Gram Positive vs Gram Negative: Key Differences

 Bacteria have cell walls made up of polysaccharides. That give them strength and rigidity. This is important since bacteria often experience variations in osmotic pressure due to the different solutions. They encounter and it is their cell walls which prevent them from shrinking or swelling. As a reminder as Osmosis is the process by which solvent molecules pass through a semipermeable membrane from a less concentrated solution to a more concentrated one equalising the concentration on either side of the membrane.

Nearly all bacterial cell walls have a peptide polysaccharide layer called peptidoglycan also known as moraine. Peptidoglycan is a polymer made up of sugars and amino acids, which forms a kind of mesh.

Bacteria can be classified based on their reaction to the Gram stain. Which identifies them as gram positive or gram-negative based on the chemical and physical properties of their cell walls.

Gram positive bacteria have a thick cell wall which consists of up to around 30 layers of peptidoglycan. this cell wall surrounds a mono derm which is a single plasma membrane.

Gram-negative bacteria have a much thinner cell wall consisting of a single layer of peptidoglycan. this layer of peptidoglycan is sandwiched between two lipid bilayer membranes called dye terms.

we can differentiate between gram-positive and gram-negative bacteria by dyeing them with crystal violet and then washing them with a decolorizing solution. Then a counter stain is added for example safranine or fruit scene.

gram-positive bacteria will retain the crystal violet dye and remain purple. while the gram negative bacteria will be stained pink.

"note" that gram positive bacteria also pick up the pink color of the counter stain. However this is not visible when they are dyed with the darker purple color of the crystal violet stain. the reason for these staining differences is due to differences in cell wall structure which is the chief difference between gram-positive and gram-negative bacteria.

the Gram stain detects peptidoglycan and since gram positive bacteria have a thick multi-layered peptidoglycan layer they retain the crystal violet dye. gram-negative bacteria do not retain the dye for two reasons.
1) They have an outer membrane getting in the way of the crystal violet and
2) They lack peptidoglycan to retain the stain.

Although both gram positive and gram-negative bacteria can be pathogenic. gram-negative bacteria are more resistant to antibodies because of their impenetrable cell wall. unfortunately these bacteria also develop resistance more quickly. Not all bacteria can be reliably classified through gram staining.

Acid-fast bacteria and gram variable bacteria for example do not respond to gram staining. acid-fast bacteria are bacteria whose cell walls retain stains particularly well although they aren't closely related to gram positive bacteria. They can't appear purple after the Gram stain test. Gram variable bacteria show a mix of pink and purple cells when stained. 

Mendelian genetics & mandelian law of inheritance

Mendelian Genetics : 
 
  Father of Genetics, Gregor Johann Mendel was born in a farmer family near Brunn in Austria in 1822. Gregor Johann Mendel conducted his historic experiments with garden pea (Pisum sativum) in the monastery garden for about nine years i.e. 1856-1864 and published his results in a less known journal - The Annual  proceedings of the Natural History Society of Brunn in 1865.  

  He chose garden pea as an experimental organism because many varieties were available that bred true for qualitative traits and their pollination could be manipulated. Mendel investigated seven variable characteristics in pea plants were - 

• seed texture (round v/s 
  wrinkled) 
• seed color (yellow vis green) 
• flower color (white v/s  
  purple) 
• growth habit (tall v/s dwarf) 
• pod shape (pinched or        
  inflated)   
• pod color (green v/s yellow) 
• flower position (axial or    
  terminal)

  Peas are normally self-pollinated plant as the stamens and carpels are enclosed within the petals. By removing the stamens from unripe flowers. Mendel could brush pollen from another variety on the carpels when they ripened. 

 After experiments, Mendel discovered that, when he crossed purebred white flower and purple flower pea plants (the parental or P generation), the result was not a blend. Rather than being a mix of the two, the offspring which is known as the F1 generation, was purple-flowered. When Mendel self-fertilized the F1 generation plants, he obtained a purple flower to white flower ratio in the F2 generation of 3 to 1. Punnett square were drawn to explain results of this cross as showed below.
  He then comes up with the idea of heredity units, which he called "factors". Mendel found that there are alternative forms of factors called genes that account for variations in inherited characteristics. For example, the gene for flower colour in pea plants exists in two forms, one for purple and the other for white. The alternative forms are now called alleles. For each biological trait, an organism inherits two alleles, one from each parent. These alleles may be the same or different from other. 

