MB 251 Exam 1

Microbiology

Study of small Biological Specimens

Bacteriology

Study of bacteria

Virology

Study of viruses

Immunology

Study of immune cells/systems

Microorganism

 Organism that can’t be seen by naked eye; Bacteria, Archaea, Viruses, Yeast

All Bacteria = Microorganisms

Not all Microorganisms = Bacteria

Colony

Pure Culture made from 1 species

Robert Hooke

 1st to describe, observe, and visualize microbes/microorganisms. Drew fruiting structures of molds and invented modern microscope

Antoni Van Leeuwenhoek

 First to describe Bacteria. Called them Animalcules. Used magnifiers to see 500x

Louis Pasteur

Found living organisms discriminate between optical isomers. Disproved Theory of Spontaneous Generation. Created vaccines for Anthrax, Fowl Cholera, and Rabies. Invented process of pasteurization. Create Swan Neck flask

Spontaneous Generation

Microorganisms arose spontaneously and Microorganisms developed from seeds/germs from air

John Needham (1745) and Lazzaro Spallanzani

Boiled broth to see if microorganisms would grow or be killed off 

Edward Jenner

Pioneer of Vaccionology. Created 1st documented Vaccine for Smallpox

Ignaz Semmelweis

Puerperal Fever (Childbed Fever). Proposed washed hands and deaths plummeted

Joseph Lister

Hypothesized infections were from contamination of wounds by living creatures. Used phenol to reduce infections. Treating infections/tools eliminated infections

Robert Koch

Linked Microbes and Infectious Diseases. Germs cause disease. Developed techniques for obtaining pure cultures of microbes

What is the first postulate of Robert Koch's postulates?

All diseased hosts must have the pathogen associated with them, and all healthy animals must be absent of the pathogen.

What is the second postulate of Robert Koch's postulates?

The pathogen must be one species of bacteria.

What is the third postulate of Robert Koch's postulates?

Cells from a pure culture of the pathogen must cause disease in a healthy animal.

What is the fourth postulate of Robert Koch's postulates?

The suspected pathogen must be reisolated and shown to be the same as the original.

Fanny Hesse (Koch Associate)

Suggested use of Agar in place of gelatin that was stable at higher temperature and not degradable by bacteria

Julius Petri (Koch Associate)

Used bell-shaped glass as a cover. Created the Petri dish

Strict Anaerobes

Anaerobic Microorganisms that do not use Oxygen (O2) for any metabolic properties. Microbes expel oxygen (toxic to them)

Largest Known Bacteria

Thiomargarita Namibiensis (750 microns)

Advantages of being a small Bacteria

More surface area relative to cell volume, grow faster, greater nutrient exchange per unit cell volume

Obligate Intracellular Bacteria

Too small to survive in the environment by themselves die to lack of needed organelles. Survive in host cells to carry out metabolic processes. Does not have necessary protein molecules

Smallest Known Bacteria

Part of Mycoplasma Genus (0.2 microns)

Magnification

Making an object larger for visualization

Resolution

Distinguish between two objects (Clarity).

Resolving Power of Light Microscope

Must be at least 0.2 Microns to see separation

Bright-Field Microscopy

Visualize differences in density/contrast between specimens/surroundings

Pros:  Easy/Live cells

Cons: Need 0.2 microns, stain sample to see structures clearly, stained samples = dead

Gram Negative

Two Layers: Outer Membrane and Thin Peptidoglycan. Outer Membrane is less selective than Cytoplasmic Membrane. Stain Red/Pink due to thin cell wall (pink color = Safrinin)

LPS/Out Membrane, Single Amino Acid Crosslinks, and Think Peptidoglycan Layer

Gram Positive

One Layer: Thick Peptidoglycan. Teichoic acids in cell wall that support/withstand pressure in cell wall. Stain Purple due to think cell wall

Amino Acid Interbridges, Teichoic Acids, and Thick Peptidoglycan Layer

Fluorescence

 Emitting light of 1 wavelength after absorbing light of a different wavelength (Can not see with naked eye)

Autofluorescence

• Natural fluorescence Chlorophyll or Pyoverdine (Siderophore)

(DAPI) and Tagged Proteins (GFP)

