Microbiology Midterm 1

what are microbes?

single-celled organisms too small to be seen with the naked eye such as bacteria, archaea, yeast, viruses, etc.

three laws of microbiology

microbes are very very small

• many different sizes: E. coli 2 micrometers, Prochlorococcus marinus 0.4 micrometers (most abundant microbe in the ocean), paramecium 150 micrometers (just visible to the naked human eye, protists that feed on smaller bacteria)

• collectively constitute the majority of biomass production on Earth (C = plants, N and P 10 x)

microbes are everywhere

• food, surfaces, soil, water, air, gut, skin, etc.

• extremophiles: survive and thrive in extreme environments

  • temperature = -20 to 121 degrees Celsius

  • pressure = 300x sea level air pressure

  • pH = 0

  • salinity = 2M

  • radiation = 15,000 Gy

    • Deinococcus radiodurans toughest bacterium, can effectively repair shattered DNA so resistant to radiation, also to dessication and vacuums

    • humans killed by 10 Gy

  • Bacillus infernus the “microbe from hell”

    • isolated from a 2-mile deep well

    • can live without light or oxygen and in high temperature and pressure

    • get their nutrients from rocks

microbes rule

• critical and unique roles, sheer mass, persistence in all habitats, metabolic diversity unique to them, and can survive without animals and plants but vice versa not true

  • 80% of the air contains nitrogen that plants cannot access on their own, so microbes in soil fixate nitrogen and convert it to nitrate and nitrite that plants can use

    • soybeans can fix nitrogen because of their bacteria symbionts

  • Prochlorococcus marinus fixates carbon in ocean via photosynthesis and is responsible for 20% of the oxygen we breathe

• 90% of life history is solely microbes

• health and medicine

  • Yersinia pestis: caused the Black Death in Europe in the 14th century that triggered the Renaissance

  • involved in obesity, autoimmune diseases, autism, and food

bacteria have tremendous diversity but very limited morphology

• coccus = sphere, bacillus = rod, spirillum = spiral

• however, morphology is not an indicator of evolutionary relationships of microbes

  • Escherichia coli and bacillus anthracis (gram-positive) are the same shape yet the first is harmless and the other is deadly

  • Must use DNA sequencing to identify bacteria, not their morphology

E. coli

• proteobacteria

• gram-negative

• stain pink/red on MacConkey media

• only make up 0.05% of gut flora (most abundant Bacteroides and Firmicutes)

tree of life

• based on small subunit rRNA sequences

• three domains of life: bacteria, archaea, eukaryotes

• humans are much more closely related to plants and fungi than proteobacteria and cyanobacteria are related to one another

• branch length = amount of evolutionary change

why is bacterial shape limited?

• determined by trade-off between swimming capabilities, maneuverability, and metabolic cost

  • long/slightly curled = better maneuverability, short = easiest to make, small/slightly curled = faster at swimming

the advantages of being small

• have more surface area relative to their volume (higher surface area/volume ratio)

  • increased sphere size = decreased SA:V (3/r)

  • maximized SA:V (smaller r) = more efficient nutrient uptake and exchange per unit of cell volume

• tend to grow faster than larger cells

  • can put less energy and materials toward growth

• easier to move things around within the cell

  • can solely rely on diffusion which does not require any energy

  • a molecule can move from one end of a bacterial cell to the other end in 1/100 of a second

  • in large cells, diffusion by itself is inadequate (takes too long and does not work well because nutrients will be used up before they can reach further parts of the cell) so moving things around requires energy

  • Thiomargarita magnifica: microbes with a nitrate vacuole that takes up the majority of space within them so that cellular contents are pushed against the cell membrane and they can rely on diffusion for the transportation of materials and nutrients because they are thin

    • Thiomargarita namibiensis: giant microbes the size of a fruit fly’s eye also filled with nitrate vacuoles

the lower limit of microbial cell size

• all the things a living cell must have: a chromosome, proteins to copy the chromosome, express genes, and make other proteins, and a minimum of 300 genes → 0.02 microns cubed

  • Pelagibacter ubique: most abundant bacteria in the ocean with a volume of 0.03 microns cubed, even smaller than Prochlorococcus marinus

the major functions of the cell membrane

1) permeability barrier- a gateway for nutrient transport into and out of the cell, prevents leakage

• cell membranes have approximately equal parts of phospholipids and proteins

• hydrophobic fatty acid tails + hydrophilic glycerol phosphate head = a phospholipid, form a bilayer structure in the cell membrane with the tails facing inward and the heads facing outward

