Prokaryote Cells Prokaryote Cells

ConclusionsConclusions

Bacterial RibosomeBacterial Ribosome

Small Sub Unit 30S 16S RNA 21 proteins

Small Sub Unit 30S 16S RNA 21 proteins

Large Subunit 50S 23S & 5S RNAs 31 proteins

Large Subunit 50S 23S & 5S RNAs 31 proteins

RibosomesRibosomes 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

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 ComplexityRibosomal Complexity

Three Dimensional image of the 30s ribosomal subunit

Vital in protein synthesis

Binds to the messenger RNA to initiate translation

Three Dimensional image of the 30s ribosomal subunit

Vital in protein synthesis

Binds to the messenger RNA to initiate translation

50s Ribosomal subunit50s 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

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. coliCulpepper Group at the Stanford School of Medicine

16 s Ribosomal subunit from E. coliCulpepper Group at the Stanford School of Medicine

InclusionsInclusions

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

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 CyanobacteriaInclusions 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.

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

Cyanophycin granules are found in the filamentous photosynthetic bacteria found in fresh water ponds that are vital to the nitrogen cycle in aquatic environments

CarboxysomesCarboxysomes 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

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

PHBPHB 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.

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.

VolutinVolutin 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.

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 VacuolesGas 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

• 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

EnterosomesEnterosomes 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

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

MagnetosomesMagnetosomes

• 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.

• 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.

MagnetosomesMagnetosomes

Movement of bacteria in a magnetic field

Movement of bacteria in a magnetic field

External StructuresExternal Structures

Fimbriae Pili Flagella

Fimbriae Pili Flagella

Bacterial piliBacterial pili

http://biophysics.bumc.bu.edu/faculty/bullitt/images/cartoon_ppili_hib.jpg

http://biophysics.bumc.bu.edu/faculty/bullitt/images/cartoon_ppili_hib.jpg

PiliPili

• 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

• 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)Pilin( Salmonella)

FimbriaeFimbriae

• 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

• 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 AdhesinsFimbriae 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

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

AdhesinsAdhesins

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.

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 gonorrheaNeisseria 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

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

Neisseria – Gram-intracellular diplococci

E. Coli and adhesionE. Coli and adhesion

http://medschool.umaryland.edu/infeMSD/Images.htm

http://medschool.umaryland.edu/infeMSD/som.html

( Donnenberg lab at University of Maryland)

http://medschool.umaryland.edu/infeMSD/Images.htm

http://medschool.umaryland.edu/infeMSD/som.html

( Donnenberg lab at University of Maryland)

Flagella MotilityFlagella Motility

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

Arrangement of flagellaArrangement 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

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 FlagellaArrangement of Flagella

The filamentThe filament

Hollow, rigid cylinder Composed of the protein flagellin Some procaryotes have a sheath

around 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

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

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 flagellumTubulin subunits of eukaryote flagellum

Tubulin dimer Tubulin dimer

Comparison of Prokaryote and Eukaryote Flagella

Comparison of Prokaryote and Eukaryote Flagella

The three parts of the flagellum

The three parts of the flagellum

3 parts filament basal body hook

3 parts filament basal body hook

Hook and Base StructureHook 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

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 bodyThe hook and basal body

Hooklinks filament to basal body

Basal bodyseries of rings that drive flagellar motor

Hooklinks filament to basal body

Basal bodyseries of rings that drive flagellar motor

Flagellar complexityFlagellar complexity

Structure of Bacterial Flagella

Structure of Bacterial Flagella

Flagellar SynthesisFlagellar 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

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 SynthesisFlagellar Synthesis

Flagellar MotionFlagellar Motion

flagellum rotates like a propeller in general, counterclockwise rotation

causes forward motion (run) in general, clockwise rotation disrupts

run causing a tumble (twiddle)

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

Caused by lowering the frequency of tumbles

Traveling away involves similar but opposite responses

Tumble and RunTumble and Run

Flagellar movementFlagellar movement

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

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

Motility and PathogenicityMotility 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.

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.

HelicobacterHelicobacter 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.

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.

ChemotaxisChemotaxis

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

Helicobacter and the Gastric Mucosa

UlcersUlcers

Other Types of MotilityOther 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 axial filaments cause flexing and

spinning movement Gliding motility

cells coast along solid surfaces no visible motility structure has been

identified

Spirochetes and motilitySpirochetes 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.

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.

ChemotaxisChemotaxis

Movement towards a chemical attractant or away from a chemical repellant

Concentrations of chemoattractants and chemorepellants detected by chemoreceptors on surfaces of cells

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

Translocation and Secretion in Prokaryote

Cells

Transport Across the Cell WallTransport Across the Cell Wall

Protein Export SystemsProtein 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

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

Proteins may be folded, unfolded, or partially folded

Some are completely assembled

TranslocationTranslocation

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)

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 NegativeMembrane 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

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

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

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 modificationIn 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

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

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 PathwayStructure of the Sec Dependent Pathway

Sec Dependent Pathway

Sec Dependent Pathway

PathwayPathway

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.

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 IIType 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

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 proteinsType II secretory proteins

Toxins( cholera toxin) Pili protein Pectinases Lipases Proteases Other enzymes to degrade

molecules in the environment

Toxins( cholera toxin) Pili protein Pectinases Lipases Proteases Other enzymes to degrade

molecules in the environment

TYPE 2TYPE 2

ABC Transporters – Type IABC 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

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 TransportersABC 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

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 structureGeneral 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.

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 TransporterATP 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.

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.

MechanismMechanism 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).

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.

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 TransportersABC Transporters

Animation for ABC transportershttp://www.cat.cc.md.us/courses/bio141/

lecguide/unit1/prostruct/active.html PMF- Proton motive forcehttp://www.cat.cc.md.us/courses/bio141/

lecguide/unit1/prostruct/pmf/pmf.html

Animation for ABC transportershttp://www.cat.cc.md.us/courses/bio141/

lecguide/unit1/prostruct/active.html PMF- Proton motive forcehttp://www.cat.cc.md.us/courses/bio141/

lecguide/unit1/prostruct/pmf/pmf.html

Type III and SecretionType 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

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

OccurrenceOccurrence

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

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 factorsExclusive to Gram NegativeType 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

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 IIIExamples of Type III

Cytotoxins Phagocytosis inhibitors Stimulators for reorganization of

the cytoskeleton Apoptosis promoters

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

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

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

ShigellaShigella

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.

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.

ArticleArticle

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

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 IVType 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

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

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

formed by some bacteria dormant resistant to numerous environmental

conditions heat radiation chemicals desiccation

Resistance toResistance to

Acids and bases Heat Radiation Reactive oxygen

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

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

CW = Vegetative cell wall

CP= Spore Coat SC= Spore Cortex EX= Exosporium

Position of endosporePosition of endospore

Spore LocationSpore 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.

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.

StainingStaining 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.

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 stainingSpore staining

Swollen terminal regionSwollen terminal region

SporogenesisSporogenesis

Normally commences when growth ceases because of a depletion of Nutrients

Sensitive low levels of Nitrogen and Phosphorus

Complex multistage process

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

Complex, multistage process

Commences in response to environmental conditions such as a lack of nutrients

StepsSteps

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

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 DNAForespore 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

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 continuedSporulation continued

Protein coats are then formed around the cortex

Maturation of the spore occurs

Protein coats are then formed around the cortex

Maturation of the spore occurs

Structure continuedStructure 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

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

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

SpoASpoA

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

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 ActivationSteps 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

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/

http://www.bt.cdc.gov/agent/anthrax/faq/