Structure, physiology and function of bacterial cell wall, cell membrane, capsule, spore, flagella, pilli, ribosome and

other cellular structure.

Smaller than 0.2m, more than 50m in diameter. Few Epulopiscium feshelsoni- upto 80m in diameter and more than 0.6 mm in length. Escherichia coli- avg size 1.1 to 1.5m wide to 2 to 6m long. Some spirochaetes-500m in length and mainly sulfur chemolithotrophs or Cyanobacteria are large.

Spherical (Coccus)

Diplococci(Neisseria) Streptococci(chain form)Lactococcus, Streptococcus Tetracocci(Micrococcus) Staphylococci -sarcinae

Rod(Bacillus)
Chain = Streptobacillus

Vibriod shapes Helical shape Spirilla and Spirochaete

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Flagella Pili Capsule Cell Wall Cytoplasmic membrane Cytoplasm Nuclear mateial Ribosomes Inclusions Vacuoles and Spores
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Organisms motile in nature possess appendages called flagella and cilia. Filamentous, slender, rigid, cytoplasmic appendages protruding through cell wall. Unbranched, long thread like structure composed of flagellin 20nm across 12-30nm in diameter 5-16m in length.
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Thin cannot be observed directly in bright field microscope stained with special technique designed to increase the thicknessstained with Phosphotungstic acid.

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Monotrichous; 1 flagella eg. Cholera vibrios Lophotrichous; tuft at one end e.g. Spirilla Amphitrichous; both ends e.g. Alkaligens faecalis Peritrichous; all around bacteria. E.g. Typhoid bacilli

Observe Picture in Micro Lab

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The Ultrastructure of Bacterial Flagella. Flagellar basal bodies and hooks in (a) gram-negative and (b) gram-positive bacteria

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Three parts a. filament b. Hook c. Basal body Filament lies external to the cell the hook-basal portion is embedded in the cell envelope. Hook-Wider region at the base of the flagellum The hook consist single type of protein function to connect the filament to the motor portion of flagellum.
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Basal body/motor is anchored in the cytoplasmic membrane and cell wall. The basal body consists of a small central rod that passes through a system of rings. In gm-ve, four ring connected to the central rod. The outer L and P ring associatec with the LPS and peptidoglycan layer. The inner M ring contacts the plasma membrane In gm+ve, two basal body ring, the inner ring connected to the plasma membrane and an outer one to peptidoglycan.
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A pair of protein called Mot is anchored in the cytoplasmic membrane and surrounds the inner ring. These protein actually drive flagellar motor causing a torque that rotate filament. Another set of protein called the Fli protein function as the motor switch reversing rotation of the flagella in response to intracellar signals;

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Individual flagellum grows not from the base, but from the tip. Synthesis of a flagellum takes place with flagellin protein molecule called self-assembly. Interestingly, all the information for the final structure of the flagellum resides in the protein subunits themselves. Flagella if broken, can continue to rotate and new flagellin passes through the filament to repair the broken off pieces.

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The rotary motion of the flagellum is imparted from the basal body, which function as a motor. Rotation of the flagellum requires energy, which is supplied by a hydrogen ion gradient across the cytoplasmic membrane. The straight line movement of bacteria is known as runs, and the turns-which occur when the bacteria stop-are called tumbles or twiddles.
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Speed of flagellar rotation is dependent upon the strength of proton motive force. Peritrichously flagellated organisms generally move in a straight line in a slow stately fashion. Polarly flagellated organisms, on the other hand move more rapidly, spinning around and dashing from place to place.

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a.

b.

Peritrichous: forward motion is imparted by all flagella rotating counter clock wise (CCW) in a bundle. Clock wise (CW) rotation causes the cell to tumble and then return to counter clock wise rotation leads the cell off in a new direction. Polar: cells change direction by reversing flagellar rotation (thus pulling instead of pushing the cell) and then return to pushing.

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Helps in motility. Acts as an antigenic substance for pathogenic species.

