Bacteriology_Module-2_Cell-Structure-Function.pptx

Module 2: Part 1&2 - Bacterial structure & function; Bacterial Cell Physiology  Bacterial Cell Structure & Function Basically, a bacterial cell consists of: 1. Cell envelope or Integument (outermost part)  Composed of: a.) Capsule b...

Module 2: Part 1&2 - Bacterial structure & function; Bacterial Cell Physiology  Bacterial Cell Structure & Function Basically, a bacterial cell consists of: 1. Cell envelope or Integument (outermost part)  Composed of: a.) Capsule b.) Cell wall c.) Cell membrane d.) Mesosome e.) Appendages - Flagellum Pilus Fimbriae 2. Cytoplasmic Component  Composed of: a.) Nuclear body b.) Ribosome c.) Endospores d.) Inclusions i.e., Granules I. CELL ENVELOPE A. Capsule  this is formed when large amounts of extracellular polymeric substances (EPS) forms a condensed, well-defined layer closely surrounding the cell.  It has a thicker, gummy consistency that gives a prominently sticky (mucoid) character to the colonies of most encapsulated bacteria. Glycocalyx- is formed when the EPS forms a loose meshwork of fibrils extending outward from the cell. - a slimy, gelatinous material produced by the plasma membrane. Slime layer - is formed when the EPS appears to be totally detached from the cell but in which the cell may be entrapped. Chemical composition:  Depending on the organism involved and the nutrient composition of the medium, the consistency of the capsule or slime layer is variable. It may consist of the following: a.) Polysaccharide -- e.g., Polymers of glucose and fructose can be synthesized by several strains of Streptococci. b. Polysaccharide complexes  polysaccharides may be complexed with various proteins.  some polypeptides occupy the spaces produced by the polysaccharide framework. c. Polypeptides  Capsules may consist of polypeptides of one or two amino acids. e.g., Bacillus anthracis produces a capsule consisting of polyglutamic acid. Site of Synthesis: Capsules, Glycocalyx and Slime layer are synthesized by enzymes located at the surface of the bacterial cell. Functions: 1. Capsules protect the cell from phagocytosis, unless they are coated with anti-capsular antibodies. 2. Capsule prevents some form of viruses to attach to bacterial cell wall. E.g., Bacteriophage, a bacterial virus, which destroys bacteria. 3. It prevents penetration of some antibiotics. 4. Glycocalyx plays a major role in the adherence of bacteria to surfaces in their environment, including cells of their plant and animal hosts. Example:  Streptococcus mutans - uses 2 enzymes in the synthesis of its glycocalyx: Glucosyl transferase and Fructosyl transferase to synthesize long-chain dextrans (poly-D-glucose) and levans (poly-D-fructose) from sucrose. These polymers are called Homopolymers. (Polymers containing more than one kind of monosaccharide are called Heteropolymers).  In some organisms, the presence of a capsule is associated with virulence (the degree to which some organisms are able to cause disease) e.g., Streptococcus pneumoniae Haemophilus influenzae Bacillus anthracis Yersinia pestis  these organisms are non-encapsulated when grown in ordinary medium but acquires a capsule if inside the animal body or humans.  Capsules are not essential for life of the microorganism, but makes survival longer due to its protective activity.  Capsules can be a source of vaccine. e.g., Bordetella pertussis Phase I cultures (killed organism) Methods of Identification:  Capsules can be readily visualized with the Wet film India Ink Method (Negative Staining Technique)  It gives a “stringy” texture to fluid culture and a moist, glistening surface to colonies grown on solid media. Bacterial Capsule Negative Staining- Principle, Reagents, Procedure and Result  The main purpose of Negative staining is to study the morphological shape, size and arrangement of the bacterial cells that is difficult to stain. eg: Spirilla.  It can also be used to stain cells that are too delicate to be heat-fixed.  It is also used to prepare biological samples for electron microscopy.  