MIC102 Midterm 1

microscopes

created by Antony Van Leeuwenhoek, magnification & resolution (shortest distance between 2 objects that can still be distinguished as 2 separate objects

light microscopy

can achieve 2000x and 1000x mag minimum

limited ability to visualize bacterial components or distinguish between bacteria without doing something extra

fluorescent microscopy

highlights different specific proteins and processes

stains and light microscopy

uses existing stains in tile making to differentiate between different bacteria 

gram positive

bacterial cell envelope that has a porous, rigid, lattice peptidoglycan that allows for diffusion of CO2 and glycerol

also contains lipoteichoic acid that touches plasma membrane

gram negative

bacterial cell envelope that has thin layer of peptidoglycan but 2 membranes

has porin to allow molecules in

transports materials twice in exchange for tougher plasma membrane

diffusion

also osmosis, movement of water from high to low water concentration

solutes & cells

hypertonic solution —> hypotonic cell —> plasmolysis

isotonic solution —> isotonic cell —> optimal growth

hypotonic solution —> hypertonic cell —> high osmotic pressure & explodes

peptidoglycan

permeable, rigid but flexible structure, peptide cross linked formed through transpeptide activity (linkages between layers

gram stain procedure

dye cells, stain with iodine, wash with ethanol, counterstain everything else

gram positive will stay purple while gram negatives will be dyed with counterstain

lipopolysaccharide structure

extra membrane support/function

o-polysaccharides

• recognized by immune system & varies between species

• used to ID by “O-antigen” type

• excludes hydrophobic compound

endotoxin

released when cell dies, historical name associated with septic shock

porin

protein used for transport across outer membrane

increases permeability of outer membrane to hydrophilic compounds like sugars, amino acids, and some ions

S-layers in bacteria and archaea

an optional structure for extra protection that occurs more in archaea

crystalline surface layer resulting from a lattice of a single repeated protein unit

provides more things based on protein properties

capsules & slime layers

an optional structure in bacteria and archaea that is expressed sometimes, most are made of polysaccharides and some with amino acid polymers

prevents dessication, limit diffusion of harmful compounds, helps avoid phagocytosis, looser in organization

glycocalyx

allows cells to adhere to surfaces and each other and creates biofilms

bacteria sticking to teeth, plaque

flagella

comprised of basal body anchored in the membrane, the hook that moves outward, and filament that helps it move, chemotaxis

basal body —> hook —> filament

filaments are extremely rigid & can be antigenic

chemotaxis

moving by physically sampling environment

axial filaments

flagella contained to periplasm spirochetes, moves in corkscrew manner

can wrap around cell wall and make it spiral shaped

bacterial pilius

structure used to move around, conjugation, and attachment (fimbria)

thinner and finer than flagella, resembles hairs

assembled from pilin monomer into a helix

nucleoid 

DNA is loosely organized into circles in bacteria, no membrane

can be detected through high density of electrons

organized into a “bottle brush” with layers of a H-NS protein core with loops around 

H-NS protein

protein that helps bind to DNA in bacteria to help form the bottle brush structure in the nucleoid

has chains with linkers, DNA and H-NS protein binding capabilities

topoisomerase

enzyme that accesses DNA in loops, transcription induces both positive and negative supercoils

relives or creates supercoiling

slight negative supercoiling promotes loop formation and makes it easier to open DNA duplex

topoisomerase 1

ATP independent, relaxes negative super coils into circular DNA 

gyrase

a type of topoisomerase that is ATP dependent, can relax positive supercoils and induce negative supercoils

specialized compartment functions

storage, limit diffusion, concentrate proteins and substrates, perform specialized functions

gas vesicles

allows gases to diffuse back and forth, common among aquatic photosynthetic bacteria like cyanobacteria

can tune buoyancy for optimum light gathering

made of protein shell permeable to gas but impermeable to water

• gas diffuses to reach equilibrium, it is not pumped in or stored higher concentrations

carboxysomes

used in bacteria that fix CO2, protein shells

enhances the function of RuBisCo by increasing concentration of it in that area

similar to enterosomes

enterosomes

found in heterotrophic bacteria that allows to metabolize other compounds with harmful intermediates, similar to carboxysomes

fructose —> propanediol —> propanaldehyde

storage granules

storage of useful materials when in abundance for later use when lacking

sulfur, calcium, phosphate, organic polymers (polyhydroxyalkanoates)

