Lecture 7v1(2)

Lecture 7v1(2) - Lecture 7 Building a prokaryo4c cell...

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Unformatted text preview: Lecture 7 Building a prokaryo4c cell Bacterial Cell Organiza4on Features found in most bacteria: •  Internal Structures –  Nucleiod •  Bacterial Envelope –  Cell Wall –  Membrane(s) * Bacterial Cell Organiza4on Features found in most bacteria: •  Internal Structures –  Cytoplasm composi4on –  Nucleiod •  Bacterial Envelope –  Cell wall –  Membrane(s) Other Features (not found in all cells): –  Internal Structures •  Gas vessicles –  External Structures •  Capsule •  Flagellum, •  Pili –  Developmental Structures •  Endospore •  Stalk Nucleoid or Nuclear region •  NOT surrounded by a membrane •  Amorphous Body located roughly in the center of the cell –  Cons4tute ~10% of the cells’ volume •  Chromosome Size –  Example: E. coli, which is 2 ­3 µm in length, has a chromosome approximately 1400 µm long. Nucleoid (DNA condensa4on) •  DNA condensa4on required –  Histone ­like proteins bind to the DNA –  The DNA is supercoiled into around 50 chromosomal domains and making it more compact. http://student.ccbcmd.edu/courses/bio141/ lecguide/unit1/prostruct/u1fig12.html Nucleoid •  Chromosomal DNA –  Most bacteria have a single circular, double ­stranded, supercoiled, DNA chromosome •  Excep4ons: –  Some have linear chromosomes: Streptomyces –  Some have mul4ple chromosomes: Rhodobacterium spherodes has two circular chromosome Bacterial Ribosomes •  Bacteria have 70S ribosomes composed on two subunits (S=Svedberg units) –  50S Large subunit: •  31 proteins •  23S rRNA and 5S rRNA –  30S Small subunit: •  21 proteins •  16S rRNA  ­ sequence used for evolu4on studies Bacterial Envelopes •  Cri4cally important structure –  Confers shape & protec4on from lysis •  Bacteria are classified based on their reac4on in a Gram stain –  Gram ­posi4ve bacteria –  Gram ­nega4ve bacteria Gram stain Bacterial Envelopes •  Comparison of the Bacterial Envelopes of Gram ­posi4ve and Gram ­nega4ve bacteria Gram posi4ve versus Gram nega4ve bacteria •  Gram ­posi*ves –  Single thick homogeneous pep4doglycan layer outside the cell membrane •  Gram ­nega*ves –  Thin pep4doglycan layer outside the cell (inner) membrane –  Outer membrane is present outside the pep4doglycan layer –  Periplasmic space between the inner and outer membranes Common feature ­ The cell membrane •  Comparison of the Bacterial Envelopes of Gram ­posi4ve and Gram ­nega4ve bacteria Bacterial Cell Membrane •  Other Names –  Some4mes referred to as the cytoplasmic membrane –  In Gram ­nega4ve bacteria, it is also called the inner membrane. •  Structure –  Fluid Mosiac similar to eukaryo4c cell or “standard” membrane –  Consists of a phospholipid bilayer and proteins Bacterial Cell Membrane Cell Membrane Func4on •  Func4ons: –  Highly selec4ve permeability barrier –  Contains proteins involved in transport, bioenerge4cs, and chemotaxis –  Site of energy genera4on and in its energized state is referred to as a proton mo4ve force (PMF) Transport •  There are at least three systems •  All of these systems require energy either from the proton mo4ve force, ATP, or some other energy rich compound Membrane spanning transporters Impact of Sterol ­like molecules on Membranes •  Examples: –  Cholesterol –  Hopanoids •  Sterol ­like molecules may strengthen the membrane and make it less flexible as a result of their rigid planar structure Archaeal cell membranes are Chemically Unique •  Bacteria & Eucaryotes =ester linkage between glycerol and the side chains •  Archaea= ether linkage between glycerol and the hydrophobic side chains Archaeal Membranes are Chemically Unique •  Archaea= ether