Microbio Exam 1

origin of earth

bacteria arose first 4 bya, then shortly after archaea did

bacteria invented anoxygenic photosynthesis (doesn’t produce oxygen)

cyanobacteria invented photosynthesis we know

pollution event due to cyanobacteria producing oxygen

• drove the origin of eukaryotic cells (2 bya)

• anaerobic bacteria died or found niches

phylogenetic tree of life

prokaryotes: bacteria and archaea

however archaea and eukaryotes are more phylogenetically similar than bacteria

last eukaryotic common ancestor

asgard archaea fused with alphaproteobacteria

before: oxygen toxic to asgard

fusion allowed archaea to metabolize oxygen due to the bacteria

gave rise to all eukaryotic organisms

origin of plants

plants took in cyanobacterium which became their chloroplasts

Eukaryotic signature proteins (ESPs)

found in asgard archaea too when originally thought to be only in eukaryotes

• endosomal sorting complex required for transport (ESCRT): drives membrane constriction and fission

• profilin: binds actin monomers to promote elongation of filaments

contribution of prokaryotes to eukaryotes

genes in todays eukaryotes can be traced to a host of bacteria

prokaryotes vs. eukaryotes

P much more inventive than eukaryotes (had many years to evove and change over time)

P right-handed supercoiled DNA, E left-handed supercoiling

E evolved size and structural genetic complexities much better than P

• due to a HUGE bottleneck (fusion)

Eukaryotes (energy and more)

able to evolve much more structural diversity and organismal size due to energetics!

100s-1000s of mitochondria (produce ATP to keep safely living in cell)

command greater energy budget: allowed genome expansion and evolution of structural complexity

have cholesterol in membranes (alters membrane fluidity)

intracellular membranes to divide workload

Prokaryotes

energy is from proton motive force across the membrane

make up for their small size by replicating very quickly (therefore frequent mutations)

bacteriophage

most abundant biological entity on earth

popularion stacked would be light years tall

bacteria in climate cycles

marine algae produce DMSP when stressed by UV

bacteria breakdown DMSP into DMS which aids in condensation and cloud formation

Diffusion

bacteria rely on diffusion because there are no intracellular organelles or transport

the larger a cell, the more inefficient

high SA to vol ratio allows for better diffusion, higher metabolic rate, higher cell numbers!

ex: Thiomargarita: huge but uses sulfur granules to decrease cytoplasm volume

bacterial shapes

cocci: spheres

bacilli: rods

spirals: comma shaped

whooping cough

bordetella pertussis

peptidoglycan (PG)

hold cell shape

dictate morphology in cell wall

polymorphic (none so loss of cell wall) - mycoplasmas

glycan portion (alternating NAM and NAG) - linkage very strong

• lactyl group of NAM attaches peptide portion of PG

• peptide portion alternating L an D isoforms with at least one diamino acid (lysine or DAP)

many abx target PG

can break down and use as an energy source

MreB

actin homolog to help determine shape

helps hold bacteria in rod shape

controls sites of extracellular PG polymerization

FtsZ

contractile protein that forms at midpoint

at cell division it contracts and pulls the cell into the middle to pinch into equal halves

find exact midpoint by detecting concentration

cytoplasmic membrane

bacteria inner membrane

phospholipid bilayer

glycerol backbone, long FA chain (hydrophobic), phosphate (hydrophilic)

can have invaginations to increase SA but keep cell small

hopanoids

most abundant molecule

regulate membrane rigidity of bacteria and archaea (evolved separately)

interdigitate between phospholipids to regulate rigidity

intraceullar compartments in bacteria

no nucleus, mitochondria, ER, lysosomes, or MEMBRANE bound intraceullar compartments

some possess microcompartments (protein or non-unit membrane bound)

ex: carboxysome! in autotrophic bacteria (syntehsize molecules from CO2)

