Chapter 4: Prokaryote (pt 1)

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Size of prokaryote

1-10 um

size of eukaryote

10-100 um

size of virus

10-100 nm

Prokaryotes Vs. Eukaryotes

Prokaryotes: -Basic building blocks: same
-Size: smaller
-Nucleus: No
-Chromosomes: Singular, circular, no histones
-Cytoskeleton: No
-Membrane enclosed organelles: No
-Ribosomes: Smaller
-Flagella: Simple
-Cell wall: complex (peptidoglycan)
-Cell Division: Binary fission
Eukaryotes: -Basic building blocks: same
-Size: larger (-10x)
-Nucleus: Yes
-Chromosomes: Multiple, linear, histones
-Cytoskeleton: Yes
-Membrane enclosed organelles: Yes
-Ribosomes: Larger
-Flagella: Complex
-Cell wall: Simple
-Cell division: mitosis

Cocci (bacterial shapes)

-tetrads
-sarcinae
-Staphylococci
-Arrangements can help in identification

Tetrads (cocci)

-divide two planes and remain in cluster of 4

Sarcinae (cocci)

-cluster of 8 (4 on bottom, 4 on top)

Staphylococci

grape-like clusters

Bacilli (bacterial shapes)

-Little staffs or rods
-bacillus refers to shape (italicized bacillus refers to genus)
-only divide vertically
-may be singles
-diplobacilli
-streptobacilli

Diplobacilli (bacilli)

set of 2 bacilli

Streptobacilli (bacilli)

chains of bacilli

Coccobacillus

-more ovular (like cocci)
-hard to distinguish
-short, stumpy rods

Spiral (bacterial shapes)

-Vibrios
-Spirillum
-Spirochetes- axial filament for movement
-axial filament= endo flagella
-2 organisms: Treponema pallidium
Barellia burgdorferi
-Most cells monomorphic
-pleomorphic

Vibrios (spiral)

-curve rods (comma shaped)
-Vibrio cholera of cholera

Vibrio cholera

-causes Cholera
-spiral
-rice water diarhea

Spirillum (spiral)

-rigid corkscrews with whip-like tail
-spirilla= plural
-usually have multiple flagella

Spirochetes

-corkscrew but are flexible and have a special axial filament for movement

axial filament (spirochetes)

-endo flagella
-wraps around the cell
-between membrane and sheath
-corkscrew movement through water
-anchored at one end of a cell beneath and outer sheath

Treponema pallidium

-causes: syphillis
-spirochetes

Barellia burgdorferi

causes: lyme disease
-spirochetes

Monomorphic (spirochetes)

-most cells
-1 normal shape
-age could affect
-pleomorphic

Pleomorphic (spirochetes)

-can appear in several shapes
-pleomorphic

Bacterial surface stuctures

-Fimbriae
-Pili
-Flagella
-all 3 composed of proteins

Fimbriae

-shorter, more numerous
-protein
-probably for adhesion to animal tissues (pathogen) or biofilms
-attaches cell to surfaces

Pili

-longer and only a few
-protein
-bacterial conjugation or attachment to tissue in some pathogens
-act as syringe
-only has one or two
-important for diseases
-movement

conjugation

-transport materical to another cells
-infect cells, inject proteins into host cells. proteins direct cell to rearrange cytoskeleton and form a pedestal

Flagella

movement

Microbial locamotion

-intentional motion: bacterium going towards stimulus
-Many bacterial cells able to move
-require energy (ATP)
-Different types of motion/structures:
-Majority have flagella (flagellum)
-Axial filament
-May glide (might involve pili)
-May float with gas vesicles
-Brownian motion: vibrations
-Streaming: floating with water (NOT locamotion)

Why be motile?

-get to or move away from stimuli
-move in response to taxes
-Taxis
-Chemotaxis
-Phototaxis
-other taxes

Taxis

movement in response to stimuli

Chemotaxis

-maority
-response to chemicals (usually addressed in regards to swimming bacteria)
-food, nutrients, toxins (chemicals)

Phototaxis

response to light

Other taxes

-oxygen
-other conditions

Types of Flagella

-Long thing appendages
-protein subunits in helical arrangement
-Arrangements:
-Peritrichous
-Monotrichous
-Lophotrichous
-Amphitrichous
-Atrichous
-arrangement indicative of cetrain organisms (ie: e.coli= peritrichous)

