MIC 102

MD #1 Content

Light Microscopy

Magnifies up to 1000x ; resolve structures around 200 nm apart ; Brightfield, Phase contrast, and Fluorescence are common techniques

Brightfield

To view pigmented or stained specimens (High Contrast)

Phase Contrast

To view non-pigmented (low contrast) specimens

Fluorescence

To view cells/ structures labeled with a fluorochrome

Electron Microscopy

Magnifies up to 500,000x; can resolve structures, estimated 0.1 nm apart; including Scanning, Transmission

Scanning EM

Scans the exterior of a specimen to reveal the topography and fine details; requires processing of the specimen

Transmission EM

Used to view the internal structures of (embedded, sliced, stained) specimens or view small specimens

How did diverse life forms arise?

Mutations, Horizontal gene transfer (between same generation), vertical gene selection(inheritance, parent, to offspring), selection

Overview of Life Forming Chart

• Hadean| Archean| Proterozoic| Phanerozoic

• Anaerobic bacteria and archaea ( O2 is toxic for organisms)

• Cyanobacteria producing O2 → buildup of it

• O2 in the atmosphere because of a buildup→ killing organisms

• Aerobic bacteria→ Selection to be able to survive oxygen

• Unicellular eukaryotes: significantly diverse and able to survive/ use oxygen

• Multicellular eukaryotes- Plants and animals, hominidsand

Phylogenetic “ Tree of life” with 3 domains

Bacteria , Archaea, and Eukarya which diverted from archaea with the engulfment of bacteria

How did the eukaryotic cell arise?

Endosymbiotic theory: An ancient endosymbiosis gave rise to organelles such as the mitochondrion and chloroplast and possibly other over time

Organellogenesis

Formation of organs during embryotic development

Evidence that mitochondria and chloroplasts are derived

• size

• single circular chromosome with similar genes

• 70s ribosomes & Methionine (formyl-met)

• Division by binary fission using homologous machinery( FtsZ rings)
  

Classifying bacteria and archaea

def: group of individuals that can reproduce naturally and produce fertile offspring

→ But this doesn’t work for prokaryotes because their asexual reproduction

So, classification is based on metabolism or physiology is possible for organisms that can be cultures

Prokaryotic Cells Structure

• Cell envelope: Membrane + layers leading to environment

• External Structure

• Internal Structures

  • Nucleoid

  • Inclusions

3 Common Morphology of Bacteria

Bacillus | Coccus | Spirllium

Bacterial and Archaeal Cell - Envelope

• capsule, slime layer

• s-layer

• ( other membrane with lipopolysaccharides)

• ( PG- associated lipoprotein)

• (teichoic acid)

• cell wall( peptidoglycan, PG)

• Cell membrane

• () means its only for some

Gram- Negative Bacterial Envelope

• Cell membrane (CM)

• Peptidoglycan( PG)→ Cell Wall Material, mesh of peptide and glycan( sugars) → thin

• Outer membrane

• lipopolysaccharides(LPS)

• PG- associated Lipoprotein (LP)→ Covalently binds to peptidoglycan and outer membrane which binds them together

Gram- Postiive Bacterial Enevelope

• Cell Membrane( CM)

• Peptidoglycan( PG)

• Teichoic Acid ( TA): Stabilizes layers or anchors weaving in and out of peptidoglycans due to x10 layers of it

  • doesn’t have an outer membrane, so it doesn’t need L,P which binds PG and Outer Membrane

Two solutes that would not cross a phospholipid bilayer by simple diffusion

• Proton: Charged

• Monosaccharides: Large + Polar which is repelled by nonpolar phosolipid tails which are nonpolar as well

Osmosis

Net movement of water from an area of higher concentration to an area of lower water concentration

Turgor Pressure

Pressure inside cell that pushes on the membrane

Osomotic Pressure

Pressure that must be applied to the solution side to stop fluid movement across a semi-permeable membrane