Carboxysome

have a hard protein shell

site of calvin cycle

very large relative to the size of microbe

carbon, CA, RuBisCO fixed within

helps maintain osmotic balance of cell (by changing bicarbonate into CO2)

hold in molecules poorly held by lipid bilayers

oxygen cannot penetrate

needs high conc. of CO2

CO2 can come in but not out

O2 cannot come in

take up bicarbonate and ribulose through diffusion

carboxysome mechanism

bicarbonate moves in since it is negatively charged, carbonic anhydrase breaks it into H2O and CO2, RuBisCO adds CO2 to ribulose 1,5 bisphosphate to make a 6 carbon molecule which is then split into 2 3-PGA which leaves to go to the glycolytic pathway or calvin cycle (more likely) to generate sugars

RuBisCO

carboxylase (take CO2 and add) and oxygenase (take O2 and add)

prefers O2

less inefficient to make PGA with O2 (1 instead of 2)

Prokaryotic Genetic Material

single circular chromosomes (haploid)

nuceloid

occasional paralogs (accidentally extra replicated copy, new genes born through them due to mutation)

semi-condensed supercoiled DNA

Bacteria supercoiling

DNA gyrase twists circular DNA so it collapses on itself (cuts one strand of DNA, passes the strand through the break and then reseals it and repeats) - (negative supercoils)

certain regions are less supercoiled than other regions to transcribe genes more easily (more accessible)

movement of replication fork causes positive supercoiling, gyrase introduces negative supercoils to fix

pseudopeptidoglycan

in archaea

diamino acid mediated cross-linking

synthesis of peptidoglycan

• occurs in cytoplasm and then moves to exterior/periplasm

• UDP shepherds NAM (ONLY NAM CARRIES PG)

-L alanine added, then D glu, then DAP (gram neg) or lysine (gram pos)

-Each are individual enzymes

-Formation is not dependent on ribosomes

-Two d alanines added at the end

-Alanine racemase: takes L alanine and turn it into D alanine

-Alanine ligase: ligate two alanines together and stick them on at the end

-UDP with NAM and AAs transfers hydrophilic molecule to a hydrophobic lipid (bactoprenol): whole thing = lipid 1

-One phosphate from UDP transferred to bactoprenol (now has 2 phosphates)

-UDP then adds NAG to NAM (lipid 2)

-hydrophobic barrier it needs to get pentapeptide (hydrophilic) across

-Bactoprenol is very hydrophobic so can cross

-Flippase (MurJ): takes lipid 2 because bactoprenol is so hydrophobic and flip it across the membrane (into periplasm for gram neg, outside of cell for gram pos)

-Flipped pentapeptide gets added to another PG

-Bactoprenol gets recycled (goes across the membrane) by using the energy from removing one phosphate to go back across the membrane (enzyme removes phosphate)

-Autolysins: class of enzymes outside of cell that break glycosidic bonds between NAM-NAG,  makes little cuts in sacculus around cell to add PG and allow cell to grow/change

-transglycosidase: enzymes outside of cell that reform glycosidic bonds

-transpeptidases crosslink PG to each other

bacitracin (neosporin)

-Inhibits enzyme that cleaves phosphate off of bactoprenol so it cannot flip back to the inside of the cell

-But Autolysins continue making cuts in the sacculus and eventually they make enough cuts that don’t fill in that the cell bursts

Vancomycin

-Binds to the terminal d alanines on pentapeptide

-Since it is so big, it blocks transpeptidase from crosslinking it to a PG and transglycosidase from reforming glycosidic bonds

-But Autolysins continue making cuts in the sacculus and eventually they make enough cuts that don’t fill in that the cell bursts

-gram negative cells naturally resistant due to outer membrane porins

-resistance is rare, must substitute d alanine for d lactate in PG

Cycloserine

-Inhibits alanine racemase and alanine ligase that add the terminal d alanines to the pentapeptide

-so PG cannot be synthesized

gram negative transpeptidation (crosslinking)

-The extra amine group from DAP allows it to form a branched peptide bond which binds to the sub terminal d alanine (second to last)

-Transpeptidase splits off terminal d alanine (energy source) which uses energy to change transpeptidase enough to form the peptide bond

-mature form of PG: 4 AAs

gram positive transpeptidation (crosslinking)