linkage between glycerol and the hydrophobic side chains •  Archaea lipids lack fafy acids and instead have repea4ng units of the five ­carbon hydrocarbon isoprene •  Major classes of archaeal lipids are glycerol diethers and glycerol tetraethers Archaeal Membranes: unique bilayers or monolayers What is the advantage of a lipid monolayer? •  Lipid monolayers are resistant to peeling apart. •  Found in hyperthermophilic bacteria can grow in boiling hot springs –  Habitat: •  The water is boiling •  High arsenic levels •  Example: Norris Geyser basin (Yellowstone) The Cell Wall or the Pep4doglycan •  Comparison of the Bacterial Envelopes of Gram ­posi4ve and Gram ­nega4ve bacteria . Importance of the cell wall Func4on: •  Provides shape and rigidity to cell •  Allows cells to withstand pressures that would otherwise cause the cell to lyse Medical Importance: •  Target of many an4bio4cs that impact Gram ­nega4ve and Gram ­posi4ve bacteria differently: –  Pencillin –  Cephalosporins –  Vancomycin Pep4doglycan (or Murein) Layer •  Enormous Polymer –  Two Sugar deriva4ves •  N ­Acetylglucosamine (NAG) •  N ­Acetylmuramic Acid (NAM) –  Four Amino Acids •  L ­Alanine •  D ­Glutamic Acid •  Either lysine or Diaminopimelic Acid (DAP) •  D ­Alanine –  Remember: D amino ­acids are never found in proteins Basic structure: NAG------NAM------NAG L-Ala D-Glu DAP (or Lys) D-Ala Basic structure simplified NAG------NAM------NAG L-Ala D-Glu DAP (or Lys) D-Ala NAG ­ ­ ­ ­ ­ ­NAM ­ ­ ­ ­ ­ ­NAG 4 AA (4 amino acids) Bacterial Pep4doglycan Layer •  Glycan backbone composed of alterna4ng NAM & NAG connected by a β ­1,4 linkage (see next slide) •  Tetrapep4de Side Chains afached to NAM –  NAG ­ ­NAM ­ ­NAG ­ ­NAM—NAG ­ ­NAM (Glycan) 4AA 4AA 4AA (Tetrapep4de) Basic structure: NAG------NAM------NAG L-Ala D-Glu DAP (or Lys) D-Ala Linkage between layers: Transpep4da4on •  Chains of Pep4doglycan Subunits are joined and results in a large, strong, flexible, network ­like molecule by transpep4dases NAM ­ ­ ­ ­ ­NAG ­ ­ ­ ­ ­NAM ­ ­ ­ ­ ­NAG ­ ­ ­ ­ ­NAM ­ ­ ­ ­ ­NAG ­ ­ ­ ­ ­ 4AA ­ ­ ­ ­4AA 4AA ­ ­ ­ ­4AA 4AA ­ ­ ­ ­ ­4AA NAG ­ ­ ­ ­ ­NAM ­ ­ ­ ­ ­NAG ­ ­ ­ ­ ­NAM ­ ­ ­ ­ ­NAG ­ ­ ­ ­ ­NAM ­ ­ ­ ­ ­ Gram ­nega4ve Pep4doglycan Layer NAG ­ ­ ­ ­ ­NAM ­ ­ ­ ­ ­ ­NAG •  Thin layer or sheet composed en4rely of pep4doglycan –  1 ­2 layers thick •  Tetrapep4des side chains are directly linked: –  Amino group of DAP to carboxyl group of the terminal D ­Ala L ­Ala D ­Glu DAP D ­Ala D ­Ala DAP Direct link D ­Glu L ­Ala NAM ­ ­ ­ ­ ­NAG ­ ­ ­ ­ ­ ­NAM ­ ­ ­ ­ Gram ­posi4ve cell wall Tetrapep4de Side Chains are linked by pep4de interbridges, the kind and number of amino acids varying from organism to organism NAG ­ ­ ­ ­ ­NAM ­ ­ ­ ­ ­ ­NAG L ­Ala Pep4de interbridge D ­Glu L ­Lys ­ ­ ­ ­ ­ ­ ­#aa ­ ­ ­ ­ ­ ­D ­ALA D ­Ala L ­Lys D ­Glu L ­Ala NAM ­ ­ ­ ­ ­NAG ­ ­ ­ ­ ­ ­NAM ­ ­ ­ ­ Gram ­posi4ve cell wall •  Composed mostly of pep4doglycan –  Can have as many as 25 layers •  Teiochoic acid may be embedded within the cell wall –  Found only in Gram ­ posi4ve cells Diversity in Pep4doglycan •  Pep4doglycan is present only in species of Eubacteria –  Archaea have “pseudopep4doglycan,” •  NAM and DAP are not found in the “cell walls” of Archaea or Eukarya •  DAP found in all Gram ­nega4ve bacteria and some Gram ­ posi4ve bacteria –  Some Gram ­posi4ve bacteria contain L ­Lys instead of DAP Impact of Lysozyme on Pep4doglycan •  Lysozyme is found in animal secre4ons –  Tears, saliva, etc. –  Line of defense? •  Breaks β ­1,4 linkage between NAG and NAM thus weakening the wall Target of Penicillin: Transpep4da4on •  Penicillin inhibits the transpep4da4on reac4on which is the final step in cell wall biosynthesis •  This explains