Cell Wall Composition of Gram Positive and Gram Negative Bacteria


  Cell wall forms a fairly rigid layer just outside the plasma membrane in most of the prokaryotes . The cell wall accounts for 10 to 40 % of the cellular dry mass depending on the growth conditions . It provides the cell support , and protection against mechanical stress or damage from osmotic rupture and lysis . The major component of the bacterial cell wall is peptidoglycan or murein . This rigid structure of peplidoglycan, specific only to prokaryotes. gives the cell shape and surrounds the cytoplasmic membrane.

Overall structure of Cell wall

1].  Light microscopic examination of bacteria, does not permit observation of differences between the cell walls of gram - positive and gram negative bacteria. It is possible only when the cell walls are examined under the transmission electron microscope.

2].  The wall of gram - positive bacteria consists of a single homogeneous layer of peptidoglycan for murein ) . that remain outside the plasma membrane. It is 20 to 80 nm thick .

3]. The cell wall of gram - negative bacteria consists of  one layer of outer membrane ( 7 to 8 nm thick ) and one layer of peptidoglycan ( 2 to 7 nm thick )

4].  A space is observed between plasma membrane and outer membrane of cell wall. It is called periplasm. Periplasm of gram - negative bacteria contains hydrolyzing enzymes. They facilitate the movement of molecules across the membrane.

5].  The gram - positive bacterial cell wall contains peptidoglycan and telchole acid as the major constituents, whereas that of gram - negative bacteria possesses peptidoglycan. lipopolysaccharide, lipoproteins and phospholipids.


Peptidoglycan

1].  Peptidoglycan ( also called murein ) is the most Important constituent of prokaryotic cell wall. It is relatively porous, elastic and somewhat stretchable. It is a complex mesh - like polymer of repeating subunits. Variation occurs from species to species in its chemical composition and structure. However, the basic structure is same in all

2]. Peptidoglycan. a hetero- polysaccharide, consists of repeating disaccharide units, to which a short peptide chain attaches and forms a complex mesh ( lattice ). The disaccharide part is formed by two acetylated amino sugars, N - acetyl glucose amine ( NAC ) and N - acetyl muramic acid ( NAM, the lactyl ether of N - acetyl glucose amine ), that are joined alternately by b-1, 4 glycosidic linkage.

3]. NAG and NAM Join linearly to form a row of 10 - 65 sugars and forms the carbohydrate backbone. A short peptide tetrapeptide - a chain of four amino acids. consisting of L - alanine, D - glutamic acid , Di amino plmelic acid ( DAP ) or L - lysine and D - alanine , attaches to the NAM residue of backbone. Tetra peptide contains alternating pattern of D- and L - amino acids.

4]. In gram-positive bacteria, these tetra peptides are Joined by cross-links in order to form a strong. mesh like polymer. Normally the carboxyl (-COOH) group of terminal D-alanine joins directly with DAP or L lysine of other tetra peptide by a penta peptide inter-bridge. Most gram-negative bacteria lack a peptide inter-bridge. Peptide inter-bridge does not always exist between all tetra peptides.

Components of gram-positive cell walls

1]. The cell walls of gram-positive bacteria are thick and made up of several layers of peptidoglycan (as many as 40). In addition, they possess a large amount of another macromolecule called teichoic acid.

2]. Teichoic acids are polymers of glycerol or ribitol joined by phosphate groups. Amino acids like D-alanine or sugars like glucose are attached to the glycerol or ribltol groups. The teichoic acids may be covalently linked with either peptidoglycan in the wall or lipids in the plasma membrane. They are respectively called wall teichoic acid or Lipoteichoic acid.

3]. Teichoic acids are negatively charged. so they contribute to the negative charge of the cell surface. They may be involved in :
a). Regulation of entry or exit of
        molecules
b). Prevention of cell lysis
c). Antigenicity
d). Attachment of
       bacteriophages

Components of gram-negative bacterial cell walls

The cell walls of gram-negative  bacteria are relatively more complex. It possesses a thin layer of peptidoglycal, surrounded by outer membrane, made of lipopolysaccharide.