It is used to view viruses, bacteria, bacterial flagella, biological membrane structures and proteins or protein aggregates, which all have a low electron- scattering power. Principle of Negative Staining Negative staining requires an acidic dye such as India Ink or Nigrosin. India Ink or Nigrosin is an acidic stain. This means that the stain readily gives up a hydrogen ion (proton) and the chromophore of the dye becomes negatively charged. Since the surface of most bacterial cells is negatively charged, the cell surface repels the stain. The glass of the slide will stain, but the bacterial cells will not. The bacteria will show up as clear spots against a dark background. Reagents of Negative Staining India ink Nigrosin Nigrosin 100 gm/L, Formalin 5 ml/L in water 4. Rest one end of the clean slide on the center of the slide with the stain. Tilt the clean slide toward the drop forming an acute angle and draw that slide toward the drop until it touches the drop and causes it to spread along the edge of the spreader slide. Maintaining a small acute angle between the slides, push the spreader slide toward the clean end of the slide being stained dragging the drop behind the spreader slide and producing a broad, even, thin smear. 6. Focus a thin area under oil immersion and observe the unstained cells surrounded by the gray stain. Results of Negative Staining (Next Slide) Results of Negative Staining  It gives a “stringy” texture to fluid cultures and a moist, glistening surface to colonies grown in solid media.  It forms a “smooth” or “mucoid” colonies; non-encapsulated organisms form “rough” colonies.  It can be removed artificially by enzyme action or naturally by mutation. Slime Layer --  The outermost component of the cell wall of a bacterium that possesses it.  Can be found in both Gram (+) and Gram (-) bacteria.  A lattice made up of protein or glycoprotein molecules.  It has an uncertain function but is believed to protect the cell from: a.) wall-degrading enzymes b.) invasion by predatory bacteria c.) bacteriophages  It is involved in cell adhesion to host epidermal surfaces. B. Cell Wall  It is found beneath the capsule, lying between the cytoplasmic membrane and the capsule.  It is the major structural component of the bacterial cell.  It is a rigid and relatively non-elastic but highly ductile, high tensile-strength structure accounting for about 10-25% of the dry weight of the cell and varies in thickness from 10-20 nm.  It contains pores of approximately 1nm in diameter which admits small molecules to the cytoplasmic membrane. It is sufficiently porous to allow diffusion of metabolites to the plasma membrane. Chemical composition:  Bacteria owes its high tensile-strength to a layer composed of a substance known as Peptidoglycan (Murein or Mucopeptide).  The chemical composition of the cell wall allows the differentiation of 2 distinct types of bacteria, namely: Gram- positive and Gram-negative. Gram-positive Bacteria -- Composition: 1.) Peptidoglycan layer (peptide – amino acid; glycan – sugar) - a complex polymer consisting of 3 parts: a.) a backbone, composed of alternating sugars: N- acetylglucosamine N- acetylmuramic acid b.) a set of identical tetrapeptide side chains, attached to N- acetylmuramic acid c.) a set of identical peptide cross bridges  The backbone is the same in all bacterial species  The tetrapeptide side chain and the peptide cross bridges vary from species to species.  Important features common to tetrapeptide side chain: a.) most have L-alanine at position 1 (attached to N-acetylmuramic acid) b.) D-glutamate at position 2 c.) D-alanine at position 4  Position 3 is the most variable e.g., in Gram (-) bacteria, diaminopimelic acid is carried at this position.  Each peptidoglycan layer is a single giant molecule.  Gram (+) bacteria - as many as 40 sheets of peptidoglycan comprising up to 50-90% of the cell wall material (10-20% of the total dry weight of the cell) Gram (-) bacteria - only 1 – 2 sheets, comprising 5-20% of the wall material. 2.) 2 Specialized components -- A.) Teichoic acid - a water-soluble polymer, containing ribitol or glycerol residues joined through phosphodiether linkages. - believed to be covalently linked to the sugar residues in the peptidoglycan chain. B.) Polysaccharides -- - composed of: 1.) Neutral sugars - Mannose; Arabinose; and Rhamnose 2.) Acidic sugars - Glucoronic acid & Mannuronic acid  The composition of the teichoic acid formed by a given bacterial species can vary with the composition of the growth medium. e.g., Repeat units of glycerol, ribitol or ribitol and glycerol joined to a sugar residue such as glucose, galactose or N-acetylglucosamine. Functions of the Teichoic Acid: 1. Together with lipoteichoic acid, it constitutes the major surface antigen of Gram (+) organisms that possess them. e.g., In Streptococcus pneumoniae, the teichoic acid bear the antigenic determinants called Forssman Antigen. 