Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Prokaryote Cells Conclusions Bacterial Ribosome Small Sub Unit 30S 16S RNA 21 proteins Large Subunit 50S 23S & 5S RNAs 31 proteins Ribosomes Complex structures consisting of protein and RNA Sites of protein synthesis Smaller than eucaryotic ribosomes procaryotic ribosomes Þ 70S eucaryotic ribosomes Þ 80S S = Svedburg unit Ribosomal Complexity Three Dimensional image of the 30s ribosomal subunit Vital in protein synthesis Binds to the messenger RNA to initiate translation 50s Ribosomal subunit The large subunit (50S) from Deinococcus radiodurans contains 33 different proteins Two rRNA chains (23S and 5S rRNA). The ribosomal rRNA Responsible for binding t RNA and the catalysis of peptide bonds for translation 16 s Ribosomal subunit from E. coli Culpepper Group at the Stanford School of Medicine Inclusions These are storage bodies in the cytoplasm of bacteria The inclusions vary with the type of bacteria Provide a supply of vital compounds or ions for metabolism Reduce osmotic pressure by tying up molecules in particulate form Inclusions in Cyanobacteria Cyanophycin granules are found in Cyanobacteria. They are large inclusion bodies composed of polypeptides comprised of arginine and aspartic acid. These store additional nitrogen for the bacteria. Inclusion bodies Cyanophycin granules are found in the filamentous photosynthetic bacteria found in fresh water ponds that are vital to the nitrogen cycle in aquatic environments Carboxysomes Cyanobacteria, thiobacilli, and nitrifying bacteria, organisms that reduce CO2 in order to produce carbohydrates, possess carboxysomes containing an enzyme used for CO2 fixation. These may be separated from the cytoplasm by internal membrane Poly- hydroxybutyrate molecules joined by ester bonds between the carboxyl and hydroxyl of adjacent molecules. These are common in purple sulfur bacteria and stain with Sudan black for light microscopy. These granules serve as storage reservoirs for glycogen and sugars necessary for energy and biosynthesis. PHB Volutin Some bacteria produce inorganic inclusion bodies in their cytoplasm, including volutin granules that store phosphate and sulfur granules that store sulfur. Volutin is a source of phosphate for DNA. Sulfur is used by purple photosynthetic bacteria that use hydrogen sulfide as a photosynthetic electron donor. Gas Vacuoles • Purple and green photosynthetic bacteria as well as some other aquatic bacteria contain gas vacuoles. These are aggregates of hollow protein cylinders called gas vesicles that are permeable to atmospheric gas, enabling the organism to regulate buoyancy. Bacteria are able to regulate the depth at which they float to regulate photosynthetic activity Enterosomes In Salmonella and E. coli have internal structures similar to carboxysomes Enterosomes contain enzymes required for the metabolism of certain molecules The existence of these molecules may be due to the necessity of dealing with toxic molecules Propanediol is a metabolite of fucose which is a sugar found on the intestinal wall of mammals that that can be degraded by intestinal bacteria – This is one of the molecules metabolized in enterosomes Magnetosomes • Some motile aquatic bacteria are able to orient themselves by responding to the magnetic fields of the earth because they possess magnetosomes, membranebound crystals of magnetite or other iron-containing substances that function as tiny magnets. Magnetosomes Movement of bacteria in a magnetic field External Structures Fimbriae Pili Flagella Bacterial pili http://biophysics.bumc.bu.edu/faculty/bullitt/images/cartoo n_ppili_hib.jpg Pili • Pili are appendages that are larger than fimbriae. Their presence is determined by genes on plasmids called sex factors. These structures function in conjugation which is a genetic exchange occurring in bacteria with these appendages Pilin( Salmonella) Fimbriae • Fimbriae are thin, hair-lie projections extending from the cell wall in Gram – bacteria. They are composed of helical protein units and designed for attachment to the host cell membranes ( mucous). • They also may contribute to types of movement in some bacteria. • These are considered to be virulence factors and induce many pathogenic effects Neisseria gonorrhea Fimbriae and Adhesins The tips of these structures have tips with adhesive proteins called adhesins They are designed to attach to a specific molecular target Fimbriae are produced in the cytoplasm and transported to the exterior of the cell Adhesins Structural polymorphism of bacterial adhesion pili. Bullitt E, and Makowski L. Bacterial adhesion pili