Microm 410 Midterm 1

Key differences between prokaryotes and eukaryotes?

Eukaryotes have:

• nucleus

• membrane bond organelles

• cytoskeletal elements

• much larger

Why was 16S rRNA used?

• Found in all cells (18S in eukaryotes)

• Performs same function in all organisms

• Not subject to horizontal gene transfer

• Highly conserved and highly variable regions

Bacteria & Archaea vs Eukaryotes

Genome structure and metabolic diversity

• circular DNA vs linear

• operons vs single genes

Archaea & Eukaryotes vs Bacteria

Transcription and translation

Bacteria & Eukaryotes vs Archaea

Membrane lipids

• ester linked fatty acids vs ether linked isoprenoids

Pathogens infecting animals/plants

• many vs none

What is an endosymbiont?

Microbes that live intracellularly within host organisms in often mutually beneficial association

What traits are associated with transition from endosymbiont to organelle?

• Reduced genome size with significant gene transfer to host nucleus

• Significant loss of metabolic versatility

• High numbers of proteins imported from host

Evidence of symbiotic origins of mitochondria and plastids?

• Multiply by binary fission

• Bacterial ribosomes

• Circular DNA genomes

• Bacterial envelope structure

Organization of bacterial cytosol?

• Genome (single, circular dsDNA) is condensed in nucleoid

• High degree of organization and structure

Examples of bacterial sub-cellular structures that have specific functions?

• Carboxysome: carbon fixation

• Gas vesicle:

• Inclusion bodies: storage (nutrients, molecules, proteins)

• Magnetosome: makes magnetic particles for movement and direction

Phospholipid composition and effect on permeability?

• No unsaturated fatty acids → lower permeability, more stable

• Many unsaturated fatty acids → higher permeability, less stable

What fills the gaps in the phospholipid membrane?

• Eukaryotes use sterols (cholesterol)

• Prokaryotes use hopanoids

How do archaeal phospholipids differ?

• L-glycerol, not D

• 1 phosphate linked, not 3

• Branched tails that can cross link and forms rings (inhibits movement → more stability)

• Ether linked, not ester linked

*can form monolayer instead of bilayer

Gram staining process

Heat fix cells → stain with crystal violet → add iodine (locks violet in G+) → destain with acetone-ethanol → counterstain with safranin

Make up and purpose of bacterial cell wall?

• Made of linked polymers called peptidoglycan

• Constrains osmotic expansion of plasma membrane

What is a peptidoglycan polymer sub-unit made of?

*What do some G+ bacteria do that G- don’t?

• Peptidoglycan = Peptide + Glycan = D amino acid peptide + NAG,NAM

• NAG and NAM disaccharides joined by B-1,4 linkage

• Lactic acid group attached to NAM is attached to a peptide w/ D amino acids

• Peptide cross-links glycan strands

*Some G+ have glycine bridge to cross-link peptides

Peptidoglycan biosynthesis steps?

1. Make subunit: add 5 amino acids to NAM → link NAG to NAM pentapeptide

2. Get across membrane: use lipid carrier bactoprenol to flip subunit into periplasm

3. Insert into existing PG: Periplasmic transglycosylases link disaccharide to existing glycan strand → transpeptidases (PBPs) cross-link pentapeptide

How are peptidoglycan polymers arranged?

• Glycan strands have high tensile strength; peptides have high elasticity

• Glycan around circumference; peptides length wise

How does expansion of the peptidoglycan sacculus happen?

• Selective formation and then cleavage of peptide cross-links

Gram + cell envelope

• Thick PG layer

• Teichoic acids (alcohol sugars w/ amino acid/alcohol sugar side group): linked to NAM of PG

• Lipoteichoic acids: link membrane lipids and PG

Gram - cell envelope

• Outer membrane surrounding cell wall

  • inner leaflet made of phospholipids

  • outer leaflet made of LPS

    • lipid A w/ polysaccharide attached

      • initial portion are core sugars - same for all

      • majority is O-antigen - varies

• Lipoproteins: connect cell wall to outer membrane

• Thin PG layer

• Periplasm: between inner and outer membrane containing enzymes and proteins

S layers composition and location

• Both G± can have S layer

• Self assembling array of proteins

• G+: in PG G-: in LPS

Glycocalyx definition, forms, function?

• Most prokaryotes have outermost external later called glycocalyx made of proteins and polysaccharides

• Capsule: compact, discrete glycocalyx

• Slime layer: less discrete, loose glycocalyx

• Attachment/adherence, protection, essential for biofilms

Archaeal cell envelopes

• Most have S layer, some have protein layer sheath

• Methanogenic have pseudomurein/PG

  • NAT instead of NAM

  • B-1,3 linkage instead of B-1,4

  • No D amino acids

Bacterial wall elongation

• PG synthesis resulting in cell elongation is mediated by scaffolding protein MreB

• MreB polymers move in circumferential direction, require active PG synthesis

Bacterial septation and division

• Septation: zonal PG synthesis

• Scaffolding element is complete ring of FtsZ

• FtsZ ring creates large complex of enzymes to form divisome

• Inward growth of cell envelope from contraction of FtsZ ring

Polar aging

Cylindrical growth and division leads to aging poles; older pole = increased chance of lysis

Bacterial flagella

• Peritrichous: flagella all over cell body

• Flagellar filament made of flagellin spins for movement

• Hook is flexible to allow filament repositioning

• Basal body resembles Type III secretion system

• Cap protein passes first and assembles hook and filament (grows from distal end)

Proton motive force

• Creates energy to spin flagellum

• Chemical and electrical gradient

• Can be used for ATP synthesis, solute transport, motility

Archaellum vs flagellum

• Basal body resembles Type IV pili

  • Pili are extended and retracted via polymerization for adherence and twitching motility

• No hook

• Filament assembled proximally

• Rotation powered by ATP hydrolysis

Chemotaxis

• Bacteria swim in straight lines (runs) and stop (tumbles) for reorientation

• No ligand → CheA autophosphorylates → phosphorylates CheY → promotes tumbling

  • CheZ phosphatase dephosphorylates CheY

• MCP binds ligand → CheA blocked → less CheY → no tumbling → longer runs