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Fimbriae. numerous shorter fimbriae are very evident in this electron micrograph of Proteus vulgaris (39,000).

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Hair like proteinaceous that extend from the cell membrane into the external environment Generally, 0.1m to 1.5m long 3 to 10nm in diameter. Found in some gram-ve bacteria and absent in gram +ve bacteria project from the cell surface as straight filaments.

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Occurrence Enterobacteriae: e.g. Proteus, Shigella, Salmonella, and Serratia Pili Generally, two types a. Common pili b. Sex pili or F(fertility)-factor In addition to these, Col I (colicin) pili is also present. Common pili are of 6 types based on their morphology, number per cell, adhesive properties and antigenic

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Common pili are adhesins that help bacteria attach to animal host cell surface, the first step in establishing infection. Sex pili-genetic material during bacterial matting i.e. conjugation. Colonization of pathogenic organisms. Staining Negatively by Phosphotungstic acid (PTA)

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Bacterial secretion, surrounding outside of the cell envelope, amorphous viscid material.

The viscid material diffuses in the surrounding medium and remain as a loose undemarcated secretion in which many cell are embedded, it is known as slime.
When more defined-capsule, less defined as a slime layer or glycocalyx.

Capsules-two category a. Macrocapsules b. Microcapsules

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Macrocapsules- 0.2m thick, seen under light microscopes. Microcapsules-narrower then true capsules and cannot be seen under light microscope. E.g. Neisseria meningitides, Streptococcus pyogenes and Haemophilus influenza. high prominent in virulent strians and its carbohydrates antigens vary greatly in stucture among strains. Quellung reaction
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Polysaccharide (pneumococcus, Klebsiella, enterobacter); polypeptide, polyglumatic acid (anthrax bacillus) or hyaluronic acid (streptococcus). 0.2m in width, 98% water, 2% solids. Homopolysaccharide are usually synthesized outside the cell from sucrose by Streptococcus mutans.

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Heteropolysaccharides are usually synthesized from sugar precursors within the cell attached to a lipid carrier molecule, transported across the cytoplasmic membrane and polymerized outside the cell. Function Protecting covering against antibacterial substance such as bacteriophage, enzymes. Capsules of pathogenic bacteria inhibit ingestion and killing by phagocytes.

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Promote attachment of bacteria to surfaces e.g, Streptococcus mutans -insoluble glycan. non-pathogenic a thick layer of cell called biofilm. Enhances bacterial virulence, certain bacteria are pathogenic only in capsulate state

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Capsular antigen in hapten in nature and specific for the bacteria. Significance They can be used for immunological distinction of closely related species. They are used as laboratory gel for filtration.

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A negative staining: capsule appears as a clear halo around the bacterium as the ink can not penetrate the capsule. Immunological method: capsular substance being antigenic- stained by serological method. After mixing a suspension of capsulated bacteria with specific antiserum, the capsule becomes refractile and visible, and appears swollen when examined under microscope. E.g. Quellung phenomenon
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Bacterial Capsules.Klebsiella pneumoniae with its capsule stained for observation in the light microscope

Bacteroides glycocalyx

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Layer, rigid lies outside the plasma membrane. Shape and protect them from osmotic lysis, and toxic substance.

Pathogenicity and site of action of different antibiotics.

Wall shape and strength-peptidoglyacan

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Gram positive-20 to 80nm thick homogenous peptidoglycan or murein lying outside the plasma membrane.

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Gram negative bacteria-2 to 7nm peptidoglycan layer, 7 to 8nm thick outermembrane. Periplasmic space Loose network of peptidoglycan Periplasm Gm ve- size of periplasmic space-1nm to 7nm Protein participate in nutrient acquisition, peptidoglycan synthesis, modification of toxic compound
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Enormous polymer composed of many identical subunits. N-acetylglucosamine and N-acetylmuramic acid, several A.a such as D-glutamic acid, Dalanine and meso-diaminopimelic acid

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Peptidoglycan Cross-Links. (a) E. coli peptidoglycan with direct cross-linking, typical of many gram-negative bacteria.