2. It binds Magnesium ion that plays a role in the supply of this ion into the cell. - Magnesium ions are required for the stability of the cell membrane as well as membrane-bound enzymes. 3. It is involved in the activation of autolytic enzymes and in determining the susceptibility of the wall to autolytic activity. - e.g., Replacement of Choline by ethanolamine as a component of the teichoic acid of Pneumococcus causes the cell to resist autolysis. 4. Teichoic acid and polysaccharides are also known to participate in the binding of bacteriophages to some gram (+) bacteria. B. Gram –negative Bacteria -- Composition: 1.) Outer membrane (OM) - convoluted, wrinkled, creviced or undulating a.) Phospholipid b.) Protein - Matrix Protein or Porins which have transport or receptor functions.  Transport - as channels for small molecules or low molecular weight – soluble substances  Receptor - for bacteriophages c.) Lipopolysaccharides (LPS)  Heat-stable, lethally toxic, pyrogenic  Mitogenic for mouse but not human lymphocytes  activates the Hageman Clotting Factor in platelets.  activates Complement  it is responsible for many of the biologic activities associated with Gram (-) bacteria  - Composition: Continued from: Lipopolysaccharides (LPS) a.) Lipid A - a complex lipid/ a toxic lipid. - consists of a chain of glucosamine disaccharide units - an endotoxin of Gram (-) bacteria which is firmly bound to the cell surface and is released only upon cell lysis. b.) Polysaccharide - constant in all Gram (-) species - represents a major surface antigen of the bacterial cell, known as “O” antigen. Functions: 1. It prevents leakage of periplasmic proteins. 2. It protects the cell from bile salts and hydrolytic enzymes of the host environment (such as in enteric bacteria) 3. It hinders the penetration of large molecules hence, the relative resistance of Gram (-) bacteria to a number of antibiotics. Periplasmic space - is actually a compartment containing a variety of hydrolytic enzymes which are important to the cell for the breakdown of large macromolecules for metabolism. These enzymes typically include proteases, phosphatases, lipases, nucleases and carbohydrate- degrading enzymes. - in the case of pathogenic Gram (-) bacteria, many of the lytic virulence factors, such as collagenases, hyaluronidases, proteases and beta- lactamase are in the periplasmic space. - It also contains components of the sugar transport system and other binding proteins to facilitate the uptake of different metabolites and other compounds. B. Lipoprotein - the most abundant protein of Gram (-) cells. Function: It stabilizes the outer membrane and anchors it to the peptidoglycan layer. C. Peptidoglycan - accounts only for 1-2% of the dry weight of the cell. D. Nutrient-binding proteins Functions of the Cell Wall: 1. It maintains the morphology of the cell. It is responsible for the cell’s constant form. - it gives rigidity and shape to the cell. 2. The cell wall provides protection from osmotic lysis. - It allows the cell to withstand osmotic pressure for up to 20 atmospheres as a result of solute concentration via active transport. Without the cell wall the bacteria succumb to the large osmotic pressure differences across the cytoplasmic membrane resulting to lysis. 3. It is the site of various antigenic determinants of the cell surface. 4. It is the site of synthesis of macromolecules. - it may elicit certain toxic symptoms of diseases caused by Gram(-) bacteria. e.g., a source of endotoxin (LPS) for Gram (-) bacteria, 5. It provides protection from some antibiotics and destructive chemicals ( in Gram (-) bacteria) 6. It determines differences in Gram – stain reaction. 7. It provides the support necessary for propulsion by flagella. 8. During infection, the peptidoglycan can interfere with phagocytosis,& is mitogenic (stimulates mitosis of lymphocytes) and has pyrogenic activity ( induces fever) Gram- Staining Procedure -- Reagent Function Gram (+) Gram (-) Crystal violet Initial Stain Blue-violet/violet Blue violet/violet Gram’s Iodine Mordant Dirty bluish-brown Dirty bluish-brown Acetone-alcohol Decolorizer Blue-violet/ violet Colorless Safranin Counter stain/ Blue-violet/ violet Red final stain Wall-deficient variants: - Wall deficient forms occur naturally or may be produced artificially in the laboratory. A. Artificially - Produced 1. Protoplast - (Gram (+) bacteria) - a wall-less, osmotically sensitive spherical body. - results from lysis of the peptidoglycan layer of the cell envelope by: a.) enzyme digestion (Lysozyme) b.) Drugs (Penicillin) - its outermost structure is the cell membrane so that these cells are poorly protected and will lyse in environments that encourage uncontrolled influx of water. - Protoplasts are susceptible to lysis by physical trauma. e.g., Shaking a broth culture of protoplasts is enough to destroy many cells. 