Outer Membrane

The outer membrane consists of lipopolysaccharides (LPS). It consists of Lipid A, Core polysaccharide and O side chain. The outer membrane is linked to inner peptidoglycan layer by a unique lipoprotein, called Braun's lipoprotein.

a). Lipid A  is not a glycerol lipid. It consists of two glucosamine sugar derivatives, each attached with three fatty acids and phosphates or pyrophosphates. The long chain fatty acids, which may be caproic acid, lauric acid, myristic acid, palmitic acid or stearic acids, join to glucosamine (GKN) phosphates through esteramine linkage. The disaccharides attaches to the core polysaccharide and the fatty acids attach to the outer membrane.

b). Core polysaccharide consists of an oligosaccharide unit attached to the glucosamine residues of lipid-A. It consists of keto-deoxyoctonate (KDO), seven-carbon sugars (heptose). glucose, galactose and N-acetyl glucosamine.

c). O-polysaccharide, also called O-side chain or O-antigen, extends from the core. It contains several peculiar sugars e.g. galactose (Gal), glucose (Gui), rhamnose (Rha) and mannose (Man) They are connected in four or five-member sequences, which are usually branched. Repetition of sugar sequences forms a long O-polysaccharide.

LPS contains porin proteins. It is tube shaped. It permits the passage of molecules smaller than 600 to 700 Daltons.

Inner Membrane

1]. Inner membrane of gram - negative bacteria consists of a thin layer of peptidoglycan . The peptidoglycan layer is non - covalently anchored to lipoprotein molecules called Braun's lipoproteins through their hydrophobic head .

2]. Sandwiched between the outer membrane and the plasma membrane , a concentrated gel - like matrix ( the periplasm ) is found in the periplasmic space . This periplasmic space contains binding proteins for transport of nutrients in to the cell . The periplasm space can act as reservoir for virulence factors and a dynamic flux of macromolecules representing the cell's metabolic status and its response to environmental factors .

Nutritional types of bacteria

Nutritional types of bacteria

Bacteria differ in their nutritional requirements. The differences among the bacteria are specic. A systematic approach for the nutritional classification of bacteria was first made by Monod. Amongst various requirements for nutrition, characteristic differences exist for the requirement of sources of energy, electron donor and carbon. Therefore, bacteria are classified in various categories on the basis of these requirements.

Nutritional types of bacteria on the basis of sources of energy, electron donor and carbon

1]. Based on source of energy :
      a) Phototrophs
      b) Chemotrophs
      c) Hypotrophs      

2]. Basesd on source of electron donor :
    a) Lithotrophs
    b) Organotrophs

3]. Based on source of carbon :
     a) Autotrophs
     b) Heterotrophs
     c) Paratrophs


1]. Classification on the basis of requirements for sources of energy

Based on requirements for sources of energy bacteria can be classifed in to three major categories,
1. Phototrophs
2. Chernotrophs
3. Hypotrophe

Phototrophs

These are the bacteria which obtain energy by using radiant energy i.e. light. These bacteria possess photosynthetic pigments and photosynthetic apparatus. With its help, they capture radiant energy and transform it into biologically utilizable form of energy i.e. ATP. Bacteria belonging to this category Include
1. Cyanobacteria,
2. Green sulfur bacteria,
3. Purple sulfur bacteria and
4. Purple non sulfur bacteria.

Chemotrophs

These are the bacteria which obtain energy by oxidizing chemicals. Upon oxidation of chemicals, chemical energy is released. It is trapped in ATP molecules during blochemical reactions and is made available for cellular processes. A wide variety of chemical compounds can be used by Chemotrophs as source of energy. They include both inorganic and organic.

Inorganic compounds, which can be oxidized by micro organisms for obtaining energy. Include ammonia, reduced sulfur compounds, ferrous salts and molecular hydrogen. e.g.
1). Thiobacillus and other sulfur oxidizers obtain energy from oxidation of reduced sulfur compounds.