Peritrichous (flagella)

all around cell

Monotrichous

-polar
-one flagella

Lophotrichous

-tuft
-several flagella on one end

Amphitrichous

-both ends

Atrichous

no flagella

Flagella structure

-3 main parts: basal body, hook, and filament
-hook allows for more movement of water
-flagella rotate around the hook (no whip-like motion like eukaryotes

Flagella movement

-organism sense chemicals
-flagella turned on to move in certain direction
-run
-tumble
-short distances

Run (flagella movement)

- all flagella go to one end of cell and rotate in one direction

Tumble (flagella movement)

-throws bacteria spin
-bacterium reaccesses situation
-changes direction of movement of flagella

Prokaryote vs. Eukaryote (flagella)

Prokaryote: -Size: 10x smaller
-Number: multiple
-Movement: circles
-Location: attached to cell wall and plasma membrane
Eukaryote: -Size: 10x larger
-Number: one or a few
-Movement: whip-like motion
-Location: inside the plasma membrane

The Glycocalyx

-polysaccharide (may have some aa) around many prokaryotes
-capsule
-slime layer

Capsule (glycocalyx)

-rigid
-excludes india ink

Slime layer (glycocalyx)

-easily deformed
-more difficult to see

Roles of Glycocalyx

-attachment of pathogenic organisms to host
-immune system evasion
-protection vs. desiccation
-example: Strep. pneumoniae
-important in disease:
-attach to things
-protect organisms from phagocytosis

Phagocytosis

white blood cell eats bacteria and destroys it

Streptococcus pneumonia

-causes: typical pneumonia
-dipplococcus
-capsule (pathogen)
-no capsule: not a pathogen
-glycocalyx

Cell envelope

- cell wall + cell membrane
-cell wall- peptidoglycan
-Complex polysaccharide layer outside of cell membrane
-Additional complexities and outer membrane if Gram negative
-more complex than eukaryote
-Cell membrane
-lipid bilayer inside cell wall

Bacterial cell walls

-Don't have cytoskeleton
-responsible for cell shape
-prevent cell from rupturing due to internal water pressure
-semi-rigid (more flexible in prokaryotes)
-Complex makeup
-peptidoglycan (not in Archaea)
-proteins

Gram + vs. Gram - Cell walls

-membrane= phospholipid bilayer
-gram +: -thick layer of peptidoglycan
-teichoic acids imbedded inside peptidoglycan
-smooth
-gram -: -thin layer of peptidoglycan
-no teichoic acids
-outer membrane (surrounds peptidoglycan)
-ruffled

Gram + (cell wall)

-thick layer of peptidoglycan
-teichoic acids imbedded inside peptidoglycan
-smooth

Gram - (cell wall)

-thin layer of peptidoglycan
-no teichoic acids
-outer membrane (surrounds peptidoglycan)
-ruffled
-periplasm

Pariplasm (gram - cell wall)

liquid in space between membranes

Pariplasmic spaces (gram - cell wall)

space between two membranes

What is peptidoglycan?

-a chain of N-acetylglucosamine (NAG, G) and N-acetylmuramic acid (NAM, M) linked by a glycolytic bond
-always NAG then NAM
-long chains of nag/nam

Glycolytic bond

bond between NAG and NAM

Building peptidoglycan- molecular level

-peptides + sugars
-peptide bridge: connects NAM
-wrap sheet around cell

Peptide bridge

connects NAM

Gram-positive cell walls: Teichoic acids and lipoteichoic acid

-teichochic acids are acidic polysaccharide chains
-strong neg charge
-2 classes: wall teichoic acids and lipoteichoic acids

Wall teichoic acids (gram + cell wall)

-imbedded in wall

lipoteichoic acids (gram + cell wall)

-go through wall
-anchored into cell membrane
-bottom is lipid

Gram-negative cell wall

-Outermembrane:
-phospholipid bilayer
-proteins
-porin
-Lipopolysaccharide

Porin (gram-neg cell wall)

pipe for H20 to flow in/out of cell

Lipopolysaccharide (gram neg cell wall)

-lipid at the bottom
-hexagons are polysaccharides (sugars)

Outer membrane (gram - cell wall)

-lipopolysaccharides (LPS) and polysaccharides
-strong negative charge
-protects against phagocytosis and some antibiotics
-LPS
-antigenic
-3 Parts
-Lipid A, Core, and O-specific

Lipid A (part of outer membrane) (LPS)