2. Spheroplasts - (Gram (-) bacteria) - cell wall is not completely destroyed; much of the outer membrane remains. - osmotically-sensitive - can be produced by growth in hypertonic environments in the presence of cell wall-synthesis inhibitors. e.g. Penicillin & Lysozyme B. Naturally – Produced -- 1. Mycoplasma - naturally exists without cell walls. - they are protected from osmotic lysis by: 1. the presence of sterols in the cell membrane 2. adopting a parasitic existence in the osmotically favorable environment of a eukaryotic cell. - grows very slowly and must be maintained in the laboratory on enriched hypertonic media 2. A group of bacteria found in marine environments that has unusual cell walls devoid of peptidoglycan. - can exist without cell wall because the salt concentration in the water prevents influx of water into the cell, thereby eliminating the danger of osmotic lysis. 3. L - forms - “L” for the Lister Institute in London where they were first discovered. - it can either be Gram (-) or Gram(+) cells that have lost all or some of their wall during growth in a natural host --- osmotically favorable environment within the human body, particularly in such areas as pus- filled wounds. - they differ in the amount of cell wall material that is left attached to the cell. - those with no cell walls are incapable of producing new cell walls. - because of their osmotic fragility, they are difficult to isolate or identify in most laboratories. - it is possible that L-forms are responsible for some infectious diseases for which no organisms can be cultured. - they may survive antibiotic therapy because of their lack of susceptibility to anti-peptidoglycan agents. E.g., Relapse of infection after completion of Penicillin therapy may result from the resumed growth of these surviving L- Anti-cell wall Agents: 1. Lysozyme - (Muramidase) - found in saliva, tears and phagocytes - it is capable of digesting the peptidoglycan of Gm(+) organisms. Action on Peptidoglycan: - Lysozyme catalyzes the hydrolysis of the bonds between the sugars in the polysaccharide chain of the peptidoglycan. - This cannot penetrate the Outer membrane of Gram (-) bacterium; pretreatment with EDTA which damages the OM, will however allow the penetration of Lysozyme, resulting to cell damage. 2. Penicillin - Affects cell wall synthesis by inhibiting the peptide bridge formation in the growing peptidoglycan. This action weakens the wall as the components of the cell wall are synthesized and is followed by bursting of the cell wall. - In Gram (-), peptidoglycan layer is less susceptible to Penicillin because of the presence of the OM. C. Cell Membrane / Cytoplasmic Membrane - A typical unit membrane; a very thin (5-10 nm.), flexible sheet molded completely around the cytoplasm. - A structure lying adherent inside the cell wall. - Accounts for some 30% or more of the cell weight. Chemical Composition: - A phospholipid-protein bilayer composed of: 60% Protein 40% Lipid - Membranes of Prokaryotes are distinguished from those of Eukaryotes by the absence of sterols. The only exception being Mycoplasma, which incorporates them into their membranes when growing in sterol-containing media. Functions: 1. Selective permeability and transport of molecules in and out of the cell. - forms a hydrophobic barrier impermeable to most hydrophilic molecules. - functions both as an osmotic link and as an osmotic barrier. - specific Protein enzymes (Permeases) are present in the cell membrane that facilitates the transport of the molecules across the membranes either through: A. Active Transport - an energy-dependent transport of specific solutes against a gradient (from low to high concentration where the cell must expend energy). - a process that requires energy (ATP).  