
  • E.g. UGA is a stop codon while it codes tryptophan in mycoplasma.
  • CUG codes for leucine while it codes threonine in mytochondria in yeasts.
  • UAA,UAG is a stop codon while it codes glycine in paramecium.

5) Unambiguous -

  • The genetic code is non-ambiguous. Thus one codon can not specify more than one amino acid.

6) Degenerate

  •  A given amino acid can be specified by more than one triplet codon. This is the code for 18 of the different codons for a given amino acid are said to be synonymous codons.
  • e.g. UUU and UUC are synonyms for phenylalanine. UCU, UCC, UCA, UCG, AGU, and AGC are synonyms for serine.

7) Polarity of genetic code -

  •   The code has definate direction for reading of message which is referred to as polarity 5' ➞ 3' direction
  • Reading of message from left to right & right to left will specify for different amino acids.
  • e.g. UUG ➞ leucine GUU ➞ valine.

Codon Bias :

  • Codon bias refers to the fact that not all codons are used equally in the genes of a particular organism.
  • Codon bias is the probability that a given codon will used to code for an amino acid over a different code which codes for the same amino acid.
  • e.g. Out of the four valine codons(GTG, GTA, GUG, GUA), human genes use GTG four times more frequently than GTA.