  An organism that has two identical alleles for a gene is said to be homozygous organisms for that gene and is called a homozygote. An organism that has different alleles for a gene is said to be heterozygous organisms for that gene and is called a heterozygote.

  Mendel hypothesized that pairs of alleles separate randomly or segregate from each other during the production of gametes i.e. egg and sperm. Because allele pairs separate during gamete production, a sperm or egg carries only one allele for each inherited trait. When at the time of fertilization sperm and egg unite, each contributes its allele, restoring the paired condition in the offspring. This is called the Law of Segregation. Mendel also found that during gamete formation, each pair of alleles segregates independently of the other pairs of alleles. This is known as the Law of Independent Assortment. 

  The genotype of an individual is made up of the many alleles. Phenotype viz. Physical appearance of individual is determined by its alleles as well as by its environment. The presence of an allele does not mean that the trait will be expressed in the individual that possesses it. If the two alleles of an inherited pair differ i.e. the heterozygous condition, then one determines the organism's appearance and is called the dominant allele; the other has no noticeable efect on the organism's appearance and is called the recessive allele. Thus, in the example above the dominant purple flower allele will hide the phenotypic effects of the recessive white flower allele. This is known as the Law of Dominance but it is not a transmission law : it concerns the expression of the genotype. Dominant alleles represents with upper case letters whereas recessive alleles represents with lowercase letters are used to represent.


Mendelian law of inheritance :       
     After experiments on garden pea (Pisum sativum) over seven years during l856-1864, he gave important laws of heredity, viz.,    
  (1) Law of Segregation, 
  (2) Law of Independent 
        Assortment, 
  (3) Law of Dominance.
These are briefly presented below. 

(1) Law of Segregation :

   According to this law, alleles segregate or separate from each other during gamete formation and pass on to different gametes in equal number. In other words, when alleles for two contrasting characters come together in a hybrid, they do not blend, contaminate or affect cach other while together. The different genes separate from each other in a pure form, pass on to different gametes formed by a hybrid and then go to different individuals in the offspring of the hybrid.

Thus main features of this law re as follows

• When both a dominant allele and a recessive allele of a gene come together in a hybrid after crossing between two plants having contrasting characters, they do not mix or blend together. 

• Both the allele separate together in pure form without affecting each other. Due to this reason, law of segregation is also known as law of purity of gametes. 

• These alleles separate into different gametes in equal number. Each gamete has only one type of allele (either A or a). 

• Separation of two alleles of a gene during gamete formation takes place usually due to the separation of homologous chromosomes during anaphase-I of meiosis, because alleles are located in the chromosomes.

• With complete dominance, segregation leads to phenotypic ratio of 3:1 in F2 for characters governed by single gene, and 9:3:3:1 ratio for characters controlled by two genes. 

• If crossing over does not take place, segregation of genes takes place during anaphase-I. If crossing over occurs, segregation of genes will take place during anaphase-II.

* Example : 
   When we make a cross between red (RR) and white (rr) flowered plants, we get red colour of flower in F1. In the F1 both the alleles R and r remain together without blending or mixing with each other, though only the effect of dominant allele is visible. In F2, allele for red flower colour and white flower colour segregate during gamete formation and pass on to the gametes in equal number, Thus two types of gametes, viz., R and r are formed. Each gamete has either R or r allele. When the F1 is self-pollinated. individuals with three genotypes, viz., RR, Rr and rr are obtained in F2 Here RR and Rr are all red and only rr individuals are white. Thus a phenotypic ratio of 3 red: 1 white is obtained. The overall mechanism is represented below.
  When selfed  seeds of RR were grown in F3, they all produced all the true breding individuals for red flower colour. The Rr individuals showed segregation in F3 similar to segregation in F2 generation. Individuals with rr genotypes were found true breeding for white flower colour when their selfed seeds were raised in F3 generation. 

(2). Law of Independen   
      Assortment :

 This is the second law of inheritance discovered by Mendel. This law states that when two pairs of gene enter in F1 combination, both of them have their independent dominant effect. At the time of gamete formation these genes are segregate, but the assortment occurs randomly and quite freely.

Main features of this law are given below : 

• This law explains simultaneous inheritance of two plant characters.
• In F1 when two genes controlling two different characters, come together, each gene exhibits independent dominant behaviour without affecting or modifying the effect of other gene. 
• During gamete formation, these gene pairs segregate independently.
• The alleles of one gene can freely combine with the alleles of another gene. Thus each allele of one gene has an equal chance to combine with each allele of another gene.
• Each of the two gene pairs when considered separately, exhibits typical 3:1 segregation ratio in F2 generation. This is a typical monohybrid segregation ratio. 
• New gene combinations are formed by random or free assortment of alleles of two genes.