why pencillin primarily kills ac4vely growing cells Target of Vancomycin: •  Vancomycins: –  Bind to the pep4des of the pep4doglycan monomers –  Block both the forma4on of gycosidic bonds between the sugars (NAM & NAG) and the forma4on of the pep4de cross ­links between layers –  This results in a weak cell wall and osmo4c lysis of the bacterium. Features found only in Gram ­nega4ve bacteria •  Comparison of the Bacterial Envelopes of Gram ­posi4ve and Gram ­nega4ve bacteria Periplasmic Space •  Located between inner (cell) and outer membranes in Gram ­nega4ve bacteria •  Contains many enzymes and proteins –  Hydroly4c enzymes: diges4on –  Binding proteins: transport –  Chemoreceptors: chemotaxis response •  Helps Gram ­nega4ve bacteria respond to osmo4c stress The outer membrane Proper4es of the outer membrane •  Asymmetric bilayer –  Outer leaflet contains lipopolysaccharide (LPS) –  Inner leaflet contains lipoproteins and not LPS •  Lipoproteins func4ons as anchors between the outer membrane and pep4doglycan layer •  Result: •  Outer membrane is less permeable than the cell membrane 49. Lipopolysaccharide (LPS) •  Found in the outer leaflet of outer membranes •  LPS is frequently toxic to animals –  Toxicity usually associated with lipid A component –  Referred to as an endotoxin LPS as an Endotoxin •  The injec4on of living or killed Gram ­nega4ve cells, or purified LPS in fairly small doses results in death in most mammals. –  Animals vary in their suscep4bility to endotoxin. –  How soon death occurs varies on the dose of the endotoxin, route of administra4on, and species of animal. •  The sequence of events follows a regular pafern: –  (1) latent period; –  (2) physiological distress (fever, diarrhea, prostra4on, shock); –  (3) death. Proper4es of the Outer membrane •  Protein composi4on –  Less diverse than inner (cell) membrane –  Contains 10 ­20 major proteins present in large amounts: 100, 000 copies/cell •  Structural proteins, nonspecific porins –  Other proteins present in lower amounts •  Enzymes, specific porins Outer membrane permeability •  The protein composi4on is impacted by many environmental signals –  Osmolarity, temperature, pH, etc. •  Outer membrane proteins also source of weakness –  Receptors for bacteriophages –  Way many an4bio4cs enter the cell 53. Porins •  Trimers of porin proteins form channels that allow small hydrophilic molecules to cross the outer membrane •  Nonspecific or specific Bacterial Envelopes •  Comparison of the Bacterial Envelopes of Gram ­posi4ve and Gram ­nega4ve Bacterial Cell Organiza4on: Quick review Primary Features: •  Bacterial Envelope Other Features –  Internal Structures •  Gas vessicles –  External Structures •  Capsule, S ­layer •  Internal Structures –  Nucleiod –  Ribosomes –  Cell appendages •  Pili and Fimbriae •  Flagellum –  Developmental Structures Components found in only some bacterial species •  Internal Structures –  Storage Polymers, Gas Vesicles •  External Structures –  Capsule or slime layer, S ­layers, Pili, Fimbriae, Flagellum •  Developmental Structures Intracellular reserve materials or storage polymers •  Prokaryotes store a variety of organic and inorganic materials as nutrients? –  Store Carbon –  Store Nitrogen –  Store Sulfur •  Appear as inclusion (granules) Carbon storage polymers (PHB) •  Appear as granules in the cell •  Can be surrounded by lipids and proteins in a membrane monolayer –  Monolayer is NOT a true “unit” membrane so granules are NOT organelles 61. Gas Vesicles •  Provide buoyancy –  Allows bacteria to move up and down a water column –  Example: Flota4on of gas vesiculate cyanobacteria from a bloom on a nutrient rich lake Gas Vesicles –  Found only in Bacteria