Narne Naga Srilakshmi

DNA replication is a fundamental process that ensures the accurate duplication of genetic information, enabling the faithful transmission of hereditary traits from one generation to the next. At the heart of this complex mechanism lie a diverse array of enzymes, each playing a crucial role in orchestrating the seamless replication of the DNA double helix. In this comprehensive guide, we will delve into the intricacies of the key DNA replication enzymes, providing a detailed exploration of their functions, kinetics, and error rates.

DNA Polymerases: The Maestros of DNA Synthesis

DNA polymerases are the primary enzymes responsible for the synthesis of new DNA strands, adding nucleotides to the growing chain in a 5′ to 3′ direction. While there are several types of DNA polymerases, each with specialized functions, the workhorse of DNA replication is DNA polymerase III.

• DNA Polymerase III: This enzyme is responsible for the bulk of DNA synthesis during replication, with an impressive speed of 500-1000 nucleotides per second in E. coli. Its high fidelity is achieved through a combination of accurate nucleotide selection and a proofreading mechanism that can correct errors at a rate of approximately 1 mistake per 10^4 to 10^6 nucleotides synthesized.
• DNA Polymerase I: This enzyme plays a crucial role in removing RNA primers and replacing them with DNA, ensuring the continuity of the newly synthesized strands. DNA polymerase I has a lower error rate of around 1 mistake per 10^5 to 10^6 nucleotides synthesized.
• DNA Polymerase II: While less well-studied, DNA polymerase II is believed to be involved in DNA repair processes, providing a backup mechanism to ensure the integrity of the genetic material.

DNA Primase: The Primer Provider

DNA primase is a specialized enzyme that synthesizes short RNA primers on the single-stranded DNA template, providing the necessary starting points for DNA polymerase to initiate DNA synthesis. The error rate of DNA primase is approximately 1 mistake per 10^3 to 10^4 nucleotides synthesized, slightly higher than that of the DNA polymerases.

DNA Helicase: The Unwinding Maestro

DNA helicase is the enzyme responsible for separating the two strands of the DNA double helix, creating the replication fork where DNA synthesis can occur. This enzyme moves in a 5′ to 3′ direction, unwinding the DNA as it progresses. The speed of DNA helicase varies among different organisms, with E. coli helicase moving at a rate of 500 to 1000 base pairs per second.

DNA Ligase: The Sealing Specialist

DNA ligase plays a crucial role in sealing the gaps between Okazaki fragments on the lagging strand, forming a continuous DNA strand. This enzyme has an error rate of approximately 1 mistake per 10^5 to 10^6 nucleotides ligated, ensuring a high degree of fidelity in the final DNA product.

Topoisomerase: The Tension Relievers

As the replication fork progresses, the DNA double helix ahead of it can become increasingly tightly wound, creating topological stress that could impede the replication process. Topoisomerase enzymes alleviate this tension by making temporary nicks in the DNA helix, allowing the strands to unwind, and then sealing the nicks to maintain the integrity of the genetic material. The rate of topoisomerase activity varies among different organisms, with E. coli topoisomerase I relaxing supercoiled DNA at a rate of 500 to 1000 base pairs per second.

In addition to these key enzymes, DNA replication also involves a host of other proteins and enzymes, such as single-stranded binding proteins, which coat the single-stranded DNA around the replication fork to prevent rewinding, and primase, which synthesizes the RNA primers complementary to the DNA strand.

The intricate dance of these DNA replication enzymes, each with its unique function and kinetic properties, ensures the accurate and efficient duplication of the genetic code, laying the foundation for the continued evolution and survival of living organisms.

References:

1. Lodish, H., Berk, A., Zipursky, S. L., Matsudaira, P., Baltimore, D., & Darnell, J. (2000). Molecular cell biology. Scientific American books.
2. Lehninger, A. L., Nelson, D. L., & Cox, M. M. (2000). Lehninger principles of biochemistry. W.H. Freeman.
3. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular biology of the cell. Garland Science.
4. Watson, J. D., Baker, T. A., Bell, S. P., Gann, A., Levine, M., & Losick, R. (2013). Molecular biology of the gene. Pearson.
5. Replication of DNA: Enzymes and the Replication Fork. (n.d.). Retrieved from https://www.nature.com/scitable/topicpage/replication-of-dna-enzymes-and-the-replication-408/
6. DNA Replication Enzymes. (n.d.). Retrieved from https://www.genome.gov/genetics-glossary/DNA-Replication-Enzymes

Active transport consumes energy and it takes place in two phases.

Active transport consumes cell energy as the molecules are transported against their concentration gradient. There are two types of active transport. They are primary active transport and secondary active transport. In this article we shall discuss active transport examples.

Examples of Primary active transport:

Examples of secondary active transport:

Sodium potassium pump:

This is the most important pump in human beings where the sodium ions (Na+) and potassium (K+) ions are transported in contrast against their concentration gradient. For the completion of every cycle of the pump three sodium are exchanged (out) against two potassium ions (in). The ATP molecule gets bound to three sodium molecules and hydrolyzed by Na+- K+ ATPase. ATP molecule gets phosphorylated and release sodium ions to outside of the cell and bring potassium ions into the cell.

Proton pump:

Proton pump is the important cycle primary active transport. It enhances the transportation of H+ ions mainly in stomach. It usually takes place in mitochondrial cell especially on the stomach surface where the protons are transported from low concentration gradient and generates electrochemical gradient at the cell membrane. Proton pump also utilises ATP molecule and hydrolysed by the enzyme called H+, K+– ATPase.

The proton pump is activated by the intracellular secondary messengers. In proton pump H+ ions are transported into or out side of the cell.

Calcium pump:

Calcium pump is usually occurs in muscle cells. In this pump the calcium ions are transported to out of the cell to reduce the calcium ion gradient around actin and myosin filaments. It helps not only the muscle relaxation but also the development of the fertilized egg.

Calmodulin is the main protein in the generation of cell signaling in the calcium pump. Calcium-ATPase plays a key role in this pump. By using this cycle the cell can maintain the inner cell calcium level 10000 times lower than extracellular concentration.  For every cycle two Ca+ ions are interchanged with two H+ ions.

Electro genic pump:

This is also an important pump in primary active transport. It uses energy from ATP molecules and leads to generation of net flow of charge of ions or protons across the membrane.

The two best examples of the Electro genic pump are Sodium potassium pump and proton pump.

Glucose symport:

The small intestine and proximal tubules of nephron cells usually perform glucose symport cycle. SGLT plays a key role in glucose symport pump.

Glucose symporters move glucose molecules in only one direction means that into or out of the cell membrane. This also helps in the renal glucose reabsorption.This cycle uses the energy in the form of electrochemical gradient that is released during the primary active transport.

Sodium – Calcium antiporters:

Sodium – Calcium antiport exchanges sodium and calcium ions in opposite directions means that it removes calcium from the cell and allows sodium into the cell. For one Ca+ ion three Na+ ions are exchanged per cycle.

Sodium- Calcium exchangers are most commonly seen in mitochondrial cell and endoplasmic reticulum. This also controls the cardiac potential by getting the signal it allows the influx of sodium and efflux calcium.

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Frequently Asked Questions

What is meant by a primary active transport?

The primary active transport is the enhance the movement of molecules, ions, nucleotides from low concentration gradient to high concentration gradient across the cell membrane.. It consumes cell energy in the form of ATP molecule.

Does active transport enhance the endocytosis and exocytosis?

Active transport enhances both exocytosis and endocytosis.

Exocytosis:

Exocytosis is a process of transfer of intracellular particles into extracellular fluid. This is enhanced by the fusion of vesicle with plasma membrane. This is the last step in the synthesis. The synthesised products like enzymes, lipids, proteins, antibodies and peptide hormones are transported to extracellular fluid by this process.

Endocytosis:

Endocytosis is the process of engulfing the extracellular molecules or particles into the cell by the cell membrane. It is divided into two types namely they are phagocytosis and pinocytosis.

Phagocytosis:

It is the process of taking large particles, part of cell or whole cell into the cell. The plasma membrane from pocket around the target particle and forms vacuole.

Pinocytosis:

Is the process of engulfing the particles into the cell in the form of fluids along with the particles in the fluids.

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Anaerobic bacteria means the bacteria that can grow in the absence of oxygen.

The bacteria that do not require in the oxygen for the respiration process are called Anaerobic bacteria.

Anaerobic bacteria examples are divided into two–

Gram Positive Anaerobic Bacteria:

Peptostreptococcus:

These are the examples of gram positive cocci. In these Peptostreptococcus is the common pathogenic anaerobic bacteria can be found on the skin or genital parts of women. It can cause inflammatory diseases in humans especially in women. Peptostreptococcus grow in small chain like structures.

Streptococcus:

Streptococcus is he common gram positive anaerobic cocci which can be found commonly in the genital parts of women and cause urinary tract infections.

Gemella:

Gemella is an facultative anaerobic species and catalyse negative gram positive cocci bacteria and it is found on the surface the skin and mucous membrane of humans and some other animals too. It may infect the tooth (dental) in the humans.

Bifidobacterium:

Bifidobacterium is commonly known to be the normal bacterial flora, present in intestine. It is a non motile bacteria.They can help humans as they act as probiotics and also absorb nutrients from food, digest food help human beings in constipation, diarrhea.

Finegoldia:

Finegoldia is an anaerobic gram positive cocci and usaually found on the skin, mouth, intestine. They may cause gastro intestinal infections and urinary tract infections.

Clostridium Botulinum:

Clostridium Botulinum is an anaerobic gram positive bacilli. This is a spore forming bacteria. This is commonly found in the surface of sea food as well as fruits and vegetables. It is toxic bacteria can cause Botulism in humans.

Lactobacillus:

Lactobacillus is an anaerobic gram positive bacilli. It is non pathogenic bacteria and acts as a probiotic. It is mostly found in intestine (digestive tract) and urinary tracts.

Listeria:

Listeria is an intra cellular pathogenic bacterium and it cause listeria monocytogenes. It is basically a food borne disease and affects the immune system.

Clostridium tetani:

Clostridium tetani is a spore forming gram positive anaerobic bacteria. It presents in soil and intestine of animals. It causes tetanus. Tetanus usually affects the neuro transmissions.

Propionibacterium:

Propionibacterium is gram positive anaerobic bacilli. This bacterium is widely used in the production of Vitamins, propionic acid and probiotics too.

Eubacterium lentum:

Eubacterium lentum is a gram positive anaerobic bacilli. It is a non spore forming bacteria. It is usually found in gastro intestinal tract. It causes Septicemia.

Cl.Novyi:

Cl.Novyi is an anaerobic gram positive bacilli and it is commonly seen in soil as well as intestinal tract. Cl.Novyi produce endo toxins.

Cl.ramosam:

Cl.ramosam is an anaerobic gram positive, non motile, spore forming bacteria. It usually a causative agent for ear infections in humans especially in young children (infants).

Cl.Histolyticum:

Cl.Histolyticum is an aerotolerant anaerobic, motile, gram positive bacteria, mostly seen in soil and feces of animals. It usually causes gas gangrene in humans as well as other animals like pig, mouse etc.

Gram Negative Anaerobic Bacteria:

Bacteroides :

Bacteroides is an obligative anaerobic bacteria and gram negative bacilli. They are friendly to humans and found in the intestine. They help in digestion of food and can produce pivotal nutrients which are needed by the body. Some times they might cause infections (Bacterial fragellis) in humans.

Escherichia coli:

Escherichia coli is an anaerobic gram negative bacilli. It is commonly found in intestine of humans. It helps the humans to keep intestine healthy. Some times it is pathogenic too and can cause diarrhea and mild fever too. It enter the body from contaminated food. It can also infect urinary tract.

Fusobacterium:

Fusobacterium is a gram negative anaerobic bacilli. It is non spore producing bacteria. It is normal bacterial flora and it is causative organism for the diseases like tonsillitis and Lemierre’s disease.

