Ultrastructure of bacterial cell Form and Function Structure

Ultrastructure of bacterial cell. Form and Function.

Structure of a Prokaryotic Cell

Bacterial Morphology and Ultrastructure • Only two types of cells are produced by all living organisms on earth. • Prokaryotes (pro. or primitive nucleus) do not have a membrane bound nucleus – eubacteria (true bacteria) – archaebacteria (ancient bacteria) • Eukaryotes (eu, or true nucleus) have a membrane bound nucleus – – – Algae fungi protozoa plants animals

Prokaryotes

Chemical Composition of Bacteria • • Water - 70% Dry weight - 30% composed of: – – – DNA - 5% MW 2, 000, 000 RNA - 12% protein- 70% found in: • • – – – Ribosomes(10, 000) – RNA Protein particles - MW 3, 000 Enzymes Surface structures polysaccharides - 5% lipids - 6% phospholipids - 4%

Prokaryote Structures: 1. Appendages- flagella, pili, fimbrae 2. Cell envelope- glycocalyx, cell wall , cell membrane 3. Cytoplasm- ribosomes, granules, nucleoid/chromosome.

Appendages

Bacterial Appendages: • Pili (pl), pilus (s) – only found in gram negative bacteria – tubulare, hairlike structures of protein larger and more rare than fimbriae. • 2 types of pili 1. atacnement pilus - allow bacteria to attach to other cells 2. sex pilus, pilus - transfer from one bacterial cell to another- conjugation.

Fimbriae • fimbriae (pl) fimbria (s) – Adhesion to cells and surfaces – Responsible for biofilms. – Pathogenesis of gonococcus and E. coli Escherichia coli.

Flagella • Flagella (pl), flagellum(s) – long appendages which rotate by means of a "motor" located just under the cytoplasmic membrane. – bacteria may have one, a few, or many flagella in different positions on the cell. • Advantages - chemotaxis - positive and negative. - motility • All spirilla, half of bacilli, rare cocci.

Structure of flagella allows for 360 degree filament rotation

Flagella Three morphological regions • Helical filament – – • long outermost region; composes up to 90% of its length contains the globular (roughly spherical) protein flagellin arranged in several chains and form a helix around a hollow core Hooked or curved area – • filament is attached; consists of a different protein Basal body – – – terminal portion of the flagellum fix the flagellum to the cell wall and plasma membrane composed of a central rod inserted into a series of rings Gram negative - 2 pairs of rings • • Outer pair - fixed to the outer membrane and peptidoglycan layer Inner pair - fixed to the plasma membrane (SM ring) Gram positive - only inner pair is present

Motility • Types of bacterial motility – – – • run or swim - when a bacterium moves in one direction for a length of time tumbles - periodic, abrupt random changes in direction swarming - rapid wavelike growth across a solid culture medium Mechanism of flagellar movement - relative rotation of the rings in the basal body of the flagellum Antigenicity – flagellar or H antigen - useful in the serological identification of serotypes of Salmonella organisms

Arrangements • Flagella vary in number and arrangement. • Polar arrangment – Monotrichious - 1 flagellum at one end • Fastest; Pseudomonas -example – Lophotrichious - tuft at one end – Amphitrichious- bipolar – Peritrichious - multiple flagella; randomly dispersed around the bacterial cell • E. coli - example

Flagellar arrangements A. Monotrichous B. Lophotrichous C. Amphitrichous D. Peritrichous E. Atrichous

Axial filaments

Axial filaments • tuft of fibrils that arise at the ends of the cell under the outer membrane and spiral around the cell • rotation an opposing of the outer membrane movement that propels the spirochetes by causing them to move like corkscrews • Found in Spirochetes and are similar to flagella, but are located between the cell wall and an outer membrane, and are attached to one end of the organism.

Evidence of motility Two ways by which motility can be demonstrated: • direct or microscopic – hanging drop preparation or wet mount preparation by dark field mycroscope – Distinguishes: • Brownian movement - when the bacteria show molecular movement • true motility - if a bacterium describes a rotatory, undulatory or sinuous movement • indirect or macroscopic – Stab inoculation of the semisolid media • nonmotile - growth is limited at the point of inoculation • motile - growth is diffuse or moves away from the line of inoculation; turbidity of the medium

Detection of Motility • Direct • Indirect Presence mobile bacteria

• Bacterial motility (Quick. Time movie) • http: //diverge. hunter. cuny. edu/~weigang/A nimations/Salmonella. Flagella-S. mov

Prokaryote Structures: 1. Appendages- flagella, pili, fimbrae 2. Cell envelope A. glycocalyx B. cell wall C. cell membrane 3. Cytoplasm- ribosomes, granules, nucleoid/chromosome.