B. Passive Transport - relies on diffusion, uses no energy and operates only when the solute is at higher concentration outside than inside the cell. 1. Simple Diffusion - requires NO ENERGY expenditure by the cell. - a passive process in which small molecules move from areas of higher concentration to areas of lower concentration (molecules move until equilibrium is reached) - provides neither speed nor selectivity e.g., Dissolve oxygen Carbon dioxide Water – through osmosis Osmosis - when the diffusing molecule is water - movement of water is from high to low concentration across a semi- permeable membrane until equilibrium is reached - since water passes freely through the membrane, its uptake or loss depends on its concentration in the environment relative to that of the cytoplasm and the available space inside the cell.  2. Facilitated Diffusion  uses no energy, so the solute never achieve an internal concentration higher / greater than what exists outside the cell.  it is however selective - channel proteins form selective channels that facilitate the passage of specific molecules.  common in Eukaryotes (E.g.,yeasts), but is rare in prokaryotes.  membrane proteins, i,e, Permeases have attachment sites for essential nutrient molecules. As these molecules bind to the Permease, they are pumped into the cell’s interior through special membrane protein channels. Example: transporting various ions i.e., sodium and iron and small organic molecules among microbes.  Glycerol is one of the few compounds that enters prokaryotic cells by facilitated diffusion. Facilitated Diffusion Active transport – Cell Membrane Permeability  C. Group Translocation (Vectorial Metabolism) - facilitates the net uptake of certain sugars (e,g.,Glucose & Mannose), the substrate becoming phosphorylated during the transport passage. - it is not active transport because no concentration gradient is involved. - this allows bacteria to use their energy resources efficiently by coupling transport & metabolism D. Endocytosis - (Phagocytosis & Pinocytosis) Phagocytosis - engulfment of solid particles through large extensions called pseudopods. Pinocytosis - fluids and/ or dissolved substances are pinocytosed into vesicles by very fine cell protrusions called microvilli. - oil droplets fuse with the cell membrane and are released directly into the cell. 2. Electron Transport & oxidative phosphorylation (in aerobic species) - Cytochrome and other enzymes and compounds of the respiratory chain, including dehydrogenases, are located in the cytoplasmic membrane.  Group Translocati on (Vectorial Metabolis m) 3. Excretion of hydrolytic enzymes - Hydrolytic enzymes excreted by the bacteria to break down or hydrolyze larger food molecules ( macromolecular organic polymers - Proteins, polysaccharides, lipids, which are sources of nutrients for microorganisms) outside the cell into smaller subunits, small enough to penetrate the cell membrane. - such extracellular digestion is mediated by enzymes released from the bacteria into their fluid environment. 4. Biosynthetic functions a.) Cell wall synthesis - it is the site of carrier lipids on which the subunits of the cell wall are assembledas well as of the enzymes of cell wall biosynthesis; biosynthesis of DNA, cell wall polymers and membrane lipids. b.) It bears the receptors and other proteins of the chemotactic and other sensory transduction system. sensory transduction - the mechanism by which a change in cell behavior is brought about in response to a change in the environment. c.) Enzymes of the phospholipid synthesis are also localized in the cell membrane. Excretion of Hydrolytic C. Mesosome - this is seen as membrane-associated cytoplasmic sacs which contain whorled, lamellar, tubular or vesicular structures and are often associated with division septa. - these are convoluted invaginations of the cell membrane; inward foldings of the cell membrane. - the genetic material (DNA) is actually connected to the mesosome and takes an active part in cell division. - they occur as irregular infoldings of the plasma membrane and as such, enhances the ability of the cell to concentrate nutrients because their folds increase the surface area of the plasma membrane. - it functions as the origin of the transverse septum that divides the cell in half and as the binding site of the DNA which will become the genetic material of each daughter cell. - It acts as an anchor to bind and pull apart daughter chromosomes during cell division. Appendages: 1. Flagella - these are threadlike appendages which are much thinner than the cilia of vertebrates or the flagella of protozoa. - Parts of a flagellum: 1. Basal body /granule - composed of a rod and 2 or more sets of encircling rings. - it is contiguous with the plasma membrane and peptidoglycan in Gm(+) bacteria and the outer membrane of the cell envelope in Gm (-) bacteria. 