Shewanella oneidensis :

Shewanella oneidensis  is anaerobic gram negative bacilli.It is normally found in aquatic regions. Scientist KEN Nealson first identified Shewanella oneidensis. It may be pathogenic to humans. Shewanella oneidensis  enters the body from unprocessed sea food.

Leptotricha Buccalis:

Leptotricha Buccalis is an anaerobic gram negative bacilli. It is usually seen in oral parts and causes dental problems.

Fragilis:

Fragilis is an anaerobic gram negative bacilli. It is commonly seen in colon and faces. It generally causes diarrhea in humans.

Porphyromonas:

Porphyromonas is an obligative anaerobic, non motile, non spore forming gram negative bacteria. It is usually found in oral cavity and causes dental problems.

Prevotella:

Prevotella are an anaerobic gram negative bacilli and can be seen in oral cavity. It is specifically a dietary fiber fermentor.

Veillonella:

Veillonella is an anaerobic gram negative bacteria and it causes dental problems and soft tissue infections.

Examples Of Pathogenic Anaerobic Bacteria:

Classification:

The bacteria can be sub classified into four groups.

Facultative aerobes:

This type of bacteria can grow in the presence or absence of oxygen.

Micro aerophilic:

This type of bacteria require very low amount of oxygen.

Obligate anaerobic:

This type of bacteria don’t require oxygen but able to with stand in the presence of oxygen. The obligate anaerobes are again sub classified basing on their tolerance towards oxygen.

If the obligate anaerobic bacteria can tolerate only ≤ 0.5% oxygen is known considered to the strict anaerobes.

If they tolerate 2 to 8% of oxygen then they can be said to be moderate anaerobes.

If they tolerate atmospheric oxygen for a limited time then they can be said to be aerotolerant anaerobes.

Examples :

Capnophiles:

This type of bacteria need more amount of carbon dioxide.

E.g. Haemophilus influenzae, Neisseria gonorrhoeae.

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Anaerobic:

The bacteria cannot grow in the presence of bacteria.

The anaerobic bacteria are first observed by the scientist Van Leeuwenhoek. He is a Dutch scientist.

The oxygen acts as a toxic agent for anaerobic bacteria. The anaerobic bacteria undergo fermentation process.

In fermentation process the anaerobic bacteria the first is the breaking down of glucose molecule. The glucose molecule is broke down two phospholipids. Anaerobic bacteria use lactic acid cycle in this process two cycles are used by them. They are tricarboxylic acid cycle and electron transport chain. The anaerobic catalyse the glucose molecule and synthesize two ATP molecules.

Types of anaerobic bacteria:

The anaerobic bacteria includes Cocci (Circle), bacilli (rod), spiral (spherical).

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Facilitated diffusion is a passive transport process that does not require energy from the cell. It is a mechanism by which molecules, such as glucose, amino acids, and ions, move across the cell membrane down their concentration gradient with the assistance of specialized transport proteins. Unlike active transport, which requires energy in the form of ATP to move molecules against their concentration gradient, facilitated diffusion relies solely on the natural tendency of molecules to move from an area of high concentration to an area of low concentration.

Biological Specification of Facilitated Diffusion

In facilitated diffusion, the transport proteins involved are either channel proteins or carrier proteins. These proteins play a crucial role in facilitating the movement of molecules across the cell membrane.

Channel Proteins

Channel proteins form a pore or channel in the cell membrane, allowing specific ions or molecules to pass through. These channels are selective, meaning they only allow the passage of certain molecules or ions based on their size, charge, and other physical properties. The presence of channel proteins increases the rate of diffusion by providing a more direct pathway for the molecules to move through the membrane.

Carrier Proteins

Carrier proteins, on the other hand, bind to the solute on one side of the membrane, undergo a conformational change, and then release the solute on the other side of the membrane. This process is known as facilitated transport or facilitated diffusion. Carrier proteins can transport molecules against their concentration gradient, but this does not require energy input from the cell.