-contains the lipids, the main problem in gram negative infections (endotoxin)
-in membrane
-attached to polysaccharide
-fever
-drop in blood pressure (may lead to shock)
-aches/pains

Core polysaccharide (part of outer membrane) (LPS)

-same sugars in all LPS

O-specific polysaccharide (part of outer membrane) (LPS)

-different sugars/ repeated differently

Endotoxin

lipids are part of outer membrane

Exotoxin

proteins that are intentionally secreted

The Gram Stain

-Developed in 1884 by Hans Christian Gram
-He was trying to find a stain to differentiate bacterial cells from eukaryotic nuclei
-Instead found way to differentiate Gram + cells from Gram - cells
-Turned out to be a very useful stain
-Entire diagnosis and treatement regimes are sometimes made based on gram stain results
-gram and shape (ie: gram - cocci)

Gram staining (process)

Step 1) Pimary stain: Crystal violet
-cells purple
-both cells affix dye
Step 2) Mordant: Iodine
-gram pos: dye crystals trapped in wall (CVI2)
-gram neg: no effect of iodine (CVI2)
Step 3) Decolorizing: Alcohol (acetone)
-gram pos: crystals remain in cell wall. cell dehydrated, smaller space, CVI2 gets stuck in cell
-gram neg: outermembrane weakened, wall loses dye. CVI2 leaks out. ends up clears
Step 4) Counterstain: safranin
-gram pos: red dye has no effect.
-gram neg: red dye stains colorless cell

Is the cell wall a good antimicrobial target?

-Yes, the cell wall is unique to bacteria.
-What if you destroy the peptidoglycan? Cell can burst
-The enzyme lysozyme can break-down peptidoglycan between the G ad M (NAG and NAM)
-Many animals secrete lysozyme (saliva, tears, etc.)-
-Found in saliva and tears (makes sense= to fight off bacterial infections)
-Weakens cell wall= water can get in
-If enough gets in, cell swells and bursts= lysis

Selectivity

-selective toxicity
-kill harmful organisms, don't kill good bacteria, don't kill host

How does Penicillin work?

-penicillin targets aa cross bridge (breaks bridges)

Would penicillin be effective against archaea?

-No
-No peptidoglycan

Would penicillin be effective against gram +?

-Yes
-plenty of peptidoglycan

Would penicillin be effective against gram -?

-No (yes with other drugs)
-LPS has strong neg. charge
-penicillin doesn't get through it

Not all cells stain well

-For those strains that do stain, cell cultures should be young, 16-25 hours old are best
- >24-36 hours: gram + looks gram - (gram variable
-mycoplasma- no cell wall
-mycobacterium- acid-fast cell wall

Cells won't stain if:

1) Cells are too old, some Gram-positive cultures will appear gram-negative (especially those in Bacillus and Clostridium genera) and may be called gram variable
2) Some bacteria do not have cell walls, such as those in Mycoplasm genera
3) Archaea do not have normal peptidoglycan, so they do not stain normally. Their cell walls are composed of polysaccharides and proteins. They do have a component similar to peptidoglycan, but it is different. (purple)
4)-Organisms in the genera Mycobacterium and Nocardium
-cells may stain Gram-positive
-Cell walls contain unique lipids and even alcohols
-Cell walls contain mycolic acids

gram variable

cells that are too old, will apparead gram neg even if they're gram pos

Mycolic Acids

-thick and waxy lipid that makes staining more complex
-makes organism pathogenic
-cell wall resistant to stain
-wax doesn't adhere dye

Acid-Fast Stain

-mycobacterium
-attaches to waxy layers
-Step 1: Primary stain (stain w/ heat): Carbolfuchsin
-acid- fast: red
-non acid-fast: red
-Step 2: Decolorizing: acid alcohol
-acid fast: red
-non acid fast: colorless
-Step 3: Counterstain: Methylene blue
-acid fast: red (mycobacteria)
-non acid fast: blue (all other bacteria)

Cell membrane

-Inside the cell wall
-Makeup
-Phospholipid bilayer
-Integral membrane proteins
-Function
-Permeability barrier, selective (some things enter, leave, excluded)
-Protein anchor
-Site of ATP synthesis (electron transport chain)
-Target for some antimicrobials

Integral protein (cell membrane)

-used to transfer molecules
-communication
-movement (flagella)
-anchors

Moving across the membrane

-passive process = no ATP for energy

Simple Diffusion (passive transport)

-Random movement of molecules from an area of higher concentration to an area of lower concentration
-oxygen and crabon dioxide movement