* Example : 
  When plants of garden pea with yellow round seeds are crossed with plants having green wrinkled seeds, we get yellow round seeds in F1. Thus yellow colour of seed exhibits dominance over green and round seeds shape over wrinkled independently. The F1 produces four types of gametes, viz., yellow round (YR), yellow wrinkled (Yr), green round (yR), and green wrinkled (yr). Selfing of F1 gives rise to all above four types of individuals in 9:3:3:1 ratio.

  The behaviour of all these genotypes was studied in F3 generation. Out of nine yellow round individuals only one (YYRR) was found true breeding in F3 generation. The other eight individuals showed segregation of various types.
  Similarly, out of 3 yellow wrinkled individuals only one (YYrr) bred true and others segregated in 3 :1 ratio. Same thing happened with green round individuals. The green wrinkled individual was also true breeding as shown in following table.
(3). Law of Dominance :

  According to Law of Dominance, recessive alleles will always be masked by dominant alleles. Therefore, a cross between a homozygous dominant and a homozygous recessive will always express phenotype of the dominant allele, while still having a heterozygous genotype. 

  This Law can be explained easily with the help of a mono hybrid cross experiment. In a cross between two organisms pure for any pair (or pairs) of contrasting traits (characters), the character that appears in the F1 generation is called "dominant" and the one which is suppressed (not expressed) is called "recessive". Each character is controlled by a pair of dissimilar factors. Only one of the characters expresses. The one which expresses in the F, generation is called Dominant. However, the law of dominance is not universally applicable.

* Mono-hybrid cross or Mono-hybrid tests : 

  In a monohybrid cross, the two parents differ through a single character. In this cross Mendel took a tall pea plant and crossed with a dwarf plant. He transferred the pollen grains of tall pea plants and placed them on the stigma of the dwarf pea plant and vice versa. To prevent self pollination he earlier removed all stamens from the flowers of the dwarf plant. Mendel noticed that all the progenies of F1 or first filial generation were tall plants. This outcome gave him the clue to state the Law of dominance.
 
 
  According to this law, from the pair of contrasting characters i.e. tallness and dwarfness one character tallness appeared in the F1 generation and the other character (dwarfness) remained hidden or suppressed. The character which appeared in F1 generation is called dominant and the other character remained hidden is called recessive character. These two contrasting characters are known as allelomorphic characters or allelomorphs or alleles. 

  Mendel now allowed the plants to self pollinate and produce F2 or second filial generation of plants. Among F2 plants he observed tall as well as dwarf plants in the ratio 3: 1. This ratio is monohybrid ratio. The recessive character (dwarfness) which was hidden in the F1 generation appeared in F2 generation. 

  Among the F2 tall plants 1/3rd were pure tall and the remaining 2/3rd were hybrid tall behaving like the plants of F2 generation. The recessive dwarf plant bred true. Thus, the F2 ratio are classified into two categories : Phenotypic ratio (3 tall : 1 dwarf) and Genotypic ratio (1 pure tall : 2 hybrid tall : 1 pure dwarf).

  In the parental generation both tall (TT) and dwarf (tt) plants have similar alleles of gene and hence called homozygous plants.The F1 plants have different alleles of the same gene (Tt) and hence known as heterozygous plants. Mendel represented the dominant factor through the capital letter and recessive factor by means of small letter. Thus, pure tall plant will be TT and dwarf plant will be represented by tt.

* Di-hybrid Cross (OR) Di-hybrid Test : 

  In a di-hybrid cross the parents differ through two characters. Mendel conducted a cross between a true breeding Round Yellow plants (RRYY) with true breeding Wrinkled Green plant (rryy). Round and Wrinkled are the shapes of seed coat whereas Yellow and Green are the colours of the seed coat.

  Mendel observed the seeds of the F1 plants were all Round and Yellow. This showed Round and Yellow were dominant over Wrinkled, Green. In the F2 generation four types of combinations were observed such as Round Yellow, Wrinkled Yellow, Round Green and Wrinkled Green. Thus the above types of offspring's of F2 generation were produced in the ratio of 9: 3: 3: 1. This ratio is called Di-hybrid ratio.
The results can be represented as follows:
  Mendel represented round character of seed by R and wrinkled by r. Similarly, he represented the yellow character by Y and green by y. 