2. Basal Hook - facilitates rotation and turning of the filament 3. Filament - acts as a propeller Composition: It is composed of a single kind of protein subunit, Flagellin, with a molecular weight of 40,000. - Contains Epsilon-N-Methyl-Lysine, an amino acid found only in Flagellin. The Structure of a Flagellum in a Gram- Negative Bacterium. (The Flagellum is composed of a filament, a hook and a basal body)  Size: 12-30 nm. in diameter 16-20 μm. in length Function: for motility purposes, they can --  migrate towards environments favorable for growth and away those that might be harmful.  increase the concentration of nutrients or decrease the concentration of poisonous materials near the bacterial surfaces by causing a change in the flow of environmental fluids.  disperse flagellated organisms to uninhabited areas where colony formation can be achieved. Movement:  Flagella propel the cell by spinning around their long axis, like a propeller.  Flagellar rotation is powered by proton current provided by ATP.  Flagellar function is governed by chemotactic responses, indicating a sensory feedback regulation system. Negative Chemotaxis: In the presence of a repellent, coordination is lost, the flagellar bundle becomes disorganized, and the cell tumbles and tends to move away from the repellent. Positive Chemotaxis: Multiple flagella rotate counterclockwise to form a coordinate bundle and affect cell movement generally in the direction of a nutrient. Attractants: sugar, amino acid Sensory transduction: the mechanism by which a change in cell behavior is brought about in response to a change in the environment. Sensory transduction is responsible for: 1. chemotaxis 2. aerotaxis - movement towards optimal oxygen concentration. 3. phototaxis- movement of photosynthetic bacteria towards light. 4. electron acceptor taxis - movement of respiratory bacteria towards alternative electron acceptors, such as Nitrate and fumarate.  Coordination of flagellar function involves chemoreceptors, known as Periplasmic Binding Proteins (they interact in membrane transport and at the methylation level of a specific plasma membrane protein). Factors affecting flagellation: 1. composition of the medium - Flagella rotate normally if the medium contains a suitable substrate for respiration or when a proton gradient is artificially established. 2. pH 3. The liquid or solid state of the medium  True motility should be differentiated from Brownian movement ( a quivering to and fro motion; caused by the bombardment of the bacteria by various molecules of the fluid in which they are suspended.) Serologic properties of flagella:  H Antigen - derived from flagellated organisms. - derived from the German word “Hauch” indicating a spreading film of growth (like breath condensing on a cold glass surface)  O Antigen - somatic antigen - derived from the German term “Ohne Hauch (without film) - present in nonflagellated organism Arrangement - a.) Monotrichous - a single polar flagellum b.) Amphitrichous - one flagellum at each pole c.) Lophotrichous - a tuft of polar flagella d.) Peritrichous - flagella are distributed around the cell Atrichous - without flagella  Flagella are not required for viability. Once removed, new ones are rapidly formed and motility is restored in 3-6 minutes. Ways to remove flagella: 1. Shaking with glass beads 2. Agitation in a blender 2. Axial Filament (Endoflagellum; periplasmic flagellum) - A flagellum-like structure - Each filament is composed of 2 fibrils identical in structure to flagella; the fibril originate at each end of the organism and extend towards the other end between 2 layers that make up the cell wall; the fibrils overlap in the organism’s mid-region. - it occurs in the periplasmic space between the inner and outer member of the cell. - it imparts a twisting, screw-like motion to the cell. ( the twisting movement causes the rigid spirochete body to rotate like a corkscrew) 3. Pilus (Plural: Pili) - Pili ( L: ‘hairs” ); hair-like microfibrils which are shorter, thinner and finer than flagella; a rigid structure. - it is composed of protein subunits known as “Pilins”. - measures 0.004-.008 μm in diameter;.5-2.0 μm in length. - they originate in the periplasmic membrane. - Pilin molecules are arranged helically to form a straight cylinder that does not rotate and lacks a complete basal body. - Minor proteins located at the tip of the pili are responsible for the attachment Axial Filament (Endoflagellum; periplasmic flagellum) 2 Classes of Pili (categorized according to functional activity) 1. Ordinary Pili - plays a role in the adherence of symbiotic bacteria to host cells. 