-Pentaglycine added to lysine in between amine group and carbon chain

-Transpeptidase does the same thing and crosslinks using the pentaglycine

-Since it doesn’t have an outer membrane, it uses the pentaglycine to keep enzymes/proteins from diffusing away (covalently linked)

transpeptidase

bind to pentapeptide of PG and split off terminal d alanine to give activation energy of enzyme

enzyme changes structure and binds to an adjacent d alanyl d alanine and forms the linkage

many different kinds

penicillin

mimics d alanyl d alanine so transpeptidase binds to it and is stuck to penicillin

Toll Like Receptors (TLRs)

-recognize Microbial Associated Molecular Patterns (MAMPS) ex: PG

-can detect multiple different kinds of MAMPS

-part of innate immune system

-once bound to ligand, trigger intracellular cascade to release cytokines that diffuse away from the site and direct wbcs to the invader (follow cytokine gradient)

beta lactam antibiotics

covalently bind transpeptidase to inactivate them

cell wall weakens and leads to lysis

resistance due to beta lactamase which cleaves beta lactam ring (gram negative)

resistance due to altered transpeptidase (gram positive)

ex: penicillin

NDM-1

enzyme that makes bacteria resistant to many beta lactam antibiotics

MRSA

gram positive

has an altered transpeptidase that is resistant to beta lactam antibiotics and augmentin

resistant to augmentin because the transpeptidase itself will not bind penicillin due to its altered state (it doesn’t fit). in other bacteria, they just break down beta lactamase so when there is a decoy, it allows penicillin to bind.

augmentin

antibiotic combination of penicillin and clavulanic acid

clavulanic acid serves as a decoy for beta lactamase so that penicillin to inactivate enough TP to cause lysis

wall teichoic acids

-signature molecule of gram positive

-made up of d alanine and d glucose

-negatively charged due to phosphate

-either linked to fatty acids or bound to PG

-allow penetration of magnesium and calcium through PG

-polymer of ribitol

Cationic Antimicrobial Peptides (CAMPs)

-part of our innate immunity

-produced by epithelial cells, among others in response to wounds/infection

-ex: defensins

• bind to lipoteichoic acid and form a pore in the cell to cause lysis

braun’s lipoprotein

only in gram negative

hold outer membrane and periplasm together (prevent swelling and separation far from cytoplasmic membrane)

bound to second amine on DAP

lipopolysaccharides (LPS) or endotoxins

signature molecule

only in gram negative

toxic molecule bound to outer membrane of the cell

does the same thing as a phospholipid

more hydrophobic than phospholipids (greater barrier to hydrophilics)

can be used as vaccine adjuvant to trigger immune response

toxic part: lipid A, analogous to phospholipids

core polysaccharide: linked to lipid A via KDO

o specific side chain: variable

bacteria can alter o specific side chain to avoid antibodies

gram neg. bacteria form vesicles to spread pathogen further or act as camouflage

detected by TLR

deplete clotting factors (internal bleeding)

porins

proteins that form channels in outer membrane into periplasm of gram negative

block vancomycin but not penicillin

outer membrane vesicles (OMVs)

vesiculation occurs during normal growth

increased during stress

used to get rid of LPS and damaged/mis-folded proteins

horseshoe crab

amebocytes form clot around LPS so that it can’t spread

assay to detect LPS: limulus amebocyte lysate (LAL)

use LAL in vaccines and more (made from limulus blood)

gram staining

gram positive: purple stays after decolorization

• acid fast stain: for mycobacterium

gram negative: purple leaves after decolorization, pink stains

glycocalyxes

capsules and slime layers made of polysaccharides

capsules

heat killed capsule bacteria mixed with no capsule bacteria affected the mouse and resulted in new active capsule bacteria

important in virulence (exclude toxins and viruses, avoid phagocytosis)

functions:

• often mediate adherence of cells to surfaces

• protect bacterial cells from engulfment

• protect from attack by antimicrobial agents of plant or animal origin

• protect cells from effects of desiccation

• reserves of carbohydrate for subsequent metabolism

surface structures of bacteria

fimbriae, pili, flagellum

fimbriae

short, numerous protein appendages

help adhesion, biofilm formation, twitching motility

grow like hair from the base and can be retracted

when retracted it is broken down inside the bacteria to be reused

pili

long rod-like protein extensions to attach to other cell

encoded by fertility plasmids and are involved in conjugation

function as receptors for bacterial viruses (bacterial std)

retractile molecules by degrading protein on cytoplasmic side

plasmid DNA is exchanged when cells are pulled together, no bacterial DNA is shared

covered in hundreds of viruses (both bacteria will end up dead)

microbial dissimilatory reduction (MDSR)

extracellularly pass on electrons from the ETC onto metal so they continue to make ATP

basis for microbial fuel cells: generate energy and break down waste products (reduce metal)

flagella

hollow, semi-rigid protein appendages

once they swarm enough, they act as one organism

CCW rigid and can move forward (default)

CW: limp and can turn/wiggle

• polar: 1 flagellum

• peritrichous: many throughout

  • CCW causes flagella to come together into a larger filament

  • CW causes flagella to come apart resulting in random movement

• amphitrichous: at each pole

• lophotrichous: tuft of flagella on one end

Flagella structure (basal body)

L ring: in outer membrane (LPS)

P ring: embedded in PG

FliG: C ring (cytoplasm)

MS ring: above C ring on cytoplasmic membrane

MotA and MotB: motor

• -Move by proton motive force, more + in periplasm, more – in cytoplasm which causes protons to move into cytoplasm, generating work/energy to rotate (torque generated by Mot proteins)

  -Protons moving through Mot protein will pass by pos/neg charges and will attract or repel to generate the torque/motion

  -Protons put into periplasm are result of ETC, protons come back through the basal body and generate torque

rotation speed depends on strength of proton motive force

prokaryotic vs eukaryotic flagellae

Prokaryote:

• -Rotate CW and CCW

  -By default rotates in a CCW direction (moving forward)

  -Generates propulsion

  -Filament connected to a hook

  -Powered by proton motive force

Eukaryote:

• -Whip back and forth

  -Generates propulsion

  -powered by ATP hydrolysis

axial filament flagella

encased in outer membrane

corkscrew motility

more powerful propulsion

Chemotaxis

response to chemicals in the environment

sense gradients via chemoreceptors (MCPs)

MCPs bound to appropriate signal start a methylation and phosphorylation cascade that results in altering the frequency of tumbling

sense temporal gradients (gradients of chemoattractants/chemorepellents)

random walk

in the absence of gradient, frequency of tumbling is random

directed movement

increase of attractant, decreased frequency of tumbles resulting in longer runs

tumbling

occurs in CW direction

random, do not pick direction, change frequency depending on the gradient

move for longer periods when moving towards attractant and shorter periods when it senses its moving away

Methyl Accepting Chemotaxis Proteins (MCP)

array present on leading pole of bacteria

send signal for which direction the flagella will rotate

signal diffuses to each of the flagella and cause change simultaneously

two parts: one in periplasm/outside and one in cytoplasm

Empty MCP mechanism

• Serine is not binding to MCP, which tells the bacteria its moving in the wrong direction

• In the absence of a ligand, MCP interacts with CheA with the help of CheW on the cytoplasmic side (CheA hydrolyzes ATP into ADP to add the phosphate)

• Causes the CheA to be active (puts phosphates on histidine)

• Autophosphorylates itself on a histidine (so adds a phosphate to itself: CheA)

• Phosphate group gets relayed to either CheY or CheB

  • CheY:

    • Gets rapidly phosphorylated by CheA (activated protein)

    • Activates with the basal body of the flagellum

    • Interacts with FliG in the basal body which causes the CCW rotation to stop and switch directions to CW

  • CheZ: removes phosphate from CheY rapidly

    • So CheY has a short half life for a short tumble

Bound MCP

Ligand being bound changes conformation on cytoplasmic side to turn off CheA kinase activity

• Tumbling still occurs but at a lower frequency

• After a certain period of time this conformation change makes the cytoplasmic side of MCP a better acceptor of CheR (methyl transferase), which increases methyl groups in the struts of glutamatic acids