  In his dihybrid cross experiment Mendel observed round and yellow characters are dominant over wrinkled and green so that all the F1 offspring's are round and yellow. In the F2 generation, he noticed an allele (dominant or recessive) of a given character freely combines with either one (dominant or recessive) of the alleles of another character. Hence, a dominant allele of a character combines, not only with the dominant, but also with the recessive allele of another character.









Bacterial Flagella : Definition, Ultra Structure and Mechanism of Movement

 Flagella are the specialized structures of bacteria that are responsible for bacterial motility. (Singular, flagellum, which means whip). Some others can move by gliding. Aquatic bacteria move through their gas-filled vacuoles. 
    Flagellum, is a long helical and thread like loco motor appendage that extends outward from the plasma membrane and cell wall. 

Structure of Flagella 

  • Bacterial flagella are long, slender, delicate and undulating structures. 
  • They measure 5 to 20 mm in length and 20 nm in diameter. The flagella of bacterial genus Bdellovlbrio ar very thick because they are enclosed by sheath of extended plasma membrane, whereas that of Vibrio cholerae has a sheath of lipopolysaccharide.
  • The flagella differ in their shapes. However, each flagellum has definite wavelength and amplitude. They can be coiled, curled normal or wavy. 
  • The flagella are so thin that they cannot be seen directly under the bright-field microscope. It can be observed only after special staining techniques.
  • These techniques make use of mordents like tannic acid that can be deposited on the surface to increase the thickness (Leifson's method and Gray's method are used). However, its ultra structure can be studied under the electron microscope only.

Arrangement of Flagella

  •  Bacterial species show differences in number of flagella and their pattern of arrangement. They can be Monotrichous, Amphytrichous, Lophotrichous or Peritrichous. It helps in identification of bacteria.   
Monotrichous : 
  • Single polar flagellation, for example Pseudomonas sp. (Mono means one and Trichous means hair).
Amphitrichous :
  • Single flagellum at each pole of a cell as seen in genus Spirillum. (Amphi means two). 
Lophotrichous : 
  • A cluster of two or more flagella at one end or both the ends of a bacterium. (Lopho means tuft) as seen in genus Spirillum.
Peritrichous : 
  • Flagella distributed on the entire surface of bacteria as seen in genera of many gram-negative bacteria like Escherichia, Proteus etc. (Peri means all around). 
Mixed flagellation : 
  • Members of genus Vibrio possess two types of flagella - polar sheathed flagella and  peritrichous unsheathed flagella. 

Ultra structure of bacterial Flagella 

 Electron microscopic studies of bacterial flagella reveal Its ultra structure. It consists of three parts.
    A. Basal body,
    B. Hook and,
    C. Filament.

Basal body

  Basal body is also called motor. It is anchored into the cytoplasmic membrane and the cell wall. It consists of a small central rod that passes through a system of rings. Basal body of gram positive bacteria is simple compared to that of gram negative bacteria. 

1]. In gram-negative bacteria (e.g. E. coli) the basal body consists of two pairs of rings, four rings.
  • a). The outer pair of rings includes the L ring and P rings that are associated with lipopolysaccharide and peptidoglycan layers respectively.
  • b). The inner pair of rings includes S and M rings that remain in contact with plasma membrane. 
2]. In gram-positive bacteria, lipopolysaccharide layer is absent. It possesses only one, inner, pair of rings. Outer one attaches to the peptidoglycan  and the inner one attaches to the plasma membrane. It is believed that the S-ring is attached to the cell wall.
  •  The basal body acts as a motor and allows movement of flagella.
  • a).The inner ring is surrounded by a pair of proteins called Mot proteins. Mot proteins drive the flagellar motor such that it causes rotation of filament.
  • b). Another protein called Fli protein also links with the flagellar motor and functions as a motor switch. It can reverse the rotation of flagella in response to the inner signals.
  • c). M-ring and the rod rotate during flagellar movement. S-ring in gram-positive bacteria does not rotate.
  • d). The P and L rings of gram-negative bacteria function as bearings for the rotating rod.

Hook

   It is a slightly wider part of the flagellum that connects basal body and the filament. It consists of a single type of protein. 

Filament 

  It is a helical structure and extends from the cell surface to the tip. It is a hollow, rigid, cylinder, which is constructed of protein subunits called flagellin. Its molecular weight ranges between 30,000 and 60,000 Daltons and varies with the bacterial species. The filament ends with a capping protein.

Mechanism of flagellar movement

  Movement of flagella like a propeller enables the bacteria to move. It spins at a rate of 270 revolutions or higher per second. The basal body functions as a motor. It imparts rotary motion to the flagellum. The required energy comes from proton motive force across the membrane through the mot complex. 
 The nature of bacterial movement depends upon the direction of flagellar rotation. 
  1. In monotrichously flagellated bacteria, the polar flagella move counter clockwise. This will thrust the cell forward and flagella trailing. The monotrichous bacteria stop and tumble randomly by reversing direction of flagellar rotation.
  2. In peritrichously flagellated bacteria, counter clockwise rotation of flagella pushes the cell forward. During this, they bend their hooks to form a rotating bundle. The bundle disrupts, when the flagella rotates clockwise and the cell tumbles. In Proteus sp.cells move in wave like motion, which is evidenced by swarming growth.




What is an Enzyme? Overview of this Amazing Biocatalyst.

Human body is a product of different chemical reactions and processes but what controls these reactions? in 1833 a french chemist, Anselme Payen was the first to discover the vital force that drove these reactions and named it "Enzyme" Enzymes are substances, proteins, or in some cases ribonucleic acid (rRNA) that speed up a biochemical reaction by modifying spesific substances called substrates.

Enzymes are supremely selective in who they binds to and modify, hence their specificity mode of action easier said but how does a tiny enzyme speed up a chemical reaction? the answer to this is simple enzymes perform this critical task by luring a reaction's activation energy that is the amount of energy needed for the reaction to begin enzymes binds to their substrates hold them, in such that chemical bond breaking and bond forming processes take place.

More easily enzymes have a spesific place in them called "active site" where the  substrate binds and real time action takes place.

Active site has a specific size, shape, and chemical behavior. Rendered to it by specific arrangements of amino acids. Thanks to these amino acids an enzyme's active site is unique only to a particular substrate in addition to the active site. Many enzymes consists of a non protein part called cofactor. Cofactors may be cations (positively charged metal ions).

Activators bound temporarily to the active site to activate the enzyme. Organic molecules vitamins or vitamin products coenzymes that joins enzyme substrate complex temporarily, prosthetic groups joins enzyme permanently. Enzyme bound many enzymes only perform their catalytic role when associated a coenzyme an inactivated enzyme, protein along with its coenzyme, non protein makes up a system called "Holoenzyme" to add to the complexity scientists terms this inactivated enzyme as "Apoenzyme" and therefore, the equation becomes Holoenzyme = Apoenzyme + Coenzyme.

Models of enzyme action lock and key hypothesis, this is the simplest model to represent how an enzyme works the substrate. simply fits into the active site to form a reaction intermediate. Just like the key fits in its specific lock the shape isn't changed. here rather the structure of the substrate absolutely compliments the structure of the enzyme like puzzle pieces.

Induced fit hypothesis in this model the enzyme, upon binding its substrate changes shape the matching between an enzyme's active site and the substrate isn't just like two puzzle pieces fitting together rather, the enzyme changes shape and binds to its substrate even more tightly. This fine tuning of the enzyme to fit the substrate is called "Induced Fit".

Types of Enzyme 

 A hydrolase,which catalyses the hydrolysis of a chemical bond, which effectively separates a molecule into two pieces,

 A lyase, which also cleaves covalent bonds, but by means other than hydrolysis.

 An enzyme with the opposite function, a ligase, is an enzyme that joins two molecules together. 

 A transferase is an enzyme that transfers a functional group from one molecule to another.

  An isomerase catalyses a spatial rearrangement of the substrate.

  An oxidoreductase is an enzyme that transfers electrons from one molecule to another.

  

  Environmental effects on enzyme function active sites are very sensitive they sense even the slightest change in the environment and respond accordingly some of the factors that affect the active site and consequently enzyme function include Temperature. The suitable temperature for enzymes to function properly is 37 degrees celcius. increasing or decreasing the temperature above 37 degrees celcius affects chemical bonds in the active site, making them less suited to bind substrates. Higher temperatures denature enzymes pH.

Amino acids present in the active site are acidic or basic fluctuation in pH, can affect these amino acids making it hard for substrates to bind extreme pH values can denature enzymes.

Enzyme concentration increasing
Enzyme concentration will increase the rate of reaction. As more enzymes will be available to bind with the substrates however after a certain concentration any increase will have no effect on the rate of reaction substrate concentration.

Increasing substrate concentration increases the rate of reaction this is because more substrate molecules will be colliding with enzyme molecules. so more product will be fought but again, this effect is valid up to a certain concentration.

Inhibition of enzyme activity some evil substances called "inhibitors" reduce or even stop the activity of enzymes in biochemical reactions they eat a block or distorts the active site. Thus inhibiting the reaction based on this there are two types of inhibitors given below
1) Competitive inhibitors occupy the active site and prevent a substrate molecule from binding to the enzyme.
2) Non competitive inhibitors attach to parts of the enzyme other than the active site to distort the shape of an enzyme.

10 Steps of Glycolysis

 Plants use sunlight as well as carbon dioxide and water to make glucose, and it is all of this glucose, among other biomolecules, that becomes the starting material for metabolic processes in our bodies. 

 This degradation of biomolecules to generate energy that cells can use is called  cellular respiration, or sometimes more specifically, aerobic respiration. looking at glucose as the substrate. 

  Aerobic respiration requires oxygen so any organism that breathes in oxygen from the atmosphere is doing so in order to facilitate aerobic respiration. 

 Glucose,which we can either consume as starch or break off from glycogen stored in the cell, can be converted through metabolic pathways in the presence of oxygen into carbon dioxide, which we breathe out, water, which is most of what we are, and energy, the energy we need to think and move.

  The electron exchanges that occur throughout these metabolic pathways utilise the electron carrier  NAD+ and it's other form, NADH. This is a dinucleotide with an interesting base,  Nicotinamide that can exist either as NAD+, with a  positively charged nitrogen  atom, or if reduced it can become NADH. 

 This transfer, facilitated by an enzyme called  dehydrogenase, helps catalyze the breakdown of glucose. Cellular Respiration happens over three major pathways. There is Glycolysis, the Citric Acid Cycle, and Oxidative Phosphorylation.


Process of Glycolysis

Glycolysis occurs in the Cytoplasm of the cell. This is the process by which glucose molecules are split into two pieces called pyruvate. This first pathway is actually anaerobic, meaning it does not require oxygen, so it is the most evolutionarily ancient metabolic pathway, occurring in even the simplest cells.

  In this pathway, one glucose molecule can yield a net of 2 ATPs. It requires 10 enzymes to happen which catalyse each of the 10 steps, as well as an investment of two ATP molecules in the preparatory phase to get 4 ATPs back over several steps in the payoff phase. 

 Steps of Glycolysis

Step 1: Phosphorylation

 First reaction is called Hexokinase reaction.The enzyme  Hexokinase phosphorylates the oxygen on carbon 6 of the glucose to make Glucose 6-phosphate. 

  The polar phosphate group traps the molecule inside the cell and also reduces the concentration of regular glucose inside the cell, which encourages more glucose to enter by diffusion. This step costs 1 ATP, which provides the necessary phosphate group for the reaction.


Step 2: Isomerisation

Glucose-6-phosphate isomerizes  to become Fructose-6-phosphate, a process which is catalysed by enzyme phosphoglucoisomerase.


Step 3: Second Phosphorylation

  That is another phosphorylation, this time on the carbon 1 hydroxyl which gives us Fructose-1,6-bisphosphate. 

This step is catalysed by  phosphofructokinase 1 and it will cost another ATP.


Step 4: Cleavage 

Now fructose-1,6 bisphosphate molecule is ready to be cleaved into two smaller ones.

 Fructose-bisphosphate aldolase is a lyase enzyme that will split fructose-1,6-bisphosphate into a molecule of Glyceraldehyde-3-phosphate, or GADP, and a molecule of Dihydroxyacetone phosphate or DHAP.


Step 5: Conversion of DHAP into GADP

The DHAP will be converted into another molecule of GADP by the enzyme Triosephosphate - isomerase, which leaves us with two molecules of GADP.

That is the end of the five-step preparatory phase, with two ATPs spent to achieve the two phosphorylations.


Step 6: Oxidation

Now it's time for the payoff phase. One of our two GADP molecules from the preparatory phase, and we see that the first thing that will happen is an oxidation to become 1,3-bisphosphoglycerate. This requires NAD+ and a free phosphate, or inorganic phosphate to occur, and the enzyme involved is called glyceraldehyde phosphate dehydrogenase.


Step 7: Dephosphorylation 

Next, a Phosphoglycerate kinase will catalyse transfer of a phosphate group to ADP to become 3-phosphoglycerate producing 1 ATP in the process. Since each of the 2 GADP molecules will make 1 ATP, that's a total of 2 ATPs, for half the total payoff of glycolysis.


Step 8: Phosphate transfer

Then, phosphoglycerate mutase transfers the remaining phosphate from this hydroxyl to the next one over to make 2-phosphoglycerate.