2). Nitrifying bacteria obtain energy from oxidation of ammonia and ammonium compounds.

3). Iron bacteria obtain energy by oxidizing Iron from Fe++ to
Fe+++ form.

4). Hydrogen bacteria can oxidize molecular hydrogen to obtain energy.

Organic compounds, which are attacked by the bacteria for the purpose of obtaining energy range from most simple substance like methane to complex substance like paraffin. eg. E. coll Bacillus and various other bacteria obtain energy from oxidation of organic carbon compounds.

Hypotrophs

These are the organisms, which cannot utilize any external source of energy. This is because of their inability to synthesis ATP. They require ready made ATP for growth. It may be obtained from other living host cells. Thus these organisms grow as parasites. e.g. Viruses and rickettsiae.


2]. Classification on the basis of source of electron donor

Bacteria show characteristic differences in the substances used as electron donor. On the basis of type of electron donor utilized, bacteria can be classified in to two categories
   1. Lithotrophs
   2. Organotrophs

Lithotrophs

These are the bacteria, which utilize Inorganic substances as electron donor. They oxidize selective Inorganic substances and generate necessary reducing power required for biosynthesis. Inorganic substances used as electron donor by organisms include reduced sulfur compounds, ferrous salts, ammonia, ammonium compounds and molecular hydrogen.

Organotrophs

These bacteria generate their reducing power from oxidation of various organic compounds.


3]. Classification on the 
basis of carbon sources

Bacteria also possess characteristic difference in their requirement of substances used as source of carbon in their nutrition. Accordingly, they can be classifed in to three categories   
  1. Autotrophs
  2. Heterotrophs
  3. Paratrophs

Autotrophs

The bacteria , which use inorganic carbon compounds as the carbon source in their nutrition are called autotrophs . They mostly use CO2. They fix CO2 and reduce it into cellular organic matter. Usually, they - fix CO2 by Calvin cycle or reductive TCA cycle. Some bacteria can also assimilate carbonates and carbon monoxide. Certain autotrophic bacteria can grow by using both CO2 and organic compounds i.e. they can live, both as autotrophs and heterotrophs. They are called facultativHeterotrophs.

Heterotrophs

These bacteria obtain their carbon from various organic substances, ranging from methane to paraffin. Majority of bacteria fall in this category.

Paratrophs

Certain bacteria lack biosynthetic abilities and are not able to use non cellular organic or Inorganic carbon compounds. Instead, they need ready made supply of organic molecules, which can be assimilated directly during their cellular synthests, such as amino acids, nucleotides etc. These organisms are classified as paratrophs.
e.g. viruses and rickettsiae.

Thus it is clear that bacteria differ in their nutritional categories drastically. Even a bacterium belonging to one category for its requirements for energy may show a marked difference in its requirements for sources of electron donor as well as carbon.

Nutritional subgroups of organisms 

Subgroups of nutritional types of bacteria on the basis of their diversity in the requirements for different sources of nutrients.

Photolithotrophs

  These are the bacteria, which obtain energy from light and use inorganic substances as electron donor. These Bacteria usually utilize H₂S or other reduced inorganic sulfur as electron donor and oxidize it to elemental sulfur.
       H₂S ➞ S + 2e+ + 2H+
Green sulfur bacteria and purple sulfur bacteria belong to this category.

Photoorganotrophs

   These bacteria obtain energy from light and generate reducing power from oxidation of organic compounds. They use substances like pyruvate, succinate, formate as electron donor and generate reducing power on oxidation of these substances. e.g.

Succinate ➞ Fumarate + 2e+ 2H+
   Purple non sulfur bacteria belong to this category.

Photoautotrophs

   These are the phototrophic bacteria, which obtain their energy from light and carbon from CO₂ They are more related to photolithotrophs.

Photoheterotrophs

   These bacteria obtain their energy from light and carbon from organic substances. Nutritionally they are close to photoorganotrophs. They can use same organic compound, both as electron donor and source of carbon.

Chemolithotrophs

   These bacteria can use the same inorganic chemical substances as the sources of energy and electron donor. They oxidize selected Inorganic chemical compounds for the purpose. The chemicals used by these organisms include:

1). H₂S and other reduced sulfur compounds
Organisms oxdize these reduced sulfur compounds, inally to SO₂ during their metabolism.
e.g. Sulfur oxidizers (Thiobacillus).

2). Ferrous compounds

Organisms oxidize ferrous iron (Fe++) compounds to ferrie (Fe+++) form and generate both energy and reducing power, e.g. Iron oxidizers (Ferrobacillus). 

3). Ammonia and ammonium compounds

  The organisms oxidize ammonia and ammonium compounds finaly to NO3 through the phenomenon of nitrosofication and nitrification, e.g. Nitrobacter

4). Molecular hydrogen

  Many bacteria can oxidize molecular hydrogen to obtain both energy and reducing power, e.g. Hydrogenbacteria.

Chemoorganotrophs

   These are the bacteria, which obtain their energy and reducing power through oxidation of same organic compound. They can oxidize a wide variety of organic substances for the purpose. Most bacteria belong to this category. Nutritionally these bacteria are more related to Chemoheterotrophs.

Chemoautotrophs

   These are the bacteria which obtain energy from oxidation of chemical compounds (usually inorganic), and carbon from CO₂. Nutritionally, they are more close to chemolithotrophs.

Chemoheterotrophs

    These are the bacteria, which obtain their energy from oxidation or chemicals (usually organic) and carbon from organic compounds Usually, they use same organic compounds for getting energy reducing power and carbon.

Mixotrophs

    Mixotrophs are the organisms which have ability to utilize either categories of substances (organic or Inorganic), as source of energy, electron donor as well as carbon.
   Certain phototrophs are able to grow even as chemotrophs under special circumstances. For example, Rhodospirillum rubrum can grow as phototroph and obtain energy from light under anaerobic conditions. But the same organisms can grow as chemotroph under aerobie condition and obtain energy from oxidation of organic compounds.
   Similarly, all chemolithotrophs usually utilize CO₂ as source of carbon and grow autotrophically. But there are some organisms which derive their energy and reducing power from oxidation of Inorganic substances, but carbon from organic substrates. These organisms are also categorized as mixotrophs.

e.g. Desulfoubrio desulfuricans obtains reducing power from axidation of hydrogen. Yet It can also use organic carbon compounds as source of both electron donor as well as carbon.

Parasites

   Parasites are the organisms, which require living host cells for their growth. This is because of the lack of their biosynthetic capabilities for ATP and other cellular molecules or their complex for nutritional requirements for growth. They are, therefore, nutritionally related to both paratrophs and hypotrophs. These organisms may be grouped in to two categories:
   a. Obligate parasites
   b. Facultative parasites

Obligate parasites are the organisms. which essentially require host for growth. They can not grow on artificial nonliving media, e.g. Treponema pellidum, Chlamydiae, Rickettsine, Bdelloribrio etc.
Facultative parasites are the organisms. which can grow even on artiflcial non living media. They may be fastidious for growth factors in their nutrition. They may also be called us fastidious heterotrophs.


Immune Cells and it's Function

 Immune Cells and it's Function


 "A protective or defense reaction against foreign substances. These foreign substances, known as antigens, release from the surface of the lymphocytes, the signals for cellular or humoral immune response."

The immune defense mechanism of the body involves the action of white blood cells, or leukocytes. Leukocytes include neutrophils, eosinophils, basophils and monocytes, all of which are phagocytic, as well as two types of lymphocyte (T-cells & B-cells), which are not phagocytics. but are critical to the specific immune response and the humoral response.

T-cells

After their origin in the bone marrow, T-cells migrate to the thymus (hence the designation "T"), a gland just above heart. There they develop the ability to identify micro-organisms' and virus by the antigens exposed on their surfaces. Tens of millions of different T-cells are made, each specialized in the recognition of one particular antigen. No invader can escape being recognized by at least a few T-cells. There are four principal kinds of T-cells.