2. Bacterial Surface Structure - cell envelope A. Glycocalyx - some extracellular material secreted by many bacterial cells in the form of: a. capsule - attached tightly to the bacterium and has definite boundaries. b. slime layer - loosely associated with the bacterium and can be easily washed off Compositions: 1. layer of polysaccharide 2. proteins - sometimes

Functions of the Capsule • • • Protection Identification Vaccine preparation Tissue attachment Antibiotic barrier

Medical Importance rapid serological identification of: • Several groups of streptococci • Meningococcus • Hemophilus influenzae • Klebsiella pneumoniae • Some of the coliforms • Yersinia and Bacillus specie •

Identification Two simple methods to distinguish the capsule India ink technique - most satisfactory method of demonstrating the capsule by Burri-Gins technique – Bacteria is suspended in diluted India ink – Stain with fuxin – Bacterial cells appear to lie in a lacunae and red cytoplasme. Quellung reaction - Homologous antibody is added to a preparation of capsule. – microprecipitation at the periphery of the capsule altering its refractive index rendering the capsule to be visible

Staining by Burri-Gins

Neisseria meningitidis - Gramnegative coccus, non-motile bacteria occur as two cells (orange) in a capsule (yellow)

Haemophilus influenza bacteria in the process of expressing polysaccharide capsules.

Cell wall Peptidoglycan (polysaccharides + protein), • Support and shape of a bacterial cell. The three primary shapes in bacteria are: • coccus (spherical), • bacillus (rod-shaped) • spirillum (spiral). • Mycoplasma are bacteria that have no cell wall and therefore have no definite shape.

Cell wall peptidoglycan (polysaccharides + protein) Components of the peptidoglycan layer: – Repeating glycan chains (N acetyl glucosamine and N acetyl muramic acid) – a set of identical tetrapeptide side chains attached to N- acetylmuramic acid – a set of identical peptide cross bridges

Peptidoglycan

Differences in Cell Wall Structure • Basis of Gram Stain Reaction – Hans Christian Gram- 1884 • Differential Stain – Gram Positive vs Gram Negative Cells • Gram Positive Cells– Thick peptidoglycan layer with embedded teichoic acids • Gram Negative Cells– Thin peptidoglycan layer, outer membrane of lipopolysaccharide.

Cell wall

Gram Stain Reaction • Hans Christian Gram- 1880 s • Divides bacteria into 2 main groups– Gram positive – Gram negative • Also- gram variable • Gram nonreactive • Gram positive bacteria – many layers of peptidoglycan and teichoic acids. – form a crystal violet-iodine-teichoic acid complex • Large complex, difficult to decolorize

Gram positive bacteria

Gram Stain Reaction • Gram negative bacteria – – Very thin peptidoglycan No teichoic acids Alcohol readily removes the crystal violet. Alcohol also dissolves the lipopolysaccharide of the cell wall. • Gram variable cells – Some cells retain crystal violet; some decolorize and take up the safranin – 4 factors • • Genetics- variable amount of teichoic acid. Age of culture- older cultures have variable amount of teichoic acid Growth medium- necessary nutrients not available Technique– smear not thin or evenly made. – Staining procedure not done correctly- decolorizer left on too long.

Gram negative bacteria

Gram stain technique

Gram stain

Gram stain

• Gram nonreactive cells – Have peptidoglycan but have very waxy- thick lipids –waterproof, dyes cannot enter either. – Examples- Mycobacterium tuberculosis and leprosy. • Alternative staining- acid fast stain

Cell wall deficient forms • L- forms ( Lister Institute where discovered) – Bacteria loses cell wall during the life cycle • Result of a mutation in cell wall forming genes • Induced by treating with lysozyme or penicillin which disrupts the cell wall – Protoplast • G + bacterium with no c. wall, only a c. membrane • Fragile, easily lysed – Spheroplast • G – bacterium loses peptidoglycan, but has outer membrane • Less fragile but weakened.

