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Prokaryote Cells . Conclusions. Small Sub Unit 30S 16S RNA 21 proteins. Large Subunit 50S 23S & 5S RNAs 31 proteins. Bacterial Ribosome. Ribosomes. Complex structures consisting of protein and RNA Sites of protein synthesis Smaller than eucaryotic ribosomes

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bacterial ribosome
Small Sub Unit

30S

16S RNA

21 proteins

Large Subunit

50S

23S & 5S RNAs

31 proteins

Bacterial Ribosome
ribosomes
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
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
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
inclusions
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
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
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
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
slide11
PHB
  • 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.
volutin
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
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
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
Magnetosomes
  • Some motile aquatic bacteria are able to orient themselves by responding to the magnetic fields of the earth because they possess magnetosomes, membrane-bound crystals of magnetite or other iron-containing substances that function as tiny magnets.
external structures
External Structures
  • Fimbriae
  • Pili
  • Flagella
bacterial pili
Bacterial pili
  • http://biophysics.bumc.bu.edu/faculty/bullitt/images/cartoon_ppili_hib.jpg
slide20
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
fimbriae
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
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
structural polymorphism of bacterial adhesion pili bullitt e and makowski l
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 host-binding 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
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
e coli and adhesion
E. Coli and adhesion
  • http://medschool.umaryland.edu/infeMSD/Images.htm
  • http://medschool.umaryland.edu/infeMSD/som.html

( Donnenberg lab at University of Maryland)

flagella motility
Flagella Motility

http://www-micro.msb.le.ac.uk/video/motility.html

arrangement of flagella
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
the filament
The filament
  • Hollow, rigid cylinder
  • Composed of the protein flagellin
  • Some procaryotes have a sheath around filament
flagellin protein structure
Flagellin( Protein structure)
  • http://www.rcsb.org/pdb/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
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
the three parts of the flagellum
The three parts of the flagellum
  • 3 parts
    • filament
    • basal body
    • hook
hook and base structure
Hook and Base Structure
  • http://molvis.sdsc.edu/atlas/morphs/flaghook/index.htm
  • http://www.umass.edu/microbio/chime/pe_beta/pe/atlas/atlas.htm
  • http://atlas.proteinexplorer.org
the hook and basal body
The hook and basal body
  • Hooklinks filament to basal body
  • Basal bodyseries of rings that drive flagellar motor
flagellar synthesis
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 motion
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
Traveling toward and Attractant
  • Caused by lowering the frequency of tumbles
  • Traveling away involves similar but opposite responses
flagellar movement
Flagellar movement
  • http://www-micro.msb.le.ac.uk/Video/motility.html
motility and pathogenicity
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
  • 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
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

other types of motility
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
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.
chemotaxis1
Chemotaxis
  • Movement towards a chemical attractant or away from a chemical repellant
  • Concentrations of chemoattractants and chemorepellants detected by chemoreceptors on surfaces of cells
protein export systems
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 for Movement out of the Cell

Proteins may be folded, unfolded, or partially folded

Some are completely assembled

translocation
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
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 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
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 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 - 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
type ii research article
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
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
Type II secretory proteins
  • Toxins( cholera toxin)
  • Pili protein
  • Pectinases
  • Lipases
  • Proteases
  • Other enzymes to degrade molecules in the environment
abc transporters type i
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
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 Gram-negative bacteria and the binding-lipoprotein-dependent transport system in Gram-positive bacteria. The transporter shows a common global organization

general structure
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
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.

Mechanism
slide80

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 transporters1
ABC Transporters
  • Animation for ABC transporters

http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/prostruct/active.html

  • PMF- Proton motive force

http://www.cat.cc.md.us/courses/bio141/lecguide/unit1/prostruct/pmf/pmf.html

type iii and secretion
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 needle-shaped
    • secreted proteins thought to move through a translocation channel
occurrence
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 and virulence factorsExclusive 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
Examples of Type III
  • Cytotoxins
  • Phagocytosis inhibitors
  • Stimulators for reorganization of the cytoskeleton
  • Apoptosis promoters
slide86

The Mxi-Spa Type III Secretory Pathway of Shigella flexneriOuter Membrane Lipoprotein, MxiM for Invasin translocationRaymond 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
  • 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
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. cornelis@mipa.ucl.ac.be
type iv
Type IV
  • Virulence Related Secretory Pathway
  • Span both membranes of the gram-negative bacterial cell or one membrane of the gram-positive
  • 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
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
Bacterial Endospores – agents of survival not dispersal
  • formed by some bacteria
  • dormant
  • resistant to numerous environmental conditions
    • heat
    • radiation
    • chemicals
    • desiccation
resistance to
Resistance to
  • Acids and bases
  • Heat
  • Radiation
  • Reactive oxygen
resistance of endospore is the result of
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
Electron Micrograph of endospore
  • CW = Vegetative cell wall
  • CP= Spore Coat
  • SC= Spore Cortex
  • EX= Exosporium
spore location
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
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.
sporogenesis
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 cell sporulation or sporogenesis
Formation of the Vegetative Cell- Sporulation or Sporogenesis
  • Complex, multistage process
  • Commences in response to environmental conditions such as a lack of nutrients
steps
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
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
Sporulation continued
  • Protein coats are then formed around the cortex
  • Maturation of the spore occurs
structure continued
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
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
slide108
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
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
CDC and Anthrax Fact Sheet
  • http://www.bt.cdc.gov/agent/anthrax/faq/