and Archaea •  Usually aqua4c and photosynthe4c –  Gas vesicles surrounded by a “membrane” composed only of protein •  Permeable to gases •  Not permeable to water or solutes External Structures •  •  •  •  Capsule or slime layer S ­layer Pili or Fimbriae Flagellum: –  Structure –  Chemotaxis . Capsule or Slime layer •  Protec*ve role: –  Against immune system –  Desicca4on –  Phage infec4on Secreted materials on surface •  Secreted material on external surface –  Usually composed of polysaccharides –  A few organisms have secreted material consis4ng of proteins Proper4es of Capsules (slime layers) •  Composi4on and proper4es vary –  Thick or thin –  Rigid or flexible •  Inclusive term is Glycocalyx –  Defined as the polysaccharide ­containing material outside the cell –  Other terms: •  If rigid and excludes par4cles (india ink)= capsule •  If flexible and easily deformed=“slime layer” •  In biofilms, it is commonly referred to EPS layer (exopolysaccharide layer) Examples of Biofilms Paracrystalline Surface Layers S ­layers •  Surface layer –  composed of a 2 ­D array of proteins –  Crystalline appearance •  Found in bacteria and archaea •  Major func4on? –  Permeability barrier? –  Defense mechanism? Cellular Appendages •  Types –  Flagellum (plural=Flagella): –  Fimbriae –  Pilus (Pili) Defini4ons: •  Flagellum (plural=Flagella): –  Long, thin appendage composed of flagellin protein and responsible for swimming mo4lity Cellular Appendages •  Fimbriae –  Composed of protein and involved in afachment –  Shorter than flaggella and more numerous •  Pili –  Composed of pilin protein –  Similar to fimbriae, but typically longer –  Only one or a few present on surface –  Involved in afachment •  Type IV fimbriae/pili –  involved in twitching mo4lity Pili and Fimbriae Pilus of E. coli is revealed by phage that adhere to it Image from: http://www.vetmed.iastate.edu/ departments/vdpam/swine/ diseases/intestines/ecoli/ Flagellum (plural Flagella) •  Thread ­like locomo4on appendage •  Terms: –  Polar Flagella4on •  Monotrichous: flagellum at one end of cell •  Amphitrichous: flagella at both ends of cell –  Lophotrichous: clustered flagella at one end –  Peritrichous: flagella covering the cell \Flagella (Lophotrichous) Flagellar Structure– •  Flagella have a helical shape •  PARTS: –  Filament –  Hook –  Basal Structure or Motor Flagellar Structure: •  Filament –  composed of flagellin protein –  Shape and wave of filament determine by flagellin protein •  Hook –  Base of flagella –  Consists of a single type of protein –  Func4ons to connect filament and basal structure Flagellar Structure: Basal Body •  Gram ­nega*ve structure is composed of four rings –  L ­ring •  (embeded in lipopolysaccharide) –  P ­ring •  (embedded in pep4doglycan layer) –  MS ring •  (embedded in cell membrane) –  C ring •  (embedded in cell membrane) Flagellar Structure: Basal Body •  Gram ­posi*ve structure –  only have the inner pair of rings –  MS ring and C ring Flagellar Structure– •  Mot proteins –  Pair of proteins –  Drive motor by causing a torque on the filament •  Fli proteins –  Func4on as the motor switch –  Respond to environmental signals The basal body: the motor Func4on of the motor: •  Energy provided by the proton mo4ve force •  Protons flow through the Mot proteins –  May exert forces on charges present in the C and MS rings, thereby spinning the motor •  Motor causes a torque on the filament Movement •  Flagella acts like a propeller on a boat – Semi ­rigid structure that does not flex •  Rotary mo4on impacted by motor –  Energy from proton mo4ve force (PMF) –  1000 protons must translocate for 1 rota4on –  Speed related to strength of PMF –  Up to 60 cell lengths/second Movement