(b) Staphylococcus aureus peptidoglycan. S.

aureus is a gram-positive bacterium.

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Peptidoglycan Structure. A peptidoglycan segment showing the polysaccharide chains, tetrapeptide side chains, and peptide interbridges.
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The backbone- alternating N-acetylglucosmine and N-acetyl muramic acid. A peptide chain of four alternating D and L aminoacids is connected to the carboxyl group of Nacetyl muramic acid. Many bacteria substitute another diaminoacid usually in the third position for meso diaminopimelic acid.

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Teichoic Acid Structure. The segment of a teichoic acid made of phosphate,glycerol, and a side chain, R. R may represent Dalanine,glucose, or other molecules
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Thick, homogenous, consist primarily of peptidoglycan. Peptide interbridge Large amount of teichoic acids (polymer of glycerol or ribitol joined by phosphate groups) or teichuronic acid (gluconic acid containing polysaccharides) A.a D-alanine or sugar attached to glycerol or ribotol groups.
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Lipoteichoic acid-the teichoic acid are connected either the peptidoglucan itself by a covalent bond with the six hydroxyl of Nacetylmuramic acid or to plasmamembrane lipids. Teichoic acid-negative charge-help to give gm+ve cell wall its negative charge. Also believed to be involeved in concentrating metal ion from the surrounding.

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Lipopolysaccharide Structure. (a) The lipopolysaccharide from Salmonella.Abe, abequose; Gal, galactose; Glc, glucose;GlcN, glucosamine; Hep, heptulose; KDO, 2-keto-3deoxyoctonate; Man, mannose; NAG, N-acetylglucosamine;P, phosphate; Rha, L-rhamnose. Lipid A is buried in the outer membrane.

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Thin, peptidoglucan layer (less than 5 to 10% of the wall wt.) Outer membrane lies outside the thin peptidoglycan layer Membrane protein (Brauns lipoprotein)covantly linked to the underlying peptidoglycan and embedded in the outer membrane by hydrophobic end. Adhesion site
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Outer membrane constitute-LPS- large, complex contain both lipid and carbohydrate and has three parts Lipid A- Contain two glucosamine sugar derivatives each with three fatty acids and phosphate. Buried in outer membrane FA like caproic, lauric, myristic, palmitic and stearic.
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The core polysaccharide joined to lipid A. Contain galactose, glucose, rhamnose, mannose and unusual dideoxy sugars such as abequeous, colitose, paratose and tyvelose. The O side chain or O antigen is a polysaccharide chain extending outward from the core

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Contributes negative charge on the bacterial surface. LPS-core of sugar-common to the one group of bacteria that may differ from one species to another within bacterial group. It provides group specificity to bacteria. Antigenic in nature and are called O-antigen Resistant to phagocytosis. Pyogenic substance.
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Protective barrier- prevent entry of bile salts, antibiotics, permits passage of small molecules like glucose and monosaccharides. Prevent loss of constituents like periplasmic enzymes. Porin protein three porin molecules cluster together and span the outer membrane to from a narrow channel through which molecules smaller than the 600 to 700 daltons can pass.

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Provides aqueous channel that provides a passive non specific diffusion of solute across membrane. Act as transporting agent of oligosaccharide and nucleotide. Prominent role in uptake of solid molecules relatively of large size across membrane.

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The cell envelope of these organisms is considerably more complex than other bacteria. Mycolic acid (long branched chained fatty acids) covantly bound via a polysaccharide to peptidoglycan.

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Bacteria without cell wall or deficient cell walls are or four types. Mycoplasma: naturally occuring bacteria which lack cell wall. L-forms: they develop either spontaneously or in the presence of penicillin or other agent that interfere with cell wall synthesis. More stable than protoplasts and spheroplasts.

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Spheroplasts: produced by the growth with penicillin and are derived from gm- ve bacteria. Protoplasts: produced artificially by lysozyme and hypertonic medium and are derived from gram + ve bacteria.

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the precursor subunit (muramyl pentapeptide attached to uridine diphosphate, UDP) is synthesized in the cytoplasm and passed to the cell membrane. The subunit is moved enzymatically from the nucleotide to a lipid carrier (bactoprenol) and built into a completed subunit (disaccharide pentapeptide with attaced bridge peptide). The completed subunits are then exported to the cell wall.
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After release of the monomer the bactoprenol is recirculated in the cell membrane and used again. The glycan backbones of the existing cell wall is enzymatically broken (by autolusins) to allow insertion of the newly synthesized subunit If these enzyme are overactive, the cell wall becomes degraded and the high osmotic pressure of the cell bursts the cytoplasmic membrane killing the cell (autolysis). Cross linking of the peptide side chain of the inserted subunit to the existing chain then occurs enzymatically (penicillin binding proteins) Chapter 4

Completed subunits of teichoic and teichuronic acids are also synthesized in the cell membrane (on lipid carriers) before transport and insertion into the existing cell wall.

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Plasma Membrane Structure. The fluid mosaic model of bacterial membrane structure shows the integral proteins (blue) floating in a lipid bilayer. Peripheral proteins (purple) are associated loosely with the inner membrane surface. Small spheres represent the hydrophilic ends of membrane phospholipids and wiggly tails, the hydrophobic fatty acid chains. Other membrane lipids such as hopanoids (pink) may be present. For the sake of clarity, phospholipids are shown in proportionately much larger size than in real membranes.
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Thin, 5 to 10nm thick Electron microscope Contain both protein and lipid. Lipid amphipathic (assymmtric with polar and nonpolar ends) Polar end- hydrophilic Nonpolar end- hydrophobic and tend to associate with one another- bilayer in membrane Phospholipid- amphipathic
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Many small globular particles called membrane protein lie within the membrane lipid bilayer. Peripheral protein loosely connected membrane 20 to 30% of total protein Easily removed Soluble in aqueous solution Integral protein- 70 to 80% Not easily extracted Insoluble in aqueous solution
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Integral protein- amphipathic Hydrophobic region buried in the lipid Hydrophilic region project from the membrane surface.

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Transport of solutes into and out of the cell Housing enzymes involved in outer membrane synthesis, cell wall synthesis and in the assembly and secretion of extracytoplasmic and extracellular substances. Generation of chemical energy (i.e ATP) Cell motility Mediation of chromosomal segregation during replication Housing molecular sensors that monitor chemical and physical changes in the environment.
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Bilayer Phospholipid Water can penetrate Flexible Not strong, ruptures easily

Osmotic Pressure created by cytoplasm

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Invagination of the plasma membrane in the shape of vesicles, tubles or lamellae. Both gm +ve (more prominent) and gm-ve. Found next to septa or cross wall in dividing bacteria Cell wall formation Chromosomal replication

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Ribosomes packed in the cytoplasmic matrix and also attached to plasma membrane. Complex, made of both protein and ribonucleic acid (RNA). Site of protein synthesis Cytoplasmic ribosomes synthesized protein destined to remain within the cell Plasma ribosomes- make protein for transport to the outside. Procaryotic ribosomes are called 70S ribosomes Dimension-14 to 15nm by 20nm, mol wt.-2.7 million Chapter 4

50S and 30S subunit

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Visible in light microscope Present in cytoplasmic matrix Used for storage (e.g. carbon compound, inorganic substance and energy) Reduce osmotic pressure by tying up molecules in particulate form. Inclusion bodies lie free in the cytoplasm- e.g phosphate granules, cyanophycin and glycogen granules
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Inclusion bodies are enclosed by a shell about 2 to 4nm thick Single layer and consist of proteins and phospholipids. Eg poly--hydroxybutyrate granules, sulfur granules, carboxysomes and gas vacuoles