Hypertonic environment

• Loss of cytoplasm volume (Plasmolysis)

• Excess solubility outside of cell . water moving out / Shirvling inside

Hypotonic environment

• Higher concertation inside cell

• Water moving inside

• Fills up until cell membrane constrains it

Peptidoglycan cell wall(Peptidoglycan = murein) Components - gram neg

• N-Acetylglucosamine (NAG)

• N-Acetylmuramic acid (NAM)

• linked by transglucosylases to form glycan strands

• Short peptides attach to NAMs lined by transpeptidases

  • this creates the multiple layers of peptidoglycan

Lipopolysaccharides- gram neg

fatty acids: If person is infected with gram negative, then this is being released and at high concentration leads to shock: also known as endotoxin

• o-antigen: immune system good at detecting this

Surface layer( S-layer)

a crystalline layer of protein; in many bacteria and nearly all archaea and it is a reinforcement of the cell and structural stability to cell wall

Glycocalyx : Sugar(glyco) ; Coat( calyx)

Slime layer and capsule; made by many species, as needed

Archaeal Envelope

Inner lipid membrane of isoprenoids linked to glycerol via more stable ether linkage( not ester) this creates more stability and resistance against extreme environmental conditions for archaea

External of Bacteria Cell Structures

Appendages

• flagella

• pili/fimbriae

Flagella

Movement for bacteria towards better environments/ bacteria can keep moving as long as there is enough energy

Flagellar structure

Anchored to envelop embedded in every layer (inner cell membrane, peptidoglycan, etc). to stabilize. Hook + Filament(10x length of cell) and is made from inside to outside( envelope to tip of filament)

Bacterial Pilus

• Greek for hair

• Pilin monomers assembled into a helical polymer

• Key functions

  • attachment (fimbriae)

  • conjugation(sex pilus) → exchanging DNA

  • Twitching motility( type 4 pili)

Attachment via fimbrae

• mostly on gram negative species

• fimbriae allow attachment to surface via adhesins at their tips which binds to the structures or cell

• reach out and drag cell if it adheres to surface

  • secures bacteria in gut if it doesnt want to leave

Which structures of a virus or immune cell could access from the outside of an intact gram-negative bacterial cell

• Capsule, Liposaccharides, S-layer, maybe not cell membrane or peptidoglycan → nuance on environment

What about Gram-positive cells?

• out membrane

Internal Bacterial Cell structures

• Cytoplasm

• cytoskeleton

• nucleoid/ chromosome

• ribosomes

• inclusions

• ( Endospore):

Do bacteria, archaea, and eukaryotes have cytoskelentons?

yes

Bacteria and Diffusion

Limited by diffusion and has not active processes , has to be small to be able to diffuse everything

Eukaryotic Cell

• x 10 larger than bacteria and has a smaller surface area to volume ratio

• Have an active process since they cannot diffuse over a larger body

3 types of major cytoskeletal proteins

Actin, Tubulin, Creatinine

Organization of bacterial genome

Chromosome

• singular, circular

• millions of base pairs

• condensed, organizes (different than histones since they don’t have histones)

May have plasmids

• circular

• thousands of base pairs

• can be high or low copy number

Inclusions(microcompartments)

Carboxysomes: enclose carbon dioxide fixation machinery inside protein shell to increase efficiency

Thylakoids: membrane stacks that increase surface area for photosynthesis light harvesting and reactions

Inclusion: Gas vesicles

gas permeable protein shells that exclude water and provide buoyancy to non-swimming organisms in a water column

Inclusion: Magnetosomes

Magnetic crystals formed inside invaginations of inner membrane allowing magnetostatic bacteria to orient to the earth’s magnetic field and swim toward the N or S pole