Theorem of Facilitated Diffusion

The theorem of facilitated diffusion can be summarized as follows:

1. Passive Transport Process: Facilitated diffusion is a passive transport process, meaning it does not require energy input from the cell.
2. Concentration Gradient: The driving force for facilitated diffusion is the concentration gradient, where molecules move from an area of high concentration to an area of low concentration.
3. Transport Proteins: Channel proteins and carrier proteins facilitate the movement of molecules across the cell membrane by providing a more direct pathway or by binding and releasing the solute.
4. No Energy Consumption: Facilitated diffusion does not directly consume ATP or any other form of energy.

Examples of Facilitated Diffusion

1. Glucose Transport in Erythrocytes: Glucose molecules are transported into erythrocytes (red blood cells) using a specific transport protein called GLUT-1. This facilitated diffusion process allows glucose to enter the cells down its concentration gradient, without the need for energy input.

2. Ion Transport in Nerve Cells: Sodium and potassium ions are transported across the nerve cell membrane using channel proteins. This facilitated diffusion of ions allows the action potential to propagate along the neuron, enabling the transmission of electrical signals.

3. Amino Acid Transport in Intestinal Cells: Amino acids are transported into intestinal cells using carrier proteins, which facilitate their movement down the concentration gradient without requiring energy.

4. Oxygen Transport in Alveoli: Oxygen molecules are transported from the alveoli in the lungs to the bloodstream through facilitated diffusion, with the help of channel proteins in the alveolar and capillary membranes.

Data Points

1. Facilitated diffusion is a passive transport process that does not require energy input from the cell.
2. The driving force for facilitated diffusion is the concentration gradient, where molecules move from an area of high concentration to an area of low concentration.
3. Transport proteins, such as channel proteins and carrier proteins, facilitate the movement of molecules across the cell membrane by providing a more direct pathway or by binding and releasing the solute.
4. Facilitated diffusion does not directly consume ATP or any other form of energy.

Conclusion

In summary, facilitated diffusion is a passive transport process that does not require energy from the cell. It relies on the concentration gradient as the driving force and utilizes specialized transport proteins to facilitate the movement of molecules across the cell membrane. This process is distinct from active transport, which requires energy input to move molecules against their concentration gradient. Understanding the principles of facilitated diffusion is crucial in various fields of biology, from cellular physiology to drug delivery and transport mechanisms in living organisms.

References

1. Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., & Walter, P. (2002). Molecular Biology of the Cell. Garland Science.
2. Lodish, H., Berk, A., Zipursky, S. L., Matsudaira, P., Baltimore, D., & Darnell, J. (2000). Molecular Cell Biology. W. H. Freeman.
3. Tortora, G. J., & Derrickson, B. (2018). Principles of Anatomy and Physiology. Wiley.
4. Purves, W. K., Sadava, D. E., Orians, G. H., & Heller, H. C. (2003). Life: The Science of Biology. Sinauer Associates.
5. Karp, G. (2010). Cell and Molecular Biology: Concepts and Experiments. Wiley.

The gram negative bacteria contain a thin peptidoglycan layer and cannot retain crystal violet stain which is used in gram staining method.

The gram negative bacteria contain a very low amount of peptidoglycan in the cell wall and appears pink colour in gram staining. In this section we shall discuss gram negative bacteria examples.

The gram negative bacteria is found almost in every environment on the earth that can support life. These are more resistant to antibiotics than the gram positive bacteria.

Examples Of Gram Negative bacteria:

Gram negative Cocci bacteria:

Gram negative bacilli bacteria:

Gram negative Spiral bacteria:

Gram negative Vibrio bacteria:

Characteristics of gram negative bacteria:

Classification of gram negative bacteria:

The gram negative bacteria are classified basing oh the morphology,( Phenotypic), Analytical characterisation and Genotypic characteriasation. The morphology classification is also known as phenotypic characterisation. It includes growth requirements, and biochemical requirements(Bio typing), serotype, antibiotic resistance patterns (MSSA Vs MRSA), size, shape and staining procedure.

Phenotypic Characterization:

In phenotypic characterization the bacteria can be classified basing on the technique like serotyping and bio typing.

Bio typing is the process of classification of bacteria in the absence of specific enzymes like lipases, proteases, nucleases.

Serotyping is the process of classification of bacteria basing on the antigen present in their cell membrane by using specific antibodies.  Serotyping is the one of the latest tools that are used for bacterial characterization.