are designed to bind specifically and maintain attachment of bacteria to target cells. Uropathogenic P-pili are sufficiently mechanically resilient to resist the cleansing action of urine flow that removes most other bacteria. P-pili are 68 A in diameter and approximately 1 micron long, and are composed of approximately 1,000 copies of the principal structural protein, PapA. They are attached to the outer membrane by a minor structural protein, PapH and are terminated by an approximately 20 A diameter fibrillus composed of PapK, PapE and PapF, which presents the hostbinding adhesin PapG. The amino-acid sequences of PapA, PapE, and PapF are similar, with highly conserved C-termini being responsible for binding to PapD, the periplasmic chaperone. Our three-dimensional reconstruction indicates that pili are formed by the tight winding of a much thinner structure. A structural transition allows the pilus to unravel without depolymerizing, producing a thin, extended structure five times the length of the original pilus. Neisseria gonorrhea To cause infection, Neisseria gonorrhoeae (inf) must first colonize a mucosal surface composed of columnar epithelial cells. Pili alow for this initial binding and, in fact, N. gonorrhoeae is able to rapidly lose pili and synthesize new ones with a different adhesive tip, enabling the bacterium to adhere to a variety of tissues and cells including sperm, the epithelial cells of the mucous membranes lining the throat, genitourinary tract, rectum, and the conjunctiva of the eye. Subsequently, the bacterium is able to make more intimate contact with the host cell surface by way of a cell wall adhesin called Opa Neisseria – Gram-intracellular diplococci E. Coli and adhesion http://medschool.umaryland.edu/infe MSD/Images.htm http://medschool.umaryland.edu/infe MSD/som.html ( Donnenberg lab at University of Maryland) Flagella Motility http://www-micro.msb.le.ac.uk/video/motility.html Arrangement of flagella monotrichous – one flagellum polar flagellum – flagellum at end of cell amphitrichous – one flagellum at each end of cell lophotrichous – cluster of flagella at one or both ends peritrichous – spread over entire surface of cell Arrangement of Flagella The filament Hollow, rigid cylinder Composed of the protein flagellin Some procaryotes have a sheath around filament Flagellin( Protein structure) http://www.rcsb.org/p db/home/home.do Search with flagellin Choose 1ucu Click on choice Choose the different image files to learn about molecular structure References on Genes and Proteins http://www.ncbi.nlm.nih.gov/ Choose structures – proteins Choose nucleotide – genes – DNA sequence Choose protein – AA sequence Cn3D – free download to study protein structure Tubulin subunits of eukaryote flagellum Tubulin dimer Comparison of Prokaryote and Eukaryote Flagella The three parts of the flagellum 3 parts filament basal body hook Hook and Base Structure http://molvis.sdsc.edu/atlas/morphs/flaghook/inde x.htm http://www.umass.edu/microbio/chime/pe_beta/p e/atlas/atlas.htm http://atlas.proteinexplorer.org The hook and basal body Hook links filament to basal body Basal body series of rings that drive flagellar motor Flagellar complexity Structure of Bacterial Flagella Flagellar Synthesis An example of self-assembly Complex process involving many genes and gene products New molecules of flagellin are transported through the hollow filament Growth is from tip, not base Flagellar Synthesis Flagellar Motion flagellum rotates like a propeller in general, counterclockwise rotation causes forward motion (run) in general, clockwise rotation disrupts run causing a tumble (twiddle) Traveling toward and Attractant Caused by lowering the frequency of tumbles Traveling away involves similar but opposite responses Tumble and Run Flagellar movement http://wwwmicro.msb.le.ac.uk/Video/motility.htm l Motility and Pathogenicity The mucosal surfaces of the bladder and the intestines constantly flush bacteria away in order to prevent colonization. Motile bacteria that can swim chemotactically toward mucosal surfaces may have a better chance to make contact with the mucous membranes, attach, and colonize. Many bacteria that can colonize the bladder and the intestines are motile. Motility probably helps these bacteria move through the mucous in places where it is less viscous. To support this, nonmotile mutants of Vibrio cholerae are less virulent than the motile wild types. Helicobacter Helicobacter pylori ,by means of its flagella, is able to swim through the mucus layer of the stomach and adhere to the epithelial cells of the mucous membranes. Here the pH is near