Step 9: Dehydration 

Then, Enolase catalyses a dehydration, resulting in the loss of hydroxyl group (OH) which will produce Phosphoenolpyruvate.


Step 10: Second Dephosphorylation

The remaining phosphate group is transferred to an ADP by pyruvate kinase, generating another ATP and the Pyruvate.

So altogether it's a10-step process. The first five steps comprise the preparatory phase, which take one molecule of glucose and produce two molecules of GADP( glyceraldehyde-3-phosphate).This will cost 2 ATP.


Then the other five steps make up the payoff phase, in which each molecule of GADP will be converted into Pyruvate, producing 2 ATP each in the process, for a total of 4 ATP, meaning the net energy production from one molecule of glucose is 2 ATP.

Certainly the main thing to remember is that in glycolysis, glucose in the cytoplasm of the cell is converted into pyruvate, which will then move on to the next stage of cellular respiration, which is TCA Cycle cycle.

Membrane Transport - Active Transport & Passive Transport accross the cell membrane


  When the organisms are allowed to grow in a nutrient environment, the concentration of solutes in the cell is usually much higher than the concentration of solutes in the extracellular environment. 

 Moreover, the composition of Intracellular solute concentration is much different than that of extracellular envtironment. This difference in the solute concentration as well as Its composition, both in intracellular and extracellular environment, is mainly due to the role cell membrane that surrounds the cell.

The selective permeabllity of the cell membrane is responsible for the transport of molecules across the cell membrane, both in and out of the cell. This transport mechanism, therefore, plays a vital role in the uptake of nutrients by the cell and helps in matntaining cell's metabolism and growth.

Membrane Transport - Active and Passive Transpprt

A variety of mechanisms operate to allow entry of nutrients in the cell. They Include:
1. Passive Transport
2. Active transport

1). Passive Transport

The Passive Transport is of two types
   a). Passive Diffusion
   b). Facilitate Diffusion

a). Passive Diffusion

This is the simplest method of transport of molecules across the cell membrane. It involves passive diffusion of small molecules through the cell membrane in favor of concentration gradient.
    A solute or substance passes through cell membrane from a high concentration to a low concentration environment till the equilibrium is attained. This phenomenon does not involve utilization of extra energy. Very small molecules such as water and gases like O₂ and N₂ are transported by this mechanism.

b). Facilitated diffusion

Certain substance, otherwise, impermeable to cell membrane, can be transported into the cell by a special class of membrane proteins. These proteins are called permeases or carrier proteins. They are located in the periplasmic region of cell or may be embedded in cell membrane.
  The activity of these permeases allows diffusion or entry of molecules into the cell, without any expenditure of energy.

Mechanism and characteristics of facilitated diffusion :

  The permeases or carrier proteins participating in the membrane transport are enzymatic in nature. They are able to bind to the substrate to be transported.
  The resulting carrier-substrate complex then undergoes a conformational change, such that the substrate now gets transported in to cytoplasm. Such a transport of molecules by role of permeases is known as facilitated difusion.

  A model explaining facilitated diffusion has been shown in figure.

Transport of nutrients in cell facilitated diffusion. a) nutrient molecules present on outer side of cell membrane bound to permease. b) binding of molecules causes conformation changes in permease and cause their release in cytoplasm.

This facilitates the diffusion of the substance through the cell membrane. The uptake by carrier proteins

Follows Michaelis-Menten kinetics. This mode of transport is further characterized as under. 

  • Transport occurs in the favor of concentration gradient.
  • It possesses a considerable degree of specificity.
  • It is often inducible i.e. cells will produce permeases in the instances, where the environment possesses the substrate to be transported.

2). Active Transport

  The transport mechanism which operates at the expense of biochemical energy is in general known as active transport.
   This mode of transport specific and can occur even against the concentration gradient. i.e. the substances may be allowed to accumulate within the cell that can be several thousand times greater than that in the environment. The metabolic energy required to drive active transport mechanism may be obtained as proton motive force or ATP.

Four different patterns of active transport mechanisms have been recognized in microorganisms. They are as under.

  1. Role of binding proteins
  2. Secondary active transport
  3. Phosphate bond linked transport
  4. Group translocation

Role of binding proteins in active transport :

  A variety of binding proteins have been recognized to occur in cell membrane of bacteria. They bind with the substrate to be transported in a highly specific manner and allow their entry Into the cell or their accumulation in the periplasmic region.
   Such accumulation requires conformational change in structure of the binding proteins such that they are carried and released inside the cytoplasm or periplasm from the outer environment. This conformational change occurs at the expense of energy. This energy may be derived from ATP or directly by utilization of proton motive force.