Artificial stains made to have color. Not natural

Fluorescent Microscopy

Traditional (2D/Flat) Fluorescent Microscopy and Confocal Scanning Laster (3D). Cells are Alive!! Fluorescence comes from cell resolution can not see individual signals/proteins

Bacteria Biofilm

Bacteria come together, secrete extracellular components (DNA/RNA/Proteins) to form mass to prevent external forces from killing them. Community of bacteria embedded in a matrix to stick to a surface. Form on tissues inside of host and non living thins

Scanning Electron Microscope (Electron Microscopy)

Uses electrons instead of photons/visible light. Can see 0.0002 microns. Samples are stained for better contrast/cover them in heavy metals; Lead Salt, Uranium, Lanthanum, gold. Cells are dead

Intact Cells; Visualization of External cell features

Transmission Electron Microscope (Electron Microscopy)

Uses electrons instead of photons/visible light. Can see 0.0002 microns. Samples are stained for better contrast/cover them in heavy metals; Lead Salt, Uranium, Lanthanum, gold. Cells are dead

Thin sections with visualization of Internal cell features

Single Molecule Imaging

Resolves fluorescent proteins. Allows us to track protein movement in cells

Traditional Fluorescent Microscopy

Can not see individual proteins (highlights whole cell)

MALDI-IMS (Matrix-Assisted, Laser Desorption, Mass Spectrometry) Microscopy

Tracks individual molecules Lipids, Proteins, Metabolites 

Cytoplasmic Membrane

Highly selective permeability barrier; enables acquisition of specific metabolites and excretion of waste. So strict it does not allow protons to pass (Must have specific protein)

Cytoplasm

Aqueous mixture of macromolecules, ions, and ribosome

Prokaryote

No membrane bound organelles; no nucleus, singular chromosomes, plasma membrane (can lose DNA and survive), Non essential functions

Eukaryotes

DNA in membrane bound organelles, large, linear chromosomes,

• Cytoplasm is where functions happen

Protein Anchors

Proteins that participate in transport, bioenergetics, and chemotaxis

Energy Conservation

Site of generation and dissipation of proton motive force

Basic Transport Events (Uniport)

1 molecule going in or out

Group Translocation (Glucose)

Chemical modification of transported substance driven by phosphoenolpyruvate

ABC Transporter (Zinc)

Periplasmic binding proteins involved and energy comes from ATP

Driven by ATP hydrolysis to power transport

Ester/Ether linkages

Stability for higher temperatures. Ester is not at stable

Archaeal Membrane Monolayers

Covalently linked membranes with no hydrophobic interactions

• Important for stability at higher temperatures

Pyrococcus Furiosus: Nerve Agent Detoxification

• Breaks down Proline-Proline Amino Acid Linkages

Periplasmic Space

Small molecules can pass. Large molecules or charged molecules can not pass. Space between Outer Membrane and Cytoplasmic Membrane. Peptidoglycoglycan of Gram-Negative bacteria located in Periplasmic Space

Embedded Proteins

ABC transporters, enzymes, and chemoreceptors

Peptidoglycan Layer

Structure that provides strength and rigidity for blueprint for cell shape. Antibiotics target this structure to kill bacteria. Layers of Polysaccharides (NAG-NAM)

Lysis

When a cell bursts open.

Transglycosylases

 Enzymes involved in linking NAG-NAM sugars in the peptidoglycan backbone. Target for Antibiotics that disrupt cell wall. Insert precursors into growing cell wall and catalyze the glycosidic bond formation between NAG-NAM

Transpeptidases

Enzymes that form amino acid links between NAM sugars of different layers. Target for Antibiotics that disrupt cell wall

B-Lactamase Enzymes

Degrade B-Lactam ring making antibiotics ineffective

Lysozyme

Destroys peptidoglycan. Cleaves bonds between NAG-NAM beta 1,4 linkages

Lipid A

Allows imbedding into phospholipid bilayer

Gram-Stain Process

Safranin (Heat Fixing): Kill cell and sticks to glass side

Crystal Violet stain: Stain both types of cells Purple

Iodine Treatment: Mordant making it harder to wash out

Destain: Acid/Alco hol mixture to wash out Crystal Violet from cell wall (7-10 seconds)

Hans Christian Gram

Developed staining method that could differentiate the two major groups of bacteria. 