with polar flagella4on Movement with peritrichous flagella4on Chemotaxis in E. coli •  When flagella rotate counterclockwise, they form a bundle that also rotates CCW –  Called a run (lasts ~1 second) •  When flagella reverses and rotates clockwise, bundle comes apart –  Called a tumble or twiddle (lasts ~0.1 second) –  Bacteria remains in place Posi4ve chemotaxis in E. coli •  Movement toward chemical or s4mulus •  Regulatory proteins controlling the motor respond to the chemical gradient –  Random movement ­higher concentra4on •  longer runs and fewer tumbles –  Random movement ­lower concentra4on •  shorter runs and more tumbles MOVIE Posi4ve Chemotaxis Nega4ve chemotaxis in E. coli •  Movement away from a chemical or s4mulus •  Regulatory proteins controlling the motor respond to the chemical gradient –  Random movement away from “bad” chemical •  longer runs and fewer tumbles –  Random movement towards “bad” chemical •  shorter runs and more tumbles Easy experiment for measuring chemotaxis •  Insert capillary with compound into a culture •  At various 4mes, remove capillary –  Afractant ­more bacteria in capillary than in culture –  Control ­same # bacteria in capillary than in culture –  Repellant ­less bacteria in capillary than in culture Results from a chemotaxis experiment Results from a chemotaxis experiment: Another Chemotaxis Assay Swarm Plates •  Low concentra4on of agar •  Large, rapidly forming swarms with thick outer rings are characteris4c of wild ­type aspartate taxis •  Strains defec4ve in chemotaxis do not grow out from the inocula4on site. Image from Weerasuriya, B. M. et al. 1998 J Bacteriol180(4): 914–920. Examples of types of taxis Chemotaxis: toward or away from chemicals Phototaxis: toward or away from light Aerotaxis: toward or away from oxygen Osmotaxis: toward or away from ionic strength •  Magnetotaxis: toward or away from a magne4c field •  •  •  •  Other types of movement •  Twitching mo4lity –  Type IV fimbriae/pili •  Gliding mo4lity –  Does not involve flagella •  Slower than flagellar movement –  Requires contact with a surface –  Mechanisms: •  Cyanobacteria: secre4on of a polysaccharide slime; adherence of slime to surface pulls cell along •  Flavobacterium: involves a rache4ng mechanism (like a tractor) •  MOVIE Bacterial Development Pictures from Google Images Biofilm on Catheter Myxobacteria Fruiting body Bacterial Development Pictures from Google Images Stalked bacteria: Caulobacter Heterocyst in Cyanobacteria Examples of developmental structures •  Biofilm forma4on: –  Many pathogenic bacteria: Vibrio cholerae •  Frui4ng bodies: –  Myxobacteria species •  Heterocyst development –  Cyanobacteria •  Stalks: –  Caulobacter crescentus •  Sporula4on: –  Endospores in Bacillus sub:lis Sporula4on: Endospores •  Formed within cell •  Occurs in Bacillus sp., Clostridium sp., and a few other Gram ­posi4ve bacteria •  Usually soil dwellers •  Highly resistant to environmental stress Spore Characteris4cs •  Highly refrac4le under phase contrast •  Resistant to: –  –  –  –  Heat UV chemical disinfectants Desicca4on •  Can survive for long periods of 4me –  One report claims spores recovered from the gut of a bee suspended in amber (25 ­40 million years old) Structure of a Spore Outside to inside •  Exosporangium: –  outermost layer made of protein •  Spore coat: –  Composed of layers of spore ­specific proteins Structure of a Spore Outside to inside •  Cortex: –  Pep4doglycan ­ membrane laminate •  Core: –  Contains core wall (old pep4doglycan layer), cell membrane, nucleoid, and ribosomes Overview of Sporula4on •  Key step: Laying down the septum vegetative germination sporulation ...
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