• The binding proteins play two roles in transport.

  1. By binding to substrate, they increase the effective concentration of the substrate in the periplasm so that carrier proteins can transport them more favorably inside the cytoplasm.
  2. They may Interact directly with carrier proteins and stimulate their transport activity.

• Two types of binding proteins are found among bacteria:

  1. Shock sensitive, which are easily dissociated by osmotic shock from the cells. These binding proteins use ATP for their activity.
  2. Shock intensitive which remain firmly bounded to the cell membrane and are not released upon osmotic shock. These binding proteins use proton motive force directly.

Secondary active transport :

   Usually proton motive force is established by transport of electrons through electron transport chain, located in cell membrane. This proton motive force is called primary proton motive force. It may also be generated by passage of charged lons across the membrane. Such proton motive force is called secondary proton motive force.

  Transport of molecules by the utilization of such secondary proton motive force is called secondary active transport.
There are three types of secondary active transport mechanisms: symport, antiport and uniport.

Symport

Simultaneous transport of two molecules by a same carrier in one direction is called symport.
   Hence entry of one substance facilitates entry jo other substance simultaneously. Usually transport of an anion is associated with simultaneous transport of a cation in the same direction.

Antiport

  Simultaneous transport of two molecules by a same carrier In opposite directions is called antiport.
  Here entry of one substance causes simultaneous exit of other substance from the cell. In this case, usually, export of an anion is associated with entry of a cation and vice versa.

Uniport

The simultaneous transport of two substances by separate carrier is called uniport.


  Here a substance (usually cation) enters the cell through one carrier. Simultaneously another substance, usually anion leaves the cell through other carrier.

Phosphate bond linked active transport :

  Transport of certain substrates requires availability of free energy released from hydrolysis of energy rich phosphor ester bonds. i.e. they need phosphate bond energy for transport. These transport mechanisms are not capable of utilization of energy from proton motive force.

Group translocation :

  This methods of transport  allows entry of molecules inside the cell without any modification. However transport mechanisms do operate in cell that primarily cause chemical modification of the substances and then they are
permitted to enter the cell.
   Such modifications in the substances are carried out at the expense of metabolic energy and transfer of a particular functional group to the substance. Therefore this mode of transport is called group translocation.

Mechanism of group translocation

A specific group translocating machinery is involved in the group translocation. These transfer systems are as under.
  a). Phosphotransferase system for transport of sugars.
  b). AcylCoA synthetase system for transport of fatty acids.
  c). Phosphoribosyl transferase system for transport of purine and pyrimidine bases.

Phosphotransferase system

  Phosphotransferase system (PTS) is the most thoroughly studied system of group translocation in bacteria. It involves transport of sugars into the cell. The system involves phosphorylation of sugar by transferring phosphate group from PEP (Phospho enol pyruvate). The phosphorylated sugar is then able to enter the cell. PTS consists of 4 proteins: HPr, Enzyme I, Enzyme Il and Enzyme III as shown in the figure.


  As shown in the figure, the components of  phosphotransferase system are arranged in specific orientation. Ell is embedded in CM. Energy rich phosphate group is transported to Ell from PEP via role of El, HPr and EIII as shown in the figure. 

  Finally, the phosphate group is transferred to the sugar molecule to be transported. Enzyme II acts as a carrier protein for sugars and permits their entry Into the cell. The PTS is mediating specific sugar transport, where role of enzyme III and enzyme ll is specific for the sugar to be transported, whereas enzyme l and HPr act in nonspecific manner for all PTS.

Acyl CoA synthetase system

   This system works for transport of fatty acids into the cells. The fatty acids are initially converted into fatty acyl CoA by transfer of CoA group from acetyl CoA and then the fatty acyl CoA is permitted to enter the cell.

Fatty acid + Acetyl CoA ➞ Fatty acyl CoA + Acetate

Phosphoribosyl transferase

  This enzyme plays role in transport of purine and pyrimidine bases. These bases are impermeable in cell membrane. However, phosphoribosyl transferase transfers of ribosyl moiety to the purine and pyrimidine bases and convert them into corresponding nucleoside monophosphates, which then enter the cell.

Purine or Pyrimidine base + Phosphoribosyl pyrophosphate (PRPP)  ➞  nucleoside mono phosphate + P-P