  • Since the unbound cytoplasmic MCP was methylated (strong bonds that cannot be removed), it cannot stop its tumbling once there is no more ligand bound to the other one because the methylation of MCP keeps CheA active

  • Overtime while CheY is getting phosphorylated, CheB is also becoming phosphorylated, which activates its enzymatic activity (methyl esterase, removes methyl groups) to reset the bacteria

• Because the unbound MCP got methylated by the bound one, it will activate CheA and therefore CheY

• So even in moderate concentrations of the ligand, it can still change direction

Accommodation

• the process by which bacteria adjust their sensitivity to chemical gradients.

• allows bacteria to maintain a constant response to stimuli despite prolonged exposure to a specific concentration.

• It is crucial for effective chemotactic behavior and survival in chemically varying environments.

Methylation adjustment causes a confirmation change on cytoplasmic side (CheA binds and can hydrolyze) and on the outer membrane side the ligand binds less easily/frequently to MCP.

If the bacteria chooses a new direction with a higher concentration, the ligand will bind more frequently since there is more of it. This changes the structure on the cytoplasmic side of MCP, causing it to release CheA so CheY phosphate is no longer produced (so less frequent tumbling)

nanobrain

array of MCPs on the leading edge of bacteria, (in periplasm for gram neg, in outer surface for gram pos)

array has all kinds of different MCPs that detect different ligands

CheW

when CheA is suppressed CheW spreads the CheA suppression signal through the whole array of MCP to prevent other MCP from trying to start a tumble since they can’t find their ligand

if MCP detects bacteria is moving towards a repellent as well as the attractant, it will tumble (repellant outweighs the attractant)

archaea

• only grown as communities because they rely on syntrophy (other microbes consume waste products and reduce oxygen levels) — most microbes are this way

• have cytoskeletons

• NO PHAGOCYTOSIS

• evolved a nucleus before taking up mitochondria (ATP production)

• L-glycerol

• chains of isoprenes called phytenyls instead of fatty acids in phospholipids

• can have lipid monolayer (thermally stable) and bilayers

• Pseudopeptidoglycan

• ETHER linkage between glycerol and isoprenes (phytanyls) (more stable at high temps)

  • Cyclopentane rings stabilize membrane at high temps

• Can live at high temps and high solute concentrations

• Have histones

• Positive supercoils (thermally stable)

bacteria

• have cytoskeletons

• NO PHAGOCYTOSIS

• D-glycerol

• fatty acids in phospholipids

• only lipid bilayers

• our cytoplasm more similar to them

• Peptidoglycan

• Actin homolog (MreB) to help determine shape

• ESTER linkage between glycerol and FA

• No histones

Unique aspects/signature molecules of bacteria:

• DNA gyrase (ciprofloxacin inhibits DNA Gyrase) 

• NAM

• Peptidoglycan

• DAP

• Gram positive: teichoic acid

• Gram negative: endoxins

Gram Positive:

• Thick cell wall (lot of peptidoglycan)

• No outer membrane

• Lysine in all

• Interbridge of glycine to crosslink

• Teichoic acid

• Mycobacterium (genus with waxy coating of mycolic acids act as outer membrane)

Gram Negative:

• Have an outer membrane (playing with idea of membrane bound organelle)

• Periplasm: membrane bound space between cytoplasmic membrane and outer membrane

  • Can do enzymatic reactions in periplasm

  • Communication between periplasm and outside environment

• Less peptidoglycan (smaller cell wall)

• DAP in all

• Directly crosslink adjacent strands of PG

• Naturally resistant to vancomycin due to outer membrane porins

• Peptidoglycan not exposed to environment but enclosed in the periplasm

• Have braun’s lipoproteins to hold outer membrane and periplasm together (prevent swelling and separation far from cytoplasmic membrane)

  • Second amine on DAP binds to braun’s lipoprotein

• endotoxins/LPS

thylakoid membrane

internal folds to increase SA of bacteria

lysozyme

enzyme breaks glycolytic bonds between NAG and NAM

more effective in gram positive since no outer membrane