2. Sex Pili - this is responsible for the attachment of donor and recipient cells in bacterial conjugation. Examples of Pili: 1. Adhesin - an adherence factor; “colonization antigens” - allows bacterium to adhere to and colonize specific host tissue cells. Example: Enteropathogenic E. coli , Neisseria gonorrheae 2. Evasin - prevents phagocytosis. 3. Aggressin - leukocidal 4. Lectins - allows a bacterium to bind to specific sugars on cell surfaces Example: α-Methyl-D-Galactose (Pseudomonas) D- Galactose (Streptococcus pyogenes) 5. Sex Pili - site of attachment of conjugating cells (cell to cell adhesion in conjugation) Example: donation of genes to recipient cell by the presence of sex pilus  Pili of different bacteria are antigenically distinct and elicit the formation of antibodies by the host.  Antibodies against the pili of one bacterial species will not prevent the attachment of another species. Some bacteria are able to make pilus of different antigenic types and thus can still adhere to cells in the presence of antibodies to its original type of pili. Example: N. gonorrheae II. Cytoplasmic Component 1. Nuclear Body - irregular, thin, fibrillar DNA network which frequently runs parallel to the axis of the cell (as seen in Electron Microscopy) - during multiplication, bacterial DNA remains as a diffused chromatin network and never aggregate to form a well- defined chromosome during cell division in contrast to the eukaryotic cell. - the nucleus is not well defined in prokaryotes because of the absence of a nuclear coat. - bacterial DNA can be detected as Nucleoids or chromatin bodies using the Feulgen stain.(It is difficult to demonstrate chromatin bodies by direct staining because of the high concentration of RNA which can be removed by pretreatment with ribonuclease.) - represents only 2% to 3% of the cell weight but occupies 10% or more of the cell 2. Ribosome  small cytoplasmic particles which are composed of approximately 30% Protein and 70% RNA.  the site of protein synthesis; workbench of protein synthesis.  the cytoplasm of the prokaryote contains thousands of these very small structures, which give the cytoplasm a granular appearance. 3. Endospores  these are highly refractile bodies formed within the vegetative cell at a certain stage of growth.  these are formed when nutritional conditions become unfavorable, especially when there is depletion of Nitrogen or Carbon or both.  It is highly resistant to desication (dryness), heat and chemical agents, such as acids, bases, certain disinfectants and even radiation.  it is released when the mother cell undergoes autolysis.  it germinates when subjected to favorable nutritional conditions and is activated.  the size, shape and the position of the spores are diagnostic.  sporulation takes about 7 hours.  the heat resistance of the spore is due in part to their dehydrated state and in part to the presence of large amounts of Calcium dipicolinate / dipicolinic acid and large quantity of Calcium salts in the core. Riboso me  Calcium ions and Dipicolinic acid contribute to the endospore’s heat resistance by stabilizing protein structures.  Germination process: 1. Activation - activated in a nutritionally-rich medium - agents that can overcome spore dormancy: a.) heat b.) abrasion c.) acidity d.) compounds containing sulfhydryl groups 2. Initiation - the germination proper; requires water and germination agents ( the amino acid L- alanine in one species or by adenine in other species.) 3. Outgrowth - occurs in the presence of adequate nutrients. - a period of active biosynthesis which terminates in cell division. - Protein and RNA are synthesized in about one hour, DNA synthesis begins → the cell now is a vegetative cell and undergoes binary fission. 