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Organic inclusion bodies- either glycogen or poly--hydroxybutyrate. Poly--hydroxybutyrate accumulate in distinct bodies- around 0.2 to 0.7m in diameter Readily stained with Sudan black for light microscopy and are seen empty hole in electron microscopy. Both inclusion bodies are carbon reserviour providing material for energy and biosynthesis.
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Cyanobacteria (photosynthetic bacteria)consist two organic inclusion bodies a. Cyanophycin granules- contain equal amount of arginine and aspartic acid Store extra nitrogen for bacteria. b. Carboxysomes contain Rubisco (ribulose-1, 5 biphosphate carboxylase) Critical enzyme for CO2 fixation, process of converting CO2 from the envt into the sugar.

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Organic inclusion body Present in photosynthetic bacteria and aquatic procaryotes such as Halobacterium and Thiothrix. Provides buoyancy Aggregates of enormous numbers of small, hollow, cylindrical structure called gas vesicles. Gas vesicles small protein

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Inorganic inclusion bodies 1. Polyphosphate granules- Many bacteria store phosphate as polyphosphate granules or volutin granules Function 1. Storage reserviors for phosphate , a component of nucleic acid 2. Energy reserve 2. Sulfur granules- store sulfur temporarily

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Chromosome is located in an irregular shaped region called the nucleiod/nuclear body/chromatin body. Usually single circle of double stranded deoxyribonucleic acid. Some linear DNA chromosome. Fibrous appearance In active growing cell- the nucleiod has projection that extend into the cytoplasmic matrix. Chemical analysis- 60% DNA, 30% RNA, 10% protein by weight.
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DNA is looped and coiled extensively. Do not use histone to package their DNA. Exception Pirellula has a single membrane Gemmata obscuriglobus two membrane

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(a) Nucleoids in growing Bacillus cells stained A model oftwo nucleoids in an actively growing E. coli using HCl-Giemsa stain and viewed with a light cell. Note that a metabolically active nucleoid is not microscope (bar 5 m). compact and spherical but has projections that extend into the cytoplasmic matrix.

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Small, double stranded DNA molecules exist independently of the chromosome. Circular and linear Less than 30 genes Cell lack them usually function normally Able to replicate normally Single copy plasmid produce only one copy per host cell plasmid integrated to chromosome and able to replicated with chromosome is called episomes. The lost of a plasmid is called curing.
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Based on the mode of existence, spread, function Conjugate plasmid-genes for the construction pilli. Eg, F factor Resistance factor- R factor typically have genes that codes for enzymes capable of destroying or modifying antibiotics. Bacteriocin encoding plasmid- eg. Col plasmid Virulence plasmid- encode factor that make their host more pathogenic.
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Metabolic plasmid- carry genes for enzymes that degrade substance such as aromatic compounds(tolune), pesticides and sugar

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Spores- specialized resistant cells that are produced by many microorganism. Bacteria produce spores either within the cellendospores. External to the cell- exospores. Endospores (endo= within) are differentiated cells formed during the process called sporulation, are highly resistant to heat, harsh chemicals, radiation and freezing. Two main genera- Bacillus and Clostridium.
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A. Gram- positive bacilli: 1. Obligately aerobic- genus Bacillus, e,g. Bacillus anthracis, Bacillus subtilis 2. Obligately anaerobic genus- Clostridia, e.g. Clostridium tetanus, Clostridium welchii, Clostridium botulinum. B. Other bacteria: Gram- positive coccus (Sporosarcina) and Gram- negative bacillus (Coxiella brunetii).
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a.
b.

c.

Spores may be Centrally located Close to one end (subterminal) Definitely terminal

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Exosporium- thin delicate covering Spore coat- lies beneath the exosporium, contain many protein, thick, impermeable, resistant to chemical. Cortex- occupy half of the spore volume, rests beneath the spore coat, made of peptidoglycan. Spore cell wall- inside the cortex and surround the protoplast or core.