Facilitated diffusion (passive transport)

-Transporter protein in membrane helps move substance across membrane
-nonspecific: particles can be dissolved in water (ions)
-specific: molecule enters, protein changes shape, molecule shoots out other side
-small molecules (sugars)

Osmosis

-Net movement of solvent molecules across a selectively permeable membrane from and area with high concentration of solvent molecules to an area of low concentration of solvent molecules= Diffusion of water (solvent) across plasma membrane
-water molecules move from an area of low solute concentration to area of high solute concentration

aquaporins

water tunnels that allow water to enter/exit cell

Solute

anything dissolved in solvent

solvent

what does dissolving (water)

Isotonic

-equal amounts of solute and solvent on both sides of the plasma membrane
-ex: most body fluids, including blood

Hypotonic

-higher conentration of solute inside the cell causes cell to swell and burst due to an intake of water (lysis)

Hypertonic

-higher concentration of solute outside the cell causes cell to shrink due to loss of water (plasmolysis)

What about active processes?

-Active transport
-diffusion is passive and very slow and may never reach high concentration
-Transport is essential to get anything across an concentration gradient or for cricial items
-Uses energy in the form of ATP

Group Translocation (active process)

-special prokaryotic transport mechanism that alters the substance as it crosses the membrane
-Does not use ATP, but other high energy phosphate compounds
Ex: glucose. too much in cell, wants to leave. glucose changed (phosphorylated). now neg. charge and can't get out

Export

-large molecules?
-In prokaryotes, large molecules usually are transported through translocases (transport proteins in the cell membrane)
-part of secretory system: sometime specific, other systems more general
-Requires energy
-Protein export key because many bacterial enzymes work outside of the bacterial cell
-Ex: Amalyse (starch) or cellulase (cellulose) which cleave starch or cellulose outside the cell in their environment

Cytoplasm (inside bacterial cell)

-Liquid medium inside the cell
-80% water + proteins (enzymes), carbohydrates, lipids, ions
-no cytoskeleton

Nuclear material (inside bacterial cell)

-One circular bacterial chromosome associated with plasma membrane
-May also have circular plasmids that replicates separately from chromosome and may carry genes associated with antibiotic resistance and/or virulance

Bacterial chromosome (nuc. material)

-nucleoid
-circular
-attached to membrane

R-plasmid

conatin genes for proteins that are resistant to antibiotics

Ribosomes (inside bacterial cell)

-sites of protein synthesis, bacterial ribosomes different than eukaryotic ribosomes (good target for antibacterial drugs. ex: streptomycin, erythromycin, etc.)
-make drugs that target bacerial ribosomes, not eukaryotes

Prokaryote vs. Eukaryote (ribosomes)

-Prokaryote: -Size: smaller
-Subunits: 30S + 50S= 70S
-Composition: proteins
-Eukaryotes: -Size: larger
-Subunits: 40S + 50S = 80S
-Composition: proteins and RNA

Sedimentation (S)

-how quickly something falls out of solvent

Unclusions/granules (inside bacterial cell)

-nutrients can accumulate tobe used later as an energy source, such as glycogen and starch polymers

Endospores

-Only produced by some bacteria, best studied in Bacillus and Clostridium genera
-form within cells during sporulation
-form when growth ceases (nutrient exhaustion)= SURVIVAL
-resistant to heat, chemicals, desiccation, radiation
-allows cell to be dormant for many years
-discovered during studies of sterilization methods
-may involve 200 genes for cellular differentiation
-Stucture= more complex than vegitative
-Layering from inside out: special core wall, then cortex of peptidoglycan, spore coat of spore-specific proteins, exosporium protein covering

Sporulation

-Step 1:spore septum begins to isolate newly replicated DNA and a small portion of cytoplasm
-Step 2: Plasma membrane starts to surround DNA, cytoplasm, and membrane
-Step 3: spore septum surrounds isolated portion, forming forespore (2 membranes)
-Step 4: Peptidoglycan layer forms between membranes
-Step 5: Spore coat (proteins)
-Step 6- endospore freed from cell

Germination

reverse process of sporulation

Endospore staining

-Endospores are resistant to normal staining procedures (resistan to chemicals)
-Endospore stain uses heat or chemicals to force stain into endospore
-endospore- green
-vegitative cell- red
-autoclave: uses steam (121*C) and pressure (15 psi); pressure delivers steam to cell

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