neutral. To also help protect the bacterium from the acid, H. pylori produces an acid-inhibitory protein that blocks acid secretion by surrounding parietal cells in the stomach. The bacterium then releases toxins that lead to excessive production of cytokines and chemokines, as well as mucinase and phospholipase that damage the gastric mucosa. Chemotaxis Positive chemotaxis is exhibited by the outer ring which are responding to serine, the second ring responding to aspartate, and the upper dot – non chemotactic The E. coli on the agar plate is responding to acetate. Acetate concentration varies from 0 to 2M at the top left Helicobacter and the Gastric Mucosa Ulcers Other Types of Motility Spirochetes axial filaments cause flexing and spinning movement Gliding motility cells coast along solid surfaces no visible motility structure has been identified Spirochetes and motility Because of their thinness, their internal flagella (axial filaments), and their motility spirochetes are more readily able to penetrate host mucous membranes, skin abrasions, etc., and enter the body. Motility and penetration may also enable the spirochetes to penetrate deeper in tissue and enter the lymphatics and bloodstream and disseminate to other body sites. Chemotaxis Movement towards a chemical attractant or away from a chemical repellant Concentrations of chemoattractants and chemorepellants detected by chemoreceptors on surfaces of cells Translocation and Secretion in Prokaryote Cells Transport Across the Cell Wall Protein Export Systems Systems are present in Archaea, Bacteria, and Eukarya Evolved independently but have many similarities Eight systems move proteins across the cytoplasmic membrane and peptidoglycans cell wall Another eight are involved in the transport of proteins across the outer membrane, LPS Proteins for Movement out of the Cell Proteins may be folded, unfolded, or partially folded Some are completely assembled Translocation Movement of a molecule from one location to another Protein export – Translocation out of the cytoplasm( compartment to compartment) Protein secretion – Translocation of proteins through all membranes into the external environment( secretion to the external environment) Membrane Systems in E. coli and Gram Negative Protein Secretion in Procaryotes numerous protein secretion pathways have been identified four major pathways are: Sec-Dependent pathway Type II pathway Type I (ABC) protein secretion pathway Type III protein secretion pathway Recognition and The Sec system for the transport of proteins Recognition by the Sec system occurs during protein synthesis While the peptide is being synthesized a portion of the molecule serves as a signal sequence which is essential for recognition This 15-30 amino acid sequence is key to the attachment to the SecA system Protein Secretion – Sec Dependent Sec A leads the attached newly synthesized membrane protein to the membrane spanning channel composed of three other Sec protein ( YEG) The channel has a hydrophilic inner surface so proteins can enter and pass through In transit another protein, SecB attaches to the protein. This is a chaperone that keeps the protein in its extended or unfolded form In transit modification In transit another molecule a signal peptidase clips off the signal sequence As the protein is passed through the Sec YEG channel An expenditure of energy is required – Both ATP and a proton motive force is required Gram - and Gram + Gram positive bacteria secrete directly into the environment Gram negative bacteria use the Sec system to transport across the cell wall( peptidoglycan cell wall into the periplasm) and a different system to move across the LPS These systems can be quite complex involving as many as 14 proteins Structure of the Sec Dependent Pathway Sec Dependent Pathway Pathway Type II( research article) Mol Microbiol. 2002 Jan;43(2):475-85.Related Articles, Links A novel type II secretion system in Pseudomonas aeruginosa. Ball G, Durand E, Lazdunski A, Filloux A. Laboratoire d'Ingenierie des Systemes Macromoleculaires, UPR9027, IBSM/CNRS, Marseille, France. Type II Transports proteins from periplasmic space across outer membrane Present in Pseudomonas aeruginosa and Vibrio cholera Observed in some gram-negative bacteria, including some pathogens Complex systems consisting of up to 12-14 proteins most are integral membrane proteins Type II secretory proteins Toxins( cholera toxin) Pili protein Pectinases Lipases Proteases Other enzymes to degrade molecules in the environment TYPE 2 ABC Transporters – Type I Also called ABC protein secretion pathway. 65 families of transporters Currently two families export large proteins and four transport peptides and small proteins General structure is two integral channel forming domains and two cytoplasmic domains that involve the hydrolysis of ATP The proteins in this system associate with two auxillary systems the MFPs, membrane fusion proteins and the OMFs, outer membrane factors MFP’s are present in Gram Positive and Gram Negative Bacteria ABC Transporters Type I The ABC (ATP binding cassette) transporter is one of the active transport systems of the cell, which is widespread in archaea, eubacteria, and eukaryotes (Higgins 1992). It is also known as the periplasmic binding protein-dependent transport system in Gramnegative bacteria and the binding-lipoproteindependent transport system in Gram-positive bacteria. The transporter shows a common global organization General structure Typically, it consists of two integral membrane proteins (permeases) each having six transmembrane segments, two peripheral membrane proteins that bind and hydrolyze ATP, and a periplasmic (or lipoprotein) substrate-binding protein. ATP and ABC Transporter The ATP-binding protein component is the most conserved, the membrane protein component is somewhat less conserved, and the substrate-binding protein component is most divergent (Tam and Saier 1993; Saurin and Dassa 1994) in terms of the sequence similarity. Mechanism The ABC transporters form the largest group of paralogous genes in bacterial and archaeal genomes (Tatusov et al. 1996), and the genes for the three components frequently form an operon (Higgins 1992). Importers and exporters represent the ABC transporters. ABC transporters include nucleotide binding domains (NBD1 and NBD2), transmembrane spanning domains (MSD1 and MSD2) and solute binding proteins (SBP1 and SBP2). In the case of exporters, the SBP domains are absent. Also inherent to the ABC transporters is the conserved organizational nature of the genes involved. •Sequences the same in black •Amino Acid Alignment data for different bacteria on the ABC Transporter •Differences in red( # in the polypeptide or protein molecule) ABC Transporters Animation for ABC transporters http://www.cat.cc.md.us/courses/bio141/lecg uide/unit1/prostruct/active.html PMF- Proton motive force http://www.cat.cc.md.us/courses/bio141/lecg uide/unit1/prostruct/pmf/pmf.html Type III and Secretion Secretes virulence factors of gram-negative bacteria from cytoplasm, across both plasma membrane and outer membrane, and into host cell Some type III secretion machinery is needleshaped secreted proteins thought to move through a translocation channel Occurrence Found in Salmonella, Pseudomonas, Yersinia, Shigella, and E. coli Contact between the bactgeria and the host cells simtulates the process Low calcium levels may be required for secretion Type III and virulence factors Exclusive to Gram Negative Type III Secretion Pathway Four different types of proteins The secretory portion, the regulators, the proteins that aid in the insertion of secreted proteins, and effectors that alter host function Examples of Type III Cytotoxins Phagocytosis inhibitors Stimulators for reorganization of the cytoskeleton Apoptosis promoters The Mxi-Spa Type III Secretory Pathway of Shigella flexneri Outer Membrane Lipoprotein, MxiM for Invasin translocation Raymond Schuch and Anthony Maurelli Invasion of epithelial cells is mediated by the Mxi-Spa, Type III secretion system The this type III secretion is activated by pathogen and host cell interaction The secretion of these factors interacts with the host cell membrane to initiate entry Regulated and mediated by invasion plasmid proteins Lyse the endosomal compartment and spread Shigella Shigella species are aerobic, nonmotile, glucose-fermenting, gram-negative rods that are highly contagious, causing diarrhea after ingestion of as few as 180 organisms. Shigella species cause damage by 2 mechanisms, invasion of the colonic epithelium, which is dependent on a plasmid-mediated virulence factor, and production of enterotoxin, which is not essential for colitis but enhances virulence. The organism is spread by fecal-oral contact; via infected food or water; during travel; or in long-term care facilities, day care centers, or nursing homes. Article Philos Trans R Soc Lond B Biol Sci. 2000 May 29;355(1397):681-93.Related Articles, Links Type III secretion: a bacterial device for close combat with cells of their eukaryotic host. Cornelis GR. Microbial Pathogenesis Unit, Christian de Duve Institute of Cellular Pathology (ICP), Universite Catholique de Louvain, Brussels, Belgium. [email protected] Type IV