Capsule

Made of Polysaccharides/Amino Acids

• Attaches to Peptidoglycan

• Works in attachment, immune evasion/cell protection (desiccation)

Fimbriae

Short Hair like appendages Visible with electron microscope. Used for attachment

EndoSpore

• Seed that recognizes threats (heat, low food) and turns into Spore

ExoSporium

• Outermost Layer that encloses spores like Bacillus (Protein)

Spore Coat

• Layers of spore proteins. Acts as a barrier to large molecules. Enzymes for germination

Dipicolonic Acid

• Only in the spore. Binds to water and used to dehydrate spore

Peritrichous

Gliding Motility

Bacterial movement across solid surface

• Does not use Flagella 

• Slower and smoother 

• Happens through excretion of polysaccharides

Twitching Motility

Allows movement on surface but uses Pilus/Pili that pulls cells across a surface

Flagellum/Flagella

• Allows movement in liquid environments

• Rotate up to 1,000 revolutions per minute

• 10-50 movements per second

• Not every bacteria produces this

• Motor powered by Proton Motive Force (PMF)

Chemotaxis

Chemical Movement. Movement of bacteria through chemical signals

• Use to direct Bacteria to more favorable environments

Attractant

Positive; Directs movement towards positive stimulus 

Repellant

Negative; less Bacteria in tub because they move away

Chemoreceptors

Methyl-Accepting Chemotaxis Proteins (MCPs)

• Proteins that sense chemical signals in environment (Uses Flagella)

• Cell encodes many sensors

• Each sensor has substrate/class they can sense

Aerotaxis

Movement in response to oxygen

Phototaxis

Movement in response to light

Microbial Growth

Increase in the number of cells (Not size of cell)

Binary Fission

• Asexual cell division, parent cell splits into two

• Each daughter cell gets chromosode and copies of all other cells needs to exist by itself (Balanced Growth)

• If a parent cell does not equally divide, possibility that one/both cells may not survive

Generation Time

• Time needed to double in number

Bacterial Growth Requirements

Media (Nutrients/Food to grow), Temperature, and Oxygen

Bacterial Growth Curve (Lag Phase)

Cells getting accustomed to environment, producing all materials to divide

Bacterial Growth Curve (Exponential Phase)

Cells actively dividing, and growing at their max rate in the specified environment (Balanced Growth)

Bacterial Growth Curve (Stationary Phase)

Growth rate and death rate are equal. The number of cells stays constant. Nutrient limitation/accumulation of toxic metabolic byproducts

Bacterial Growth Curve (Death Phase)

No nutrients remain and waste products remain. Population dies out 

Theoretical Growth

Increase in population continues if there is a constant supply of new nutrients/removal of waste byproducts

Fts Proteins: Filamentous temperature sensitive proteins

• Needed for cell division in all Bacteria (most Archaea)

• Proteins interact to form divisome (cell division apparatus)

• FtsZ: Forms ring around center of cell; related to tubulin

Min System (C,D, & E)

 Ensure cell division occurs in the middle of a cell.

C: Direct inhibitor that drives cell Division

D: ATPase that binds to inner cell membrane recruiting MinC to block cell division

E: Oscillates from pole to pole. Clears C and D allowing FtsZ to assemble in the center of the ring

FtsZ

Responsible for cell dividing, forming a ring at the center of the cell

Bacteria Cell Morphology

Morphology = Cell Shape that is genetically coded

MreB

Rod Shaped Morphology determining factor in Prokaryotes

• Forms cytoskeleton in Bacteria

• Forms spiral shaped bands around inside of cell, underneath cytoplasmic membrane

• Not found in coccus shaped bacteria

• Localizes synthesis of new peptidoglycan and other cell wall components to locations in cylinder of rod shaped cell during growth

• Localize peptidoglycan to elongate a cell. Does not have MreB protein would be coccus

Autolysins

• Small openings in the wall that begin at the FtsZ ring. Linkages in preexisting peptidoglycan

Bactoprenol

•  Carrier molecule involved in transport of NAG-NAM peptidoglycan precursors across cytoplasmic membranes. Makes precursors hydrophobic to cross cytoplasmic membrane

Transpeptidation

•  Final step in cell wall synthesis (Ftsl). Forms peptide cross links between muramic acid residues in adjacent glycine chains

Monotrichous

1 tail