(A) Helper T-cells (Th) : Commander of the immune response, detects infection and sounds the alarm, initiating both T-cell & B-cell responses.

(B) Inducer T-cells :
immediate response to infection, mediates the maturation of other T-cells in the thymus i.e. oversce the development of T-cells in thynus.

(C) Cytotoxic T-cells (Tc):
(cells-poisoning) Detects and kill infected body cells, recruited by helper T-cells.

(D) Suppressor T-cells :
They terminate the immune response. Dampen the activity of T and B- cells, scaling back the defense after the infection has been checked.


B-cells

Unlike T-cells, B-cells do not travel to thymus, they complete their maturation in bone marrow (B-cells are so named because they were originally characterized in a region of chickens called the bursa). From the bone marrow, B-cells are released to circulate in the blood and lymph. Individual B-cells, like T-cells, are specialized to recognise particular foreign antigen. When a B-cell encounters the antigen to which it is targeted, it begins to divide rapidly, and its progeney differentiate into plasma cells and memory cells,

Plasma cells

Biochemical factors devote to the production of antibodies (proteins) directed against specific foreign substance. Antibodies produced stick like flags to that antigen where ever it occurs in the body, making any cell bearing the antigen for destruction. The immunity that Pasteur observed resulted from, such antibodies and from the continued presence of the B-cells that produced them.

The humoral immune response, carried out by B-cells, protects the body from bacteria and other invading cells, by labeling these cells for destruction.

The B-cell lymphocytes that carry out this response produce and secrete antibodies that circulate in the blood plasma, lymph and other extracellular fluids, hence the term HUMORAL (relating to fluid) is used in describing their immune responses. In response to antigen exposure, a B-cell divides to produce plasma cells that serve as short lived antibody factories, and to produce long-lived memory cells.

Mast cells

Initiator of the inflammatory response, which aids the arrival of leukocytes, at a site of infection, secrete histamine and are important in allergic responses.

Monocyte : Precursor of macrophage.

Macrophage :
The body's first cellular line of   defense, also serves as
antigen-presenting cell to B & T-cells  engulfs antibody covered cells.

Killer cells

They recognise and kill infected body cells, natural killer cells (NK) detect and kill cells infected by a broad range of invaders, killer (K) cells attack only antibody coated cells.

(Above mentioned are various type of cells of the Immune system).

'Basic Structure of an Antibody Molecule


 In our body there are two different modes of immune system, one is the cell mediated immune system, another one is known as a humoral immune system.

  Cell-mediated immunity involves T-lymphocytes. Cell mediated immunity fights against intracellular antigens. They kill infected cells tumor cells etc.

  But there are many pathogens which multiply in the extracellular spaces of the human body. These extracellular spaces are protected by B-lymphocytes. Which are responsible for humoral immunity.

Role of B lymphocytes in the basic structure of antibody

  B lymphocytes originated in bone marrow and they also complete their maturation. In the bone marrow after the maturation stage these cells are released into the blood. Mature B-cells keep recirculating between lymph, blood and secondary lymphoid tissues.

  When these mature B-cells recognise specific antigens they get activated. This recognition occurs via specific receptors present on B cells, which are known as B-cell receptors.

  Once activated these B cells proliferate and differentiate into  Memory b-cells and Plasma cells which are antibody secreting cells. Plasma cells produce and secrete antibodies specific to the antigen.

Antibodies

  Antibodies are glycoproteins. The basic structure of antibody resembles Y-shaped molecule having two antigen binding sites and a stock known as FC region.

   These antibodies circulate in the lymph and blood from there they reach the site of invasion by the pathogen. Antibodies bind to the pathogens or antigens and activate defense mechanisms that lead to the destruction of the pathogen.

  Antibodies are also known as immunoglobulins. this is because they belong to a group of glycoproteins known as globulins. The term immune reflects that these glycoproteins play major role in immunity.