magnetosomes

membrane-bound, iron containing structures 

several crystals chained together act as a compass needle

Fe₃O₄ or Fe₃S₄

thylakoids

enhances light-gathering abilities of photosynthetic bacteria by greatly increasing membrane SA (more proteins and photosynthesis)

stacks of membrane sac with a shared lumen, similar structures made by chemolithotrophs

binary fission

makes 2 of everything and divide evenly, main method of growth in bacteria

minimal medium

growth media only contains the absolutely necessary components necessary for growth, uses purified substances and not the faster

defined medium

growth media where every component and its quantity is known, grows faster

undefined medium

growth media where some components/quantities are not known exactly (yeast extract)

rich medium

growth media where it contains components allowing for high growth rates that are not strictly necessary for basic growth

lag phase

initial phase of growth where cells are adapting to the new environment

exponential phase 

second phase of growth where cells grow and divide exponentially at a consistent rate

consistent repeatable results for replication, cell division, metabolism

used to maximize protein/DNA synthesis in lab

stationary phase

third phase of growth where cells are not actively growing

nutrients are used up, toxic metabolites build up, cells undergo changes to survive

• growth becomes more balanced and “flat”

• competition can arise, bringing upon subpopulations that compete with each other

death phase

last phase of growth where cell population lowers exponentially

optical density 

used when you want to measure fast cell growth, describes how much a material reduces the power of light passing through it through absorption or scattering

hemocytometer

used when you want a slow method for measuring cell growth (see who is producing colonies)

flow cytometry

used when you want to find the population or ratio of population of cell growth, measures multiple parameters at the same time

not used to measure concentration

serial dilution

used to count viable cells that can be used for growth

calculation for bacterial growth

\frac{\log_{10}N-\log_{10}No}{0.301}=n , where n = # of generations

g=\frac{t}{n}, where g= generation time and t = time elapsed

temperature effects on growth 

high temp = proteins denature

lowe temp = weakened hydrophobic interactions, alters protein conformation/assembly

decimal reduction time

time a treatment takes to reduce population by one log

cell division

even segregation of cellular components

FtsZ 

a protein where its polymerization is essential for cell division

goes from monomers —> chains —> bundles —> rings 

helps determine the position of where to divide

Min proteins

C & D polymerizes and concentrates at the poles of cells and prevents FstZ monomers from turning into polymers

E keeps them there

growth metabolism

life generating processes needed to grow

maintenance metabolism 

life sustaining processes needed to function

fueling —> precursor metabolites (metabolism)

precursor metabolites made from initial carbon and energy sources (ATP, proton motive force)

• some precursors may be available in environment

energy is generated (redox reactions)

reducing power generated (NADH & NADPH)

precursor metabolites —> building blocks (metabolism)

building blocks become more reduced than precursor metabolites

most microbes can synthesize all building blocks with 13 precursor metabolites, energy, and reducing power 

building blocks —> macromolecules (metabolism)

polymerization of building blocks (except for lipids)

specific macromolecules vary by species 

high energy cost like protein synthesis, DNA replication/repair

macromolecules —> cell structure (metabolism)

macromolecules turn into the structure of the cell

some structures require enzyme-catalyzed reactions for assembly

translocation from manufacture site to final location

microbial metabolism

enzyme catalysis

• speeds an unfavorable/slow process & reduces energy needed for it

• important in breakage (hydrolysis) and synthesis of chemical bonds

redox

• respiration, harvesting energy

membranes and ion gradients

• transducing energy into different forms

reaction coupling

• couple unfavorable and favorable reactions together

chemical energy transfer

stored in chemical bonds, coupled carbon and energy metabolism, drives unfavorable reactions

light energy

harvested to split H2O, uncoupled wtih carbon metabolism, drives unfavorable reactions

NADH

reduced molecule gained from fueling, used to transfer electrons in reactions that ultimately makes ATP

can use transhydrogenases to convert to NADPH

NADPH

reduced molecule needed for biosynthesis (add a phosphate to ribose)

can use transhydrogenases to convert to NADH

fermentation

when respiration cannot occur, recycles NADH —> NAD+, ATP payoff is lower than glycolysis