• ethanol can dissolve cell membranes and kill bacteria

• a permeability barrier for polar and charged molecules- small uncharged (hydrophobic) molecules (O2, H2, CO2) can diffuse through but most nutrients are charged (hydrophilic) and must enter and exit via specific protein transporters

2) protein anchor- site of many proteins involved in transport, bioenergetics, and chemotaxis

• there are 20 amino acids with different R groups/side chains that can be nonpolar (hydrophobic) and polar neutral/acidic/basic (hydrophilic)

  • the surfaces of cytoplasmic proteins are hydrophilic because 70% of cells is made of water

  • proteins than span the cell membrane have hydrophobic inner regions and hydrophilic outer regions that lock them into the membrane

• membrane protein functions: structural support (flagella), detection of environmental signals (sensors), secretion of virulence factors and signal molecules, substrate transport, generate energy

3) energy conservation site of generation and use of the proton motive force

• proton gradient- more H+ ions outside the cell than inside the cell, used to generate or conserve energy, unique to bacteria (mitochondria in eukaryotes ancestors of bacteria)

cell membrane fluidity

• bacteria change the lipid composition of their cell membranes in response to changes in the environment

• different types of phospholipid fatty acid tails

  • unsaturated fats: plant-derived, C=C, naturally kinked = loose, more fluid membrane

    • cis = kinked and fluid

    • trans = straight and packed (artificially made)

    • oleic acid can be cis or trans

  • saturated fats: mostly animal-derived, C-C, naturally straight = solid, more tightly packed membrane

    • palmitic acid

    • cyclopropane fatty acid (derived from plants)

• hopanoids: bacterial version of cholesterol (5 rings instead of 4) that stabilizes the cell membrane and makes it less fluid

archaeal membrane

• ether links between glycerol and fatty acids more stable than ester in bacteria

• stiffer due to covalent bond-joined fatty acid tails (diglycerol tetraethers) that form a lipid monolayer

• modulate biphytanyl to adjust the fluidity, rigidity, or proton permeability of the membrane

• unique chemistry allows them to grow at 121 degrees Celsius → cannot be killed by an autoclave (high pressure water cooker that sterilizes)

bacterial cell wall keeps cell from exploding

• the inside of a cell is packed full of components, meaning that cells in high water environments experience osmotic stress (the pressure of water on the cell due to the high intracellular solute concentration and low water concentration)

• the bacterial cell wall keeps osmotic stress at bay so that the cells do not explode

  • made of glycan chains cross-linked by peptide bridges that form a porous, mesh-like, single-linked molecule called peptidoglycan

    • glycan chains made of N-acetylglucosamine (G) and N-acetylmuramic acid (M), peptide bridges linked to M

    • peptide bridges made up of L-Alanine, D-Glutamic acid, m-Diaminopimelic acid, and 2 D-Alanines, cross-link formed by releasing terminal D-Alanine so that the m-Diaminopimelic acid of the other bridge can bind to the remaining D-Alanine

    • D-version of amino acids only found in the cell walls of bacteria

    • penicillin antibiotic that blocks the release of the terminal D-Alanine by binding to transpeptidase so that the cell wall is unable to make new peptide bridges and is destroyed, only effective on actively growing, non-dormant bacteria (a persister not a resister) and gram-positive bacteria (cannot get through the outer membrane in gram-negative)

    • vancomycin antibiotic that binds to D-Alanine-D-Alanine to prevent the removal of the terminal D-Alanine, blocking cross-bridge formation and destroying the cell wall

    • lysozymes can degrade and destroy the cell wall

• not all bacteria have a cell wall- intracellular pathogens, endosymbionts, mitochondria and chloroplasts are in a balanced environment so do not need cell walls

penicillin

• an antibiotic that blocks the release of the terminal D-Alanine by binding to transpeptidase so that the cell wall is unable to make new peptide bridges and is destroyed, only effective on actively growing/non-dormant, gram-positive bacteria

  • gram-negative bacterial outer membrane blocks access of penicillin to cell wall because penicillin is hydrophobic and cannot pass through outer membrane porins, so it must be modified into ampicillin or amoxicillin that are hydrophilic and capable of passing through the outer membrane porins

  • persistence = bacterial cells that become dormant to avoid antibiotics vs. resistance = bacterial cells that can grow in spite of antibiotics

vancomycin

an antibiotic that binds to D-Alanine-D-Alanine to prevent the removal of the terminal D-Alanine, blocking cross-bridge formation and destroying the cell wall

gram staining

• gram-positive = thick cell wall (traps crystal violet)