Basic Antibody Structure

  All antibodies have a same core structure. It consists of four polypeptide chains
  - Two identical heavy chains designated as H chains and
  - Two identical light chains designated as L chains.

  Heavy chains of Antibody are the longer ones and light chains are the shorter ones. The term heavy and light refers to their molecular weights. Heavy chains have more molecular weights than light chains.

   Antibodies are polypeptides, so the N terminal of this polypeptide chain is present at the tip and C terminal is present at the base of each polypeptide chain.

Structure of an Antibody
   As per figure, image these chains are assembled into a Y-shaped structure.

• Each light chain is connected to a heavy chain via a disulfide bond.

• The heavy chains are connected to each other via two disulfide bonds.

• In the mid region besides these disulfide bonds a number of non covalent bonds are also present, which keep these chains together.

• The mid-region of antibody has considerable flexibility this region is known as the hinge region. Hinge region make possible the rotation and bending of antibody molecule. This hinge region of Antibody is reach in prolin amino acid.

• The stalk of this Y shaped antibody molecule is the stem region also known as FC region.

•  To each heavy chain short carbohydrate chains are attached. These carbohydrate chains serve many additional functions such as increasing the solubility of immunoglobulins.

Light Chains

   Light chains are the two shorter subunits of basic antibody molecule.
• Each light chain has a molecular weight of about 25 kD.
• Each chain contains about 220 amino acids.
• In humans there are two types of light chains
  -  Lambda(λ) chain and
  -  kappa(K) chains
Kappa chain is encoded on chromosome 2 and lambda chains are encoded on chromosome 22.
• Each antibody molecule produced by a B-cell will either have Kappa or lambda light chain but never both.
• In human 60% of light chains are Kappa and 40% are lambda.

Heavy Chains

heavy chains are the longer subunits of the antibody structure.
• Each heavy chain has a molecular weight of about 50 to 70 kD and each heavy chain contains about 440 amino acids.
• There are five types or classes of heavy chains in humans all encoded on chromosome 14.
• These five classes are designated by lowercase Greek letters gamma, alpha, mu, Delta and Epsilon. They are also returned as γ, α, μ, δ and ε respectively.

   Each light and heavy chain contained two distinct regions variable regions and constant regions.

Variable (V) region

• variable region refers to the first 110 amino acids of the N-terminal region
• In each heavy and light chain these regions are so called because the amino acid sequences in these regions have great variability.
• These regions are designated as VL in each light chain and VH in each heavy chain.
• It is the variable region of a light chain and a heavy chain, Which together form the antigen binding site
•  So there are two antigen binding sites in a core antibody molecule.
•  Variability of amino acid sequences is precisely organised.
•  These areas are called  hypervariable regions or complementarity determining regions (CDRs).
•  This is because these regions together form a structure which is complementary to the shape of specific antigen bound by the antibody.
• Scientists have found that there are three CDRs in the variable region of each chain. These are designated as CDR1, CDR2 and CDR3.
•  The intervening sequences between CDRs or hyper variable regions are known as framework residues. These intervening sequences have restricted variability. 
•  Antibodies can recognise diverse types of antigens because of these hypervariable regions

Constant (C) Region

   The region beyond the variable region of both heavy and light chain is known as constant region.
• They are so called because the amino acid sequence in these regions shows little variation among antibodies.
•  There is a single constant region in each light chain which is designated as CL.
•  Light chains are of two types Kappa chain and lambda chain. These chains differ from each other by minor differences in the constant region of light chain.
• Constant regions in heavy chains vary form 3 to 4.
• This depends on the antibody class and it is the constant region of heavy chain which forms the basis of this antibody classification.
•  In a particular class of antibody all antibodies have almost same constant region but constant region of one antibody class is different from the another class.
•  The constant regions of the heavy chains are designated as CH 1, CH 2, CH 3 and CH 4 starting from the N-terminal of the chain.
   Each variable and constant region in an antibody molecule also has at least one disulfide bond these are internal disulfide bonds.