Virulence Related Secretory Pathway Span both membranes of the gram-negative bacterial cell or one membrane of the grampositive Agrobacterium tumefaciens transports DNA into plant cells But Bordetella pertussis( whooping cough) uses a similar system to transfer the pertussis toxin into host cells Insertion of Proteins in the Cell Membrane The Oxal family consists of membrane insertases. In E. coli, these proteins function primarily to insert proteins into membranes Bacterial Endospores – agents of survival not dispersal formed by some bacteria dormant resistant to numerous environmental conditions heat radiation chemicals desiccation Resistance to Acids and bases Heat Radiation Reactive oxygen Resistance of endospore is the result of Calcium (complexed with dipicolinic acid) Acid-soluble, DNA-binding proteins Dehydrated core Spore coat DNA repair enzymes Electron Micrograph of endospore CW = Vegetative cell wall CP= Spore Coat SC= Spore Cortex EX= Exosporium Position of endospore Spore Location The position of the endospore differs among bacterial species and is useful in identification. The main types within the cell are terminal, subterminal and centrally placed endospores. Terminal endospores are seen at the poles of cells, whereas central endospores are more or less in the middle. Subterminal endospores are those between these two extremes, usually seen far enough towards the poles but close enough to the center so as not to be considered either terminal or central. Lateral endospores are seen occasionally. Examples of bacteria having terminal endospores include Clostridium tetani, the pathogen which causes the disease tetanus. Bacteria having a centrally placed endospore include Bacillus cereus, and those having a subterminal endospore include Bacillus subtilis. Sometimes the endospore can be so large the cell can be distended around the endospore, this is typical of Clostridium tetani. Staining Visualising endospores under the light microscope can be difficult due to the impermability of the endospore wall to dyes and stains. While the rest of a bacterial cell may stain, the endospore is left colourless. To combat this, a special stain technique called a Moeller stain is used. The allows the endospore to show up as red, while the rest of the cell stains blue. Another staining technique for endospores is the Schaeffer-Fulton stain, which stains endospores green and bacterial bodies red. Spore staining Swollen terminal region Sporogenesis Normally commences when growth ceases because of a depletion of Nutrients Sensitive low levels of Nitrogen and Phosphorus Complex multistage process Formation of the Vegetative CellSporulation or Sporogenesis Complex, multistage process Commences in response to environmental conditions such as a lack of nutrients Steps The nucleoid lengthens forming a structure called the axial filament ( axial filament formation can be induced by exposure in early exponential growth phase by the antibiotic, chloramphenicol. Inward folding of the cell membrane to enclose part of the DNA and produces the polar septum. The larger product is the mother cell, the smaller product is the forespore Forespore and DNA Upon formation – only 30% of the DNA is in the forespore – the remainder enters prior to the formation of the septum The mother cell sends out pseudopods that act in the same way a phagocyte to surround the forespore The two cells face each other and a murein wall is laid down between Sporulation continued Protein coats are then formed around the cortex Maturation of the spore occurs Structure continued Dipocolinic acid and Calcium ions are accumulated - the forespore dehydrates Outside of the cells a thick protective coat is synthesized This thick layer is known as the cortex It may be surrounded by a membrane known as the exosporium Quorum sensing and Sporulation Sporulation is controlled by a complex series of molecular communications known as quorum sensing The number of cells must reach a certain population and secrete peptides that trigger sporulation SpoA Sporulation is initiated by the signals that initiate phosphorylation and activation of SpoA which is a DNA binding protein The cascade of events occurs as a result of the SpoA phosphorelay system Further regulation of SpoA is through kinases and phosphatases Steps in Activation Activation prepares spores for germination often results from treatments like heating Germination spore swelling rupture of absorption of spore coat loss of resistance increased metabolic activity Outgrowth emergence of vegetative cell CDC and Anthrax Fact Sheet http://www.bt.cdc.gov/agent/anthrax /faq/