• no terminal electron

• still has carbon source

• produces acids, gases, or alcohols

photosynthesis

instead of getting electrons from a reduced compound, use energy from light to excite electrons

chlorophyll has pigments that collect light and uses light for photosystem (reaction center) and makes ATP with H2O as terminal electron accepter

large diversity of bacteriochlorophylls; archaea not so much

• most photosynthetic bacteria are autotrophs —> much greater need for reducing power

building blocks

makes up macromolecule polymers

• nucleotides —> nucleic acids

• amino acids —> proteins 

if a microbe can’t make one, they must get it from environment 

trade-off between making your own or getting from environment 

• getting from environment is easier but more dependent on it

can all prokaryotes synthesize all essential building blocks

no

e.coli: microbial jack of all trades; synthesizes all essential building blocks

s. agalactiae: specializes in some building blocks

both lives in the guts

precursor metabolites

needed to make the building blocks

entry into cell —> feeder pathways —> central pathways —> 13 precursor metabolites

ion-coupled transport (entry-active transport)

transport is coupled to another ionic gradient (proton, sodium)

energy used to set up this gradient

transport can go either direction

ABC (ATP binding cassette) (entry-active transport)

binding proteins bring solute to membrane protein complex

ATP hydrolysis used to open channel and bring in solute

very common in bacteria

entry-group translocation

phosphotransferase system (PTS) transports a solute while phosphorylating it (usually a sugar)

Pi often comes from phosphoenolpyruvate (PEP)

phosphorylating a sugar traps it inside a cell

energy cost savings since first step in sugar metabolism is going to be phosphorylation anyway

siderophores

chelates ions in the environment

ion-siderophore complex recognized and actively transported inside

• multi-protein complex

• can be found in both gram negative and positive

feeder pathways

heterotrophs 

• convert organic compounds into intermediate molecules 

• functionally very complicated 

• potentially need to be able to use dozens of different compounds 

• polymer breakdown and isomerization 

autotrophs 

• carbon fixation 

calvin cycle

most autotrophs use RuBisCo to fix carbon

• adds CO2 to a 5-carbon sugar-phosphate

addition of ATP and NADPH fuels creation of fructose-6P and regeneration of ribulose-1,5 biP

6 CO2 + 12 NADPH + 18 ATP —> 1 sugar + 12 NAD + 12 ADP + 17 Pi

energetically expensive and needs a lot of reducing power

entner-doudoroff

an auxiliary pathway that is an alternative link between pentose phosphate pathway and glycolysis

auxiliary pathways

alternative pathways that help microbes perform metabolic functions, provides additional links to the central pathways (skip steps) to make more efficient/flexible reactions

evolved due to poor nutrients and carbon sources (harsh environment)

entner-doudoroff, glyoxolate shunt, fermentation

glyoxalate shunt

an auxiliary pathway that skips over TCA cycle steps that would release CO2 —> saves carbon when growing on acetate directly

flexibility in pathways

some pathways can make too much unnecessary energy —> recycles resources or run reactions in the opposite direction 

best yield & energy efficient pathways 

nitrogen metabolism

denitrification (anaerobic respiration)

• NO3- —> N2

nitrogen fixation

nitrogen fixation

N2 —> 2NH3, adds hydrogens to break triple bond

extremely energy intensive 

16 to 24 ATP to make 2 molecules of NH3

microbes are the only thing that can do this (mainly bacteria and some archaea)

essential to nitrogen cycle (plants give food to microbes, microbes gives nitrogen)

nitrogen assimilation

nitrogen can be assimilated onto organic compounds as ammonia/ammonium (NH3/NH4+) into glutamate & glutamine (1 more NH2 than glutamate) synthesis

NO3 is common nitrogen source that can be easily absorbed

can only assimilate as ammonia

needs reducing power & ATP

glutamate/glutamine functions

glutamate is key to making other amino acids by transferring its amino group

glutamine is used for other building blocks like nucleotides by transferring the amino group

sulfur assimilation

occurs in plants, fungi, and many bacteria

sulfate-reducing bacteria can use multiple forms, converting to S2-

SO₄^2- —> S^2- —> cysteine

amino acids

building blocks of proteins

different families of amino acids come from different precursors

can predict source of some mutations in central mutations in central pathways by which families of amino acids can’t be made and must be supplied 

regulation of biosynthetic pathways

end product often regulates its own production with allosteric regulation (enzyme activity is regulated by binding to a site other than the active site)

binds to the enzyme that makes the first intermediate (doesn’t allow precursor —> intermediate a by binding to enzyme 1)

nucleotides

building blocks for nucleic acids (DNA, RNA)

made in the activated state, ready for polymