Microbiology

Microbiology

1. Eukaryotes

   •  ~100 microns   
   • Protozoa
   • Fungi

2. Prokaryotes

   •  ~1-2 microns   
   • Bacteria
   • Archaea
   • Cyanobacteria
   • Mycoplasmas
   • Prokaryotic Cell Structure
     • Capsule/Slime Layer
       • All bacteria secrete some sort of glucocalyx, an outer viscous polysaccharide and polypeptide covering forming a gelatinous material.
       • If tightly bound to the cell wall, it is referred to as a capsule, if loosely bound, it is a slime layer
       • This protects the cell from other host cells and allows groups of prokaryotic cells to stick together as a colony for further protection. Glycocalyx also inhibits opsonisation (the process by which a cell is marked for ingestion and destruction by a phagocyte)
     • Cell Wall
       • Physical barrier that protects against mechanical damage and excludes certain substances May contain peptidoglycan
     • Cell Surface Membrane
       • Controls movement of substances in/out of the cell The membrane performs the same functions as eukaryotic membranes, but also the site of electron transport ADP phosphorylation (electron acceptors: O2, SO4-, NO3-, CO3 2-) 
       • The cell membrane follows the Fluid-Mosaic model: *Fluid because the phospholipid molecules can move relative to each other, giving the membrane a flexible structure, constantly changing in shape *Mosaic because other proteins embedded in the phospholipid bilayer are of different shapes and sizes, thus resembling a mosaic. 70% of the proteins are integral/intrinsic, completely spanning the bilayer and are mostly transporters and channels
       • The cell surface membrane also has deep invaginations called mesosomes => these were recognized to form after chemical fixation techniques were used on bacteria to chemically fix them in preparation for electron microscope => thus mesosomes are no longer considered to be part of normal bacterial cell structure. They are considered to be artifacts
     • Nucleoid
       • An irregular-shaped region visible in micrographs. Contains the single bacterial chromosome
     • Cytoplasm
       • Packed with ribosomes, proteins and DNA, but lacking a cytoskeleton and membrane-bound organelles
     • Flagellum
       • Aids movement of the cell due to its rigid, corkscrew shape and rotating base, helping it spin through fluid Motility allows positive and negative response to stimuli (chemotaxis) May be restricted singly to one pole of the cell (polar), in tufts (lophotrichous) or distributed in general over the whole cell (peritrichous). Flagella are strongly antigenic, and thus an important target for antibody responses
       • In spirochetes such as *Treponema pallidum* which causes syphilis, the flagella run between the inner and outer membrane in the periplasmic space, which is sometimes known as axial filaments
     • Granules
       • Also, referred to as 'inclusion bodies' These may be stores of glycogen or oil droplets, used for energy storage and osmoregulation.  These may be bound by protein or lipid proteins
     • Plasmids
       • Small circular strands of extra-chromosomal DNA often containing genes that provide antibiotic resistance
     • Pilus
       • Pili are proteinaceous hair-like appendages and function in attachment => either to other prokaryotic cells (the sex pili) involved in conjugation and plasmid transfer, or to host cells (the common pili)
     • Cell Envelope
       • This is the cell surface membrane and cell wall, plus an outer membrane if present. The main structural component of the cell wall is the peptidoglycan (a mixed polymer of N-acetylglucosamine => NAG, and N-acetylmuramic acid => NAM) The alternating NAG and NAM residues are linked by a beta-1,4 glycosidic linkage. Attached to the N-acetylmuramic acid is a peptide chain of 3-5 amino acids, which differ between Gram positive and Gram negative bacteria
       • Gram positive
         • In Gram positive bacteria, the peptidoglycan forms a thick layer (20-80 nm) external to the cell membrane and may contain other macromolecules. The layer is often found as multiple layers and helps prevent osmotic lysis of the cell. There is no periplasmic space and no storage of digestive enzymes => digestion is extracellular.  The polar polysaccharides and amino acids in the peptidoglycan layer make if very hydrophilic. This property allows Gram-positive bacteria to resist the activity of bile in the small intestine
         • The amino acids attached to N-acetylmuramic acid (NAM) are: *L-Alanine *D-Glutamine *L-Lysine *D-Alanine *D-Alanine These peptide chains can then form crosslinks between two linear chains which is called an interpeptide bridge
         • Lipotechoic Acid (LTA)
           • A major component of Gram-positive cell walls. It has: *highly antigenic properties, evoking an immune response *anchors the cell surface membrane to peptidoglycan layer since it traverses the cell wall *Structures of LTA varies between different species
       • Gram negative
         • The peptidoglycan forms a thin layer (5-10 nm) external to the cell membrane and again may contain other macromolecules.  There is a space between the peptidoglycan layer and the cell surface membrane known as the periplasmic space. Here, digestive enzymes are stored. It is overlaid by an outermembrane, anchored to lipoprotein molecules in the peptidoglycan layer. This outer membrane principally contains lipoproteins and lipopolysaccharides and is the main permeability barrier giving an intrinsic resistance to certain antiobiotics like Vancomycin
         • The amino acids attached to N-acetylmuramic acid (NAM) in the peptidoglycan layer are: *L-Alanine *D-Glutamic acid *meso-Diaminopimelic acid *D-Alanine Entry of hydrophilic molecules such as sugars and amino acids is necessary for nutrition; and is achieved through porins, which are special channels or pores in the outer membrane
         • Lipopolysaccharides (LPS) and Gram-negative bacteria
           • Lipopolysaccharides are the major component of the outer membrane of the Gram-negative bacteria, providing  structural integrity to the membrane. Their structure consists of: *O-antigen *Core compound, which split into inner and outer core *Lipid A
           • O-antigen
             • Composition varies from strain to strain Highly antigenic and in normal animals evokes an immune response Full length O-chains render the LPS smooth; the absence or shorter O-chains render the LPS rough Bacteria with rough LPS are my penetrable by hydrophobic antibiotics, since a rough LPS is more hyrdophobic
             • Core Component
               • Genus/family specific Consists of an inner and outer core of polysaccharides Directly attaches to Lipid A
               • Lipid A
                 • Responsible for much of Gram-negative cell's toxicity Little variation between species Consists usually of a phosphorylated glucosamine disaccaride, decorated with fatty acids.  The fatty acid domains are anchored in the outer membrane, and the rest of the LPS projects from the cell surface It is an endotoxin
       • Mycobacterial Cell Envelope
         • The mycobacteria (i.e. *Mycobacterium leprae* and *Mycobacterium tuberculosis*) have a distinct peptidoglycan-arabinoglycan-mycolic acid cell envelope, which is not typical of either Gram-positive or Gram-negative
         • The cell wall contains mycolic acids, giving the cell wall a waxy characteristic and making it highly hydrophobic. The cell wall allows it to survive within macrophages and other cells and against natural antimicrobials
         • The cell wall gives the mycobacteria specific staining properties (acid-fast bacteria)
         • Also contains porins to allow entry of hydrophilic molecules such as amino acids and sugars
         • Pseudoperiplasmic space between the cell surface membrane and cell wall
         • Glycolipids function as energy stores and also serve as markers for cellular recognition
   • Survival of bacteria in adverse conditions: endospores
     • Under conditions of environmental stress, where the cell cannot grow and divide, some bacteria are able to form highly resistant, dormant spores within their cells, known as endospores
     • The spore has a complex multi-layer surrounding a new bacterial cell, containing large amounts of calcium and dipicolinic acid, which are thought to confer the endospore's high resistance
     • Spores are dehydrated structures which may survive for centuries and have resistance to extreme temperatures and chemicals.  When environmental conditions improve, the endospores rapidly reconvert to normal existance as new bacterial cells
     • Endospores are abundant in soils and those of the Clostridium (i.e. *Clostridium tetani*) and Bacillus are particularly hazardous. Tetanus and anthrax are both associated with endospores infection of wounds
   • Cell Morphology of bacterial cells
     • Coccus (cocci) => e.g. *Staphylococcus aureus* Bacillus (bacilli) => e.g. *Bacillus subtilis* Vibrio (vibrios) => e.g. *Vibrio cholerae* Spirochetes/Spirillum => e.g. *Treponema pallidum*
   • The Gram stain
     • Developed by Hans Christian Gram in 1884, it is a means of differentiating bacteria into two large categories: Gram-positive bacteria and  Gram-negative bacteria However, note that not all bacteria can be classified by this technique
     • The technique is: *Apply crystal violet to a heat-fixed smear of a bacterial culture (purple stain) *Add iodine, which binds to crystal violet and traps it within the cell *Add alcohol or acetone => this washes out the crystal violet if the sample is a Gram-negative bacterium, whereas Gram-positive bacterium will retain its purple colour *Apply counter-stain of safranin => this gives a pink/reddish colour to Gram-negative bacteria
   • More methods of classifying bacteria
     • *Measuring total genome size (i.e. the molecular weight of DNA present) *Determining the ratio of the bases Guanine to Cytosine in the DNA, i.e. mol% = (G+C)/(G+C+A+T) x100 *rRNA sequencing *Dna Homology => the extent to which DNA sequences vary, as measure by:    =>Genome sequencing    => Genetic fingerprinting    =>Hybridisation
   • Taxonic Ranks
     • -Kingdom -Phylum -Class -Order -Family -Genus -Species

3. Viruses

   •  ~0.1 microns   
   • Viruses
   • Bacteriophages

4. Sub-Viral Particles

   •  ~0.01 microns   
   • Viroids
   • Prions
     • Supposedly are infectious proteins => proteins that are naturally found in the brain => some change in the folding of the protein leads them to cause formation of plaques within the brain => destroys tissues and impairs the function of the brain

5. Comparing Eukaryotic and Prokaryotic Cells

   • We can target bacteria and kill them and not our own cells because of the major differences between the two cells  => in bacteria, have a single, circular chromosome which is not protected by a nucleus => have different types of ribosomes => thus why you can target protein synthesis with antibiotics => have a cell wall (we don't) => if we attack the cell wall, it's not a metabolic process that affects us => bacterial membranes also tend to be less rigid than ours because they don't have sterols (e.g. cholesterol)
   • Compartmentalisation
     • Eukaryotic cells are compartmentalised => they contain membrane bound organelles, i.e. ER/Golgi Apparatus/Mitochondria and a distinct nucleus bound by a nuclear envelope
     • Prokaryotic cells are not compartmentalised => no ER, Golgi Apparatus, mitochondria, lysosomes, or distinct nucleus
   • DNA
     • DNA is present in Eukaryotic cells in chromosomes
     • DNA is present in Prokaryotic cells only as a single, circular chromosome
   • Ribosomes
     • Ribosomes in Prokaryotic cells are smaller, of the 70s (Svedberg unit, a measure of sedimentation velocity). Ribosomal subunits are 30s and 50s
     • Ribosomes in Eukaryotes are larger, of the 80s type. Ribosomal subunits are 40s and 60s
   • Membrane
     • Membrane of a prokaryotic cell does not usually contain sterols (e.g. cholesterol) However, note: there are tiny bacteria (mycoplasma) that don]t have a cell wall but do have cholesterols => rigidified by that
     • Membrane of a eukaryotic cell does contain sterols (e.g. cholesterol)

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