4. Inclusions – Granules: - indicates accumulation of food reserves. Types: 1. Metachromatic Granules the stored form of phosphate (Polyphosphate); generally formed by cells that grow in phosphate-rich environments. they stain red with certain blue dyes such as, Methylene Blue & Toluidine Blue. collectively called Volutin granules e.g., Babes Ernst granules Found in Algae, Fungi, Protozoans & Bacteria 2. Polysaccharide granules - composed of glycogen and starch - can be demonstrate with the application of stains, e.i.,Iodine. glycogen - reddish brown starch - blue 3. Lipid granules - commonly found as storage material. - poly-β-hydroxybutyric acid – a polymer that is unique to bacteria - Can be demonstrated by the use of fat soluble dyes.i.e., Sudan dyes - Appear in various species of Mycobacterium, Bacillus, Azotobacter, Spirillum Granu les 4. Sulfur granules - serve as energy source - certain bacteria known as “Sulfur bacteria” which belong to the Genus Thiobacillus, derive energy by oxidizing sulfur and sulfur-containing compounds. 5. Carboxysomes - these are polyhedral or hexagonal inclusions that contain the enzyme Ribulose 1,5 – diphosphate carboxylase. e.g., Nitrifying bacteria, Cyanobacteria & Thiobacilli 6. Gas vacuoles - these are hollow cavities found in many aquatic Prokaryotes, including: Cyanobacteria, Photosynthetic bacteria and Holobacteria - each vacuole consists of rows of several individual gas vesicles which are hollow cylinders constructed of proteins. - Function: to maintain buoyancy so that the cells can remain at the depth in the water appropriate to their receiving sufficient amount of O2, light & nutrients. 7. Plasmids - these are self-replicating circles of DNA that are extrachromosomal elements. That is, they are not connected to the main bacterial chromosome and replicate autonomously. - plasmids may be gained or lost without harming the cell. - most commonly found in Gram (-) bacteria - although not essential for cellular survival, they provide a selective advantage: many confer resistance to one or more antibiotics. Types of Plasmids: 1. Conjugative Plasmids - genes for carrying out their transfer to another cell ( and for transferring the bacterial chromosome or other plasmids). 2. Dissimilation Plasmids - have genetic coding for enzymes that catalyze the catabolism of certain unusual sugars and hydrocarbons. e.g., Some species of Pseudomonas can actually use such substances as Toluene, camphor and the high molecular weight hydrocarbons of Petroleum as primary carbon and energy sources. 3. Bacteriocinogenic Plasmids - contain genes for the synthesis of bacteriocins (toxic proteins that kill other bacteria) 4. Resistance Factor (R factor) - Carry genes that determine the resistance of their host cell to antibiotics, heavy metals or cellular toxins. - Many R factors are conjugative plasmids that contain 2 groups of genes: 1.) One group has the Resistance Transfer Factor and includes genes for replication and conjugation. 2.) R genes - codes for resistance and contains genetic instructions for the production of enzymes that inactivate certain drugs. - Different R factors, when present in the same cell, readily recombine to produce R factors with new combinations of R genes. - widespread use of antibiotics has led to the preferential survival of bacteria that have R factors. Transposons - These are pieces of DNA that move readily from one site to another, either within or between the DNAs of bacteria, plasmids and bacteriophage. - Also named “Jumping genes” because of their unusual ability to move. - Moves from one chromosome site to another: from a chromosome to a plasmid or, from plasmid to a chromosome. - Transposons contain DNA that codes for enzymes needed to remove and reintegrate the transposon to another site in the genome. - transposons code for drug resistance enzymes, toxins or a variety of metabolic enzymes. - It can either cause mutations in the gene into which they insert or alter the expression of nearby genes. - In contrast to plasmids or bacterial viruses, transposons are not capable of independent replication; they replicate as part of the recipient’s DNA. - More than one transposon can be located in the DNA. - Over-all effect of Transposon: to scramble the genetic language. - This effect can beneficial or adverse, depending upon such variables as a.) where insertion occurs in a chromosome. b.) what kinds of genes are relocated c.) the type of cell involved. - In bacteria, transposons are known to be involved in a.) changes in traits such as colony morphology, pigmentation and