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Core- contain ribosomes and nucleoid but metabolically inactive. Contain dipicolinic acid complexed with calcium ion. Dipicolinic acid- spore heat resistant Calcium- resistant to wet heat, oxidizing agent Calicium dipicolinate often stabilizes spore nucliec acids. Small, acid soluble DNA- binding proteins- protect from heat, radiation, dessication and chemicals.
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Dehydration of protoplast- very important in heat resistance. Spore coat- protect against enzymes and chemicals such as hydrogen peroxide. Spore contain some DNA repair enzyme

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1. 2.

3.

4.

Commences when growth ceases due to lack of nutrients. Complex process and divided into seven stages An axial filament of nuclear material forms. Followed by an inward folding of the cell membrane to enclose part of the DNA and produce the forespore septum. The membrane continues to grow and engulf the immature spore in a second membrane. Net cortex is laid down in the space between the two membranes and both calcium and dipolinic acid are
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5. Protein coats then are formed around the cortex. 6. Maturation of the spore occur. 7. Finally, lytic enzyme destroy the sporangium releasing the spore. 8. 10 hrs in Bacillus megaterium

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1. 2.

3.

Transformation of dormant spores into active vegetative cells. Activation Germination Outgrowth

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Activation treatment like heating. Germination- the breaking of spores state. - Spore swelling, rupture and absorption of spore coat. - Loss of resistance to heat and stresses - Loss of refractility - Release of spore component - Increase in metabolic activity. Outgrowth- spore protoplast makes new component, emerges from the remains of spore coat. - Develop again into an active bacterium.

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Withstand boiling upto 3 hrs, Dry heat at 150 0C for one hour. Destroyed by autoclaving at 121 OC for 15-20 mins and 1% aqueous solution of iodine in few hours.

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Determine shape of bacteria Strength prevents osmotic rupture 20-40% of bacteria Unique to bacteria Some antibiotics effect directly

Penicillin

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Gram + only Glycerol, Phosphates, & Ribitol Attachment for Phages

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Endotoxin or Pyrogen

Fever causing Toxin nomenclature

Endo- part of bacteria
Exo- excreted into environment

Structure

Lipid A Polysaccharide
O Antigen of E. coli, Salmonella

G- bacteria only

Alcohol/Acetone removes

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Functions

Toxic; kills mice, pigs, humans

G- septicemia; death due to LPS

Pyrogen; causes fever

DPT vaccination always causes fevers

Adjuvant; stimulates immunity

Heat Resistant; hard to remove Detection (all topical & IV products)

Rabbits (measure fever) Horse shoe crab (Amoebocytes Lyse in presence of LPS)
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Appearance of Colonies

Mucoid = Smooth (lots of LPS or capsule) Dry = Rough (little LPS or capsule) 2,000 different O Ags of Salmonella 100s different O Ags of E. coli
E. coli O157

O Antigen of Salmonella and E. coli

O Ags differ in Sugars, not Lipid A

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A number of gm+ve bacteria can form a special resistant, dormant structure called an endospore. Develop within vegetative bacterial cell of many genera such as 1.obligate aerobic-Bacillus anthracis, Bacillus subtilis 2. obligate anaerobicClostridium tetanus, Clostridium botulism and other Sporosarcina and Coxiella brunetii Resistant to environment stress such as heat, ultraviolet radiation, chemical disinfectants and dessication.

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Resistant structure

Heat, irradiation, cold Boiling >1 hr still viable

Takes time and energy to make spores Location important in classification

Central, Subterminal, Terminal Used for quality control of heat sterilization equipment Used in biological warfare
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Bacillus stearothermophilus -spores

Bacillus anthracis - spores

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G+

Thicker cell wall Teichoic Acids

Endotoxin - LPS

Which are more sensitive to Penicllin? Alcohol/Acetone affects which more?

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Cell Wall Teichoic Acids LPS Endospores Circular DNA Plasmids

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Eukaryote Cell Structure

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