Analytical characterization:

This is a big process and requires special equipment.

Usually this is performed in special laboratories.

The analytical characterisation Includes Whole cell lipid analysis, cell wall fatty acid analysis, Whole cell protein analysis, enzymes and electrophoresis.

The whole cell protein analysis is performed by using mass spectroscopy.

Genotypic Characterization:

In genotypic characterisation bacterial DNA is used for classification.

Earlier scientist used to identify Guanine and Cytosine ratio (G-C ration).

Now a day’s scientist are also using DNA – DNA hybridisation.

 Nucleic acid sequence analysis is also one of the most useful method now a days.

 In nucleic acid sequence analysis the known sequence is used for identification of unknown sequence.

The growth of gram negative can be prevented by subjecting them to antibiotics.

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Frequently Asked Questions:

What is the difference between gram positive and gram negative bacteria?

The gram positive bacteria contain a thick peptidoglycan layer where as it is thin in gram negative bacteria.

The gram positive bacteria contain a thick peptidoglycan layer and appears violet in color in gram staining and gram negative bacteria contain thin peptidoglycan layer and appears pink in color.

Does E.Coli come under gram negative bacteria?

E.coli comes under gram negative bacteria and it causes urinary tract infections in humans.

What are the common diseases caused by gram negative bacteria in humans?

Cholera, Plague, Typhoid fever, Meningitis and Urinary tract infection are common bacterial diseases in humans.

Why are gram negative bacteria more harmful than gram positive bacteria?

The gram negative bacterial cell wall is hard and it does not susceptible to antibiotics easily and can release endotoxins when they are subjected to antibiotics.

What are the secretion system in gram negative bacteria?

The gram negative bacteria have broad enclosed secretion system to shift tiny molecules, DNA, Amino acids, Proteins.

What is meant by gram negative blood stream infection?

The blood stream infection means the attack of blood by the microorganisms like bacteria. In this case the bacteria are resistant to antibiotics and might cause fever. Some times the blood stream infection is relieved with out any treatment.

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The gram positive bacteria appear purple in colour in gram staining because of the presence of the thick peptidoglycan layer.

The gram positive bacteria retains purple or violate color  which is used for gram staining process as they have thick peptidoglycan layer. Here we shall discuss more on gram positive bacteria examples.

In this article we shall discuss in detail about gram positive bacteria.

Gram Positive Cocci:

1. Coagulase positive: e.g. S.aureus
2. Coagulase negative: e.g. S. epidermidis

Gram Positive Bacilli:

History:

Gram staining technique was developed by Hans Christian Gram. The technique is known as Gram staining. By this technique we can differentiate bacterial species into two types Gram-positive bacteria and Gram-negative bacteria.  The crystal violate stain is used in gram staining.

When observed through an optical microscope, the bacteria which contains mesh-like cell wall made of thick peptidoglycan layer takes crystal violet and appears in purple. They are named Gram-positive bacteria. Whereas the bacteria which contains thin cell wall takes saffranin and appear in pink are called gram negative bacteria.

Because of the presence of the thick peptidoglycan layer and absence of outer membrane the gram positive bacteria are more susceptible to antibiotics than gram negative bacteria.

Cell structure:

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Frequently Asked Questions:

What are the common infections caused by gram positive bacteria?

Anthrax, Diphtheria, Diarrhea, Meningitis, Nausea, Skin infections Urinary tract infections.

What are the most common gram positive bacteria?

streptococcus pyogenes, Streptococcus pneumoniae, Staph. epidermidis, Staph. saprophyticus and Enterococcus faecalis.

How the gram positive bacteria replicate?

The gram positive bacteria undergo replication with the help of self replicating plasmids.

The plasmids present in bacterial cell involve in the replication of bacteria. The transcriptional repressor protein and anti sense RNA control the replication process.

What is a plasmid and its role?

A Plasmid is small circular double stranded DNA present in bacterial cells.

A plasmid is circular DNA present in bacterial cells naturally. It helps the bacteria in the development of antibiotic resistance.

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