  • Bacillus anthracis, streptococcus pneumoniae

  • blue/violet in a gram stain

  • teichoic acid connects peptidoglycan layers

  • have porous S-layer of proteins

• gram-negative = thin cell wall (crystal violet can pass through)

  • E. coli, Salmonella

  • red/pink in a gram stain

  • have an outer membrane

  • have porous S-layer of proteins

• how to perform a gram stain: fix cells onto a slide using methanol → add crystal violet dye → add iodine (binds stain to increase stain retention in gram-positive) → wash with ethanol for 10 seconds (removes stain from gram-negative cells), → add safranin (pink counterstain that makes gram-negative cells visible)

gram-negative cell envelope

• 2nd protective barrier containing porins that transfer small molecules

• periplasm = space between inner and outer membrane, where peptidoglycan layer is found

• lipopolysaccharide (LPS) = fatty acid and sugar chain inserted into outer membrane

  • lipid A + core oligosaccharide + polysaccharide chain (called the O-antigen)

  • function is to mask cell surface from immune system antibodies and food poisoning macrophages

  • up to 160 different types of LPS O-antigens, including 157 in O157:H7 for E. coli

  • different types of toll-like receptors in immune cells can recognize and bind with LPS in gram-negative bacteria and lipoteichoic acid in gram-positive bacteria

  • LPS is an endotoxin within the bacterial cell that can trigger a strong immune reaction that causes septic shock vs. an exotoxin released outside of the bacterial cell to cause damage to the host

capsules

• polysaccharide layers that assist in attachment to surfaces, aid in evasion of the immune system, resist desiccation (loss of moisture), and trap nutrients by absorbing water

  • Xanthomonas campestris- bacteria that is slimy and smooth because of capsules, causes black rot disease in plants, used to make xanthan gum that increases the viscosity and homogeneity of various foods and is digested by bacteria in our gut

stalks

• Caulobacter crescentus- bacteria that uses a stalk to stick to places like rocks in streams and the inside walls of water pipes

• made of polysaccharides (holdfast at end of stalk meditates adhesion) and proteins

• 1 square inch can lift 10,000 pounds

fimbriae and pili

• filamentous protein structures that enable organisms to stick to surfaces or to each other to form biofilms or pellicles (aggregation in test tubes that looks like foam)

• Porphyromonas gingivitis stick to our gums and cause gum disease

• Pili facilitate genetic exchange between bacterial cells (bacterial sex, called conjugation)

bacterial flagella

• allow bacteria to swim, move, and avoid dangerous situations

• bacterial swimming- 60 body lengths per second (3x faster than a cheetah) and can rotate 18,000 rpm (2-3x a car)

• structure of the gram-negative flagellum

  • hollow tube made of flagellin polymers

  • hook structure that connects the flagellum to the motors is made of 4 rings: L ring in outer membrane, P ring in peptidoglycan, MS ring in cell membrane, and 2 C rings in cytoplasm (anchors)

  • C rings interact with Mot A and Mot B, which use proton motive force to drive rotation

  • Fli proteins like FliG can switch the direction of rotation

• flagellum rotation in archaea is driven by a sodium gradient

• H = flagellum type, like H7 in O157:H7 for E. coli

microbial taxes

• taxis: directed movement in response to chemical or physical gradients

  • chemotaxis = chemicals, phototaxis = light, aerotaxis = oxygen, osmotaxis = ionic strength, hydrotaxis = water

• chemotaxis and biased random walk

  • counterclockwise rotation moves a flagellated bacterial cell towards an attractant

  • clockwise rotation stops forward motion so that the cell tumbles and changes direction

  • are more likely to stay heading in the right direction towards the attractant when attractant receptors are bound to more attractant and are more likely to tumble and change direction when attractant receptors are bound to less attractant

bacterial swarming

• when a population of bacteria move across a solid surface and their flagella bundle together

  • the flagella allow them to glide to edges because they secrete surfactant that facilitates gliding

nanotubes

pili that can conduct electricity by ionizing water molecules from the air along with a proton gradient

bacterial cytoskeleton

• FtsZ (Z-ring) facilitates bacterial cell division in all morphologies, homologous to cytoskeleton proteins in eukaryotes