Surface structures continued: • Outer membrane – This lipid bilayer is found in Gram negative bacteria and is the source of lipopolysaccharide (LPS) in these bacteria – LPS is toxic and turns on the immune system. – Not found in Gram positive bacteria.

Lipopolysaccharide

C. Cell membrane • Located just under cell wall • Very thin • Lipid bilayer, similar to the plasma membrane of other cells. Transport of ions, nutrients and waste across the membrane • Typical – 30 -40% phospholipids – 60 -70% proteins • Exceptions– Mycoplasma- sterols – Archaea- unique branched hydrocarbons

Mesosome Extension of cell membrane – Folding into cytoplasm – internal pouch – Increases surface area. • Gram-positive bacteria-prominent • Gram negative bacteria- smaller, harder to see. • Functions– Cell wall synthesis – Guides duplicated chromosomes into the daughter cells in cell division.

Functions of Cell Membrane • Carries out functions normally carried out by eukaryote organelles. • Site for energy functions • Nutrient processing • Synthesis • Transport of nutrients and waste • Selectively permeable • Most enzymes of respiration and ATP synthesis • Enzyme synthesis of structural macromolecules – Cell envelope and appendages • Secretion of toxins and enzymes into environment.

Prokaryote Structures: 1. Appendages- flagella, pili, fimbrae 2. Cell envelope A. glycocalyx B. cell wall C. cell membrane 3. Cytoplasm A. B. C. D. Nucleoid/chromosome Plasmid Ribosomes Granules

3. Cell cytoplasm • Encased by cell membrane • Dense, gelatinous • Prominent site for biochemical and synthetic activities • 70 -80% water- solvent • Mixture of nutrients- sugar, amino acids, salts – Building blacks for cell synthesis and energy

A. Bacterial chromosome • Singular circular strand of DNA • Aggregated in a dense area- nucleiod • Long molecule of DNA tightly coiled around protein molecules.

B. Plasmids – Nonessential pieces of DNA • Often confer protection- resistance to drugs – Tiny, circular – Free or integrated – Duplicate and are passed on to offspring – Used in genetic engineering

Types of plasmid • Fertility-F-plasmids. They are capable of conjugation (transfer of genetic material between bacteria which are touching). • Resistance-(R)plasmids, which contain genes that can build a resistance against antibiotics or poisons and help bacteria produce pili. • Col-plasmids, which contain genes that determine the production of bacteriocins, proteins that can kill other bacteria. • Degradative plasmids, which enable the digestion of unusual substances, e. g. , toluene or salicylic acid. • Virulence plasmids, which turn the bacterium into a pathogen (one that causes disease).

Cell division in Prokaryotes • Prokaryotes use a relatively simple form of cell division - binary fission. • The diagram at 1. shows a bacterial cell. • The cell wall and membrane are in red, • the bacterial chromosome in blue, • the cytoplasm in light green, • the yellow dot represents a point of attachment of the chromosome to the cell membrane.

C. Ribosomes • Site of protein synthesis • Thousands – Occurs in chains –polysomes • 70 S – 2 smaller subunits – 30 S and 50 S

D. Inclusions • If nutrients abundant- stored intracellularly • Granules – Crystals of inorganic compounds not enclosed by membranes • Polyphosphate- corynebacterium • Sulfur granules- photosynthetic • Metachromatic- Mycobacterium

Bacterial Internal Structures • Endospores – inert, resting, cells produced by some G+ genera: Clostridium, Bacillus and Sporosarcina • have a 2 -phase life cycle: – vegetative cell – metabolically active and growing – endospore – when exposed to adverse environmental conditions; capable of high resistance and very long-term survival » Features of spores- size, shape, location=identification – sporulation -formation of endospores • • hardiest of all life forms Forms inside a cell- functions in survival not a means of reproduction withstands extremes in heat, drying, freezing, radiation and chemicals – germination- return to vegetative growth

Endospores • Resistance linked to high levels of calcium and dipicolinic acid • Dehydrated, metabolically inactive thick coat • Longevity verges on immortality - 25, 250 million years. • Resistant to ordinary cleaning methods and boiling • Pressurized steam at 120 o. C for 20 -30 minutes will destroy

Bacterial Shapes, Arrangements, and Sizes • Variety in shape, size, and arrangement but typically described by one of three basic shapes: – coccus - spherical – bacillus – rod • coccobacillus – very short and plump • vibrio – gently curved – spirillum - helical, comma, twisted rod, • spirochete – spring-like

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