How do bacteria grow

By binary fission which is the division of one cell doubling in the next generation 1→ 2→ 4→ 8 → 12

batch culture growth curve

Lag phase: acclimating to media/ metabolizing but not dividing

Exponential Phase: rapid growth/ division

Stationary Phase: certain point where no change due to decreasing in division bc food is becoming limited and waste is increasing/ trying to find balance

Death Phase: too poor of conditions, too much waste

• growth and death are in logs, x10

Calculating population size

Nt= No x 2n

Nt: population size at time t

No: initial population size

n= number of generations

Calculating # of generations (n)

1) n=log10Nt-log10No/.301

2)g=t/n

Culture Organisms Nutrients

Macronutrients: CHONPS

Culture Organisms Needs

• Incubation Conditions

  • Light or dark

  • atmosphere

  • Temperature

  • pH

  • Solute Concentration

  • Hydrostatic Pressure

Extremophiles are adapted to environments most organisms find inhabitable - examples

• thermophiles: heat loving

• halophiles: salt loving

• amido/alkaliphiles: acid / basic loving

• barophiles: pressure loving

Optimal temperature for Extremophiles

Psychrophiles: 10 C, best at low conditions

Mesophiles: 37 C, mild conditions

Thermophiles: 50+ C

Hyperthermophiles: 80 C +

Why does growth increase as the temperature goes up from the minimum?

For every 10C increase the reaction will increase of 2-3 because it allows for my energy to be used/ made

Why does growth decrease at temperatures above optimum?

Because of the denaturation of macromolecules and cell structures

How are thermophiles adapted to high temperatures

Challenges with solutions

• DNA strands denature(separate)→ additional twist (positive supercoiling) and compaction

• Proteins denature (unfold)→ stabilize with more intramolecular bonds

• Membranes melt (too fluid/ permeable), glycerophosphate head of phospholipid monomers is hydrolyzed →archaea only, isoprenoid acyl side chains, monolayer membrane, ETHER linkage between acyl group and phosphoglycerol

Unsaturated v Saturated

Unsaturated cis fatty acid: kinks in membrane

saturated: no kinks

Pattern of microbial death: log reductions over time

90% of population killed per log scale

Catabolism- breaking it down

• Energy, electron, and carbon sources

• Entry, feeder pathways

• Fueling (making of precursor metabolites, reductant, ATP)

Anabolism( build it up)

• Autotrophy

• Photosynthesis

• Biosynthesis (of building blocks)

What is Metabolism?

Series of chemical reactions performed by living to make energy, build cell material, and maintain homeostatic

Image of Cell- gram negative bacteria

11 Components - Pili, outer membrane, capsule, wall, periplasm, cell membrane, flagella, nucleoid, cytoplasm, polysomes, vesicles

How expensive is it to make a cell

Type of -Troph

Energy source: Photo, Chemo

Carbon: Auto, Hetero

Electron: Chemo, Hetero

Metabolism Overview

CO2 (autotrophs/ carbon fixation) →or Organic macromolecules (feeder pathways for heterotrophs)→ Org C→ Fueling products Building Blocks→ Macromolecules → Cell

Fueling Products

• Energy

• Reductant

• Precursor

• Metabolites

Building Blocks

• Animo Acids

• Nucleotides

• Sugars

• Lipids

Macromolecules

• Proteins

• Nucleic Acids

• Polysaccharides

Entry Barrier: Semi Permeable membranes

• extracellular enzymes to break down larger macromolecules

• Porins, transporters

• Proteins?

High concentrations of solutes inside the cell

Hypotonic: Molecules at a higher concentration outside the cell can diffuse down their concentration gradient (passive transport)

Hypertonic: Molecules at higher concentrations inside the cell require energy to be transported against them concentration gradient (active transport)

Entry, Feeder pathway example: Lactose

Lactose→ (permease)feeder pathway→ glycolysis→ Central metabolism

Fermentation

• Produces energy during glycolysis

• uses organic molecules

• Regenerates NAD+

• does not require oxygen, does not use the TCA cycle or electron transport chain