Download Prokaryote Cells – Part 2,week 2

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/