  • FtsZ only in coccus

  • FtsZ + MreB in bacillus

  • FtsZ + MreB + crescentin-like protein in spirilla

  • FtsZ (tubulin) + MreB (actin) + crescentin (intermediate filament) in vibrio

specialized structures of cyanobacteria (photosynthetic bacteria)

• thylakoids: extensively folded intracellular membranes that increase surface area for photosynthesis

• gas vesicles: increase buoyancy

• carboxysomes: polyhedral bodies packed with the enzyme rubisco for CO2 fixation

why do we culture bacteria?

in nature, bacteria typically exist in mixed communities composed of multiple species, so to study the properties of individual species in the lab, they must be grown separately in pure culture

culture media

• nutrient-rich substances used to grow and maintain microorganisms in the lab for studying

• broth: liquid used to grow a lot of cells quickly by shaking it but cannot be used for isolating cells

• agar plate: solid made from seaweed that can isolate bacterial colonies and separate cells but takes more time, invented by Robert Koch (father of microbiology who studied anthrax, tuberculosis, and cholera)

• complex media: nutrient-rich but poorly defined (no idea what or how much of what is in it)

  • enriched media: complex media plus specific blood components, for bacteria that need blood to survive, chocolate agar

• synthetic/defined media: precisely defined (know exactly what and how much of what is in it) but not as nutrient-rich

  • minimal defined media: contain only the nutrients essential for growth, used when we do not know and want to find out what a cell needs to grow

• selective media: favors the growth of one organism over another; differential media: show differences between two species that grow equally well

  • MacConkey media is both selective and differential:

    • selective because crystal violet and bile salts kill gram-positive bacteria (crystal violet inhibits cell wall formation and bile salts absorb fat that destroys cell membrane)

    • differential because lactose-fermenting cells lower the pH of the medium around them and turn red, while non-lactose-fermenting cells remain uncolored

  • Mannitol salt agar is both selective and differential

    • selective because of its high NaCl concentration so only halotolerant cells will grow (like Staphylococcus epidermis that live on sweaty skin and Staphylococcus aureus the human pathogen)

    • differential because mannitol-fermenting cells produce acid that lowers the pH of the added phenol red and causes the die to turn yellow

      • Staphylococcus aureus = yellow, Staphylococcus epidermis = red

how to isolate bacteria species?

• spreading: used when the concentration of CFUs is unknown

  • perform a serial dilution with 1 ml CFUs and 9 mL of water and increasingly dilute it ten-fold (10^1, 10^2, 10^3, etc.) → take out diluted CFUs from each test tube and spread each onto its own agar plate → count CFUs on plate

    • math example: 3 CFUs / 0.1 × 10^6 mL = 3 × 10^7 cells/mL, using plate count

    • microscopic count: suspend sample in liquid, put in in a chamber of known volume in a glass slide, put under microscope, count #, #/V = density

    • confluent growth: too many CFUs so uncountable

• streaking: flame a metal loop, let it cool down, spread bacteria on plate in a zig-zag pattern, reflame loop before streaking again or changing direction

why agar plate is not right for every species?

• different organisms have different growth conditions

• one plate can support the growth of only a fraction of the population

• the culture medium is selective/biased toward microbes that grow fast, can grow to high density, can grow in isolation, and can tolerate high concentrations of nutrients

• some organisms have adapted so well to their natural habitat that we still do not know how to grow them in the lab

  • Rickettsia prowazekii grows only within the cytoplasm of eukaryotic cells

  • 99.9% of bacterial species cannot be grown in the lab at all

symbiosis

• Nanoarchaeum equitans the world’s smallest living cell attach to the surface of another archaea Ignicoccus and has to be co-cultured with the host

  • has one of the smallest genomes of 552 genes

  • deficient in biosynthetic capacity: lacks many essential metabolic genes involved in biosynthesis of of macromolecules and lost many biomolecular pathways like glycolysis

• Micavibrio aeruginosavorus: “vampire bacterium” that latches onto and feeds off of other bacteria such as Pseudomonas aeruginosa

  • lost genes for making amino acids

binary fission

• microbial asexual reproduction during which one parent cell splits into two genetically identical daughter cells

• cell elongation → septum formation → formation of walls → cell separation

• generation time (a.k.a. doubling time) = how long it takes for a cell to double

• bacteria have a limited life span

  • observed by flowing nutrients across the tops of test tubes that each contain an anchored mother cell at the bottom and see how many generations form before the mother cell dies and the rest of the cells are washed away

  • an E. coli cell can divide for hundreds of generations before dying

• bacteria have one replication origin

what are the genes and proteins involved in bacterial cell division?

• Fts genes are essential for cell division in all bacteria and archaea

• Fts mutant: filamentous temperature sensitive mutants do not divide normally and form long filaments

• FtsZ: induces the formation of the divisome complex that combines with scaffolding proteins to form a ring complex responsible for septum formation, peptidoglycan synthesis, and inner/outer membrane assembly

  • homologous to tubulin in eukaryotic cells

  • only cell shape-determining protein in cocci

• PBP: penicillin binding proteins like transglycosylase and transpeptidase needed to make new cell wall for division

• MreB: major shape-determining factor found in bacillus, spirilla, and vibrio that determines sites of cell wall synthesis at the points where it touches the cell wall and causes bacteria to extend in length

  • homologous to actin in eukaryotic cells

• crescentin: shape-determining protein produced by vibrio-shaped cells of Caulobacter crescentus that organizes into 10 nm-wide filaments on the concave face of the curved cells

  • homologous to intermediate filament

peptidoglycan synthesis and cell division

• pre-existing peptidoglycan needs to be cut to allow newly synthesized peptidoglycan to form

  • small openings in the wall are created by autolysins and new cell wall materials is added across the openings

    • lysozymes break the sugar bond between NAG and NAM

    • endopeptidase breaks up peptide cross-links

• add peptides to NAM sugar that is attached to UDP (racemase converts L-Alanine to D-Alanine) → transfer molecule from UDP to bactoprenol (moves within membrane, hydrophobic tail + phosphate head) → add NAG to NAM → transporter flips completed precursor molecule to outside of membrane → transglycosylase joins NAG to NAM of another precursor molecule → transpeptidase forms peptide cross-links between the two molecules → pyrophosphatase removes a phosphate group from bactoprenol so it can be flipped back inside and reused

  • bacitracin inhibits pyrophosphatase so that bactoprenol is depleted and the cell wall cannot form

bacterial growth curve

1) lag phase- bacteria are preparing their cell machinery for growth

2) log phase- growth approximates an exponential curve (straight line on a logarithmic scale)

• doubling time is constant

3) stationary phase- cells stop growing and shut down their growth machinery while turning on stress responses to help retain viability

4) death phase- cells die with a “half-life” similar to that of radioactive decay (a negative exponential curve)

the math of growth

• Nt = No x 2^n

  • Nt = cell number at time t, No = original cell number, n = number of generations (t / generation time)

bacterial growth rate varies

• microbes have both the fastest and slowest growth rates of known organisms

• some hot spring bacteria double in 10 minutes, whereas deep sea sediment microbes may take as long as 100 years

• these differences are determined by intrinsic and external factors

  • external: nutrients, oxygen, temperature, water availability

  • internal: # of rRNA genes (more rRNA = more ribosomes = more proteins = more growth)

biofilms

• specialized, surface-attached communities: plaque on teeth, sinks, contact lenses, gut linings, medical implants, rocks in streams

• attachment to monolayer by flagella → microcolonies → exopolysaccharide (EPS) production → mature biofilm → dissolution and dispersal

• can consist of one or of multiple species

• increased antibiotic tolerance, possible due to dormancy (persistence) and difficulty antibiotics have penetrating the EPS capsule

• communicate via quorum sensing (bacteria can detect and respond to cell population density and form coordinated responses by regulating their genes)

cystic fibrosis (CF)

• genetic disorder: a mutation in a human ABC transporter that transports chloride and causes thick, sticky mucus to build up in the lungs that Pseudomonas aeruginosa will colonize and form biofilms in

  • Micavibrio aeruginosavorus “living antobiotics” that will attack and eat Pseudomonas aeruginosa

• life threatening as it is one of the most common chronic lung diseases in children and young adults

extremophiles

• microbes living in extreme environments

  • minority of bacteria

• growth rate is affected by the availability of nutrients and external factors

  • optimal growth condition classification: temperature, pH, osmolarity, oxygen, and pressure

oxygen

• aerobes: require oxygen

  • obligate = oxygen required, soil/dust

  • microaerophilic = low levels of oxygen required

• anaerobes: do not require oxygen

  • obligate = oxygen is harmful

  • aerotolerant = oxygen does not affect them

  • facultative = grow better with oxygen but do not require it

• oxygen-related growth zones in a test tube

  • thioglycolate added to medium to destroy oxygen → oxygen in air penetrates top of test tube over time → bacterial gradient forms (aerobes at top → anaerobes at bottom)

• why is oxygen toxic?

  • reactive oxygen species (ROS) damage DNA, RNA, proteins, and lipids

  • ROS form when oxygen accepts the final electron from FAD at the end of the ETC → generates ROS “radicals” → superoxide dismutase forms hydrogen peroxide (H2O2, can be converted back into radicals) → catalase and peroxidase convert H2O2 into water (no longer a ROS)

    • obligate anaerobes do not have catalase and peroxidase so ROS from O2 kills them

  • aging caused by decrease in function of the mitochondria since they produce ROS in humans

temperature

• higher temperature = higher growth rate until temperature passes optimum temperature (maximum enzymatic reaction rate) and proteins begin to denature, membranes collapse, and the cell undergoes thermal lysis

  • minimum temperature is when the cell membrane gels and the cell is dormant because transport processes are so slow (does not kill cells like going past max temp does)

• optimum growth temperature groups

  • mesophile = normal, middle, most common, 37 degrees Celsius

  • thermophile = hot, 50 degrees Celsius

  • extreme thermophile = extremely hot, 80 degrees Celsius

  • psychrophile = cold, (0-20 degrees Celsius, 10 degrees Celsius optimum)

pasteurization

the process of exposing bacteria to 72 degrees Celsius for 15 seconds to reduce the number of viable pathogens in food and drink so they are unlikely to cause diseases in humans

Pseudomonas syringae

• gram-negative, rod-shaped, aerobic plant pathogen (tomatoes, rice, tobacco)

• possess ice-nucleating proteins (INPs) that align H2O molecules to promote ice formation and contribute to frost damage to plants so they can infect them and take their nutrients

• promotes cloud, rain, and snow formation in the air and used for snowmaking at ski resorts since INPs require less energy

thermophiles

• get their energy from chemicals

• can live in 300 degrees Celsius vents

• how do they cope with high temperatures?

  • make more stable proteins and enzymes

    • more ionic interactions at protein surfaces

    • a few critical amino acid mutations at a few locations allow for more stable folding

    • chaperones help refold denatured proteins

  • make more stable cell membranes

    • lipids rich in saturated fatty acids (more tightly packed and thus stabler membrane)

  • more stable DNA

    • DNA binding proteins prevent damages to DNA

• example: Taq DNA polymerase isolated from Thermus aquaticus that lives in the hot springs in Yellowstone for PCR so the high temperatures used to denature and separate the DNA strands do not denature it

water availability/salt

• problem in high salt environments is losing too much water → plasmolysis

  • this is how food preservation works: add lots of sugar or salt so bacteria dehydrate and cannot grow

• halophiles: can live in high salt environments

  • make or accumulate compatible solutes that can bind to water and compete with extracellular salt for the water but do not interfere with normal metabolism

    • include small sugars, polar/charged amino acids, potassium

pH

• groups

  • neutralophiles: grow at pH 5-8, most pathogens

  • acidophiles: grow at pH 0-5, acid mine drainage

  • alkaliphiles: grow at pH 9-11, soda lakes (Na2CO3)

    • cyanobacterium Spirulina has a high concentration of carotene that makes flamingoes pink and a high concentration of phycocyanin used as a natural blue food coloring

• maintain relatively neutral inner pH

  • change cell wall and membrane compositions so they are stiffer and stronger

  • ion pumps to pump out protons if inside becomes too acidic

  • decarboxylase to remove acid groups and increase pH

endospore

• nature’s ultimate survival package

• components:

  • exosporium and spore coat = extra layers of protective proteins

  • core wall = tougher cell wall

  • small acid soluble proteins (SASP) = stabilize DNA

• almost zero metabolic activity, dormant stage, highly resistant to harsh conditions (nutrient depletion, heat, harsh chemicals, desiccation, and radiation)

• only present in some gram-positive bacteria: Bacillus and Clostridium

  • best studied in Bacillus subtilis (gram-positive model organism)

• over 200 genes involved

• sporulation: asymmetric division → mother cell engulfs smaller cell to give it an extra layer → mother cell loses all of its DNA → forespore develops a peptidoglycan cortex layer → mother cell releases spore

  • triggered by starvation or harsh environment, can reverse when conditions are good again

• Bacillus anthracis endospores sent to government and news outlets in envelopes in 2001 attack

  • spores not only are tough but are also tiny and light so can spread easily

  • when they are inhaled they can get into the lungs because of their small size → taken up by macrophages → multiply and produce exotoxins lethal and edema factor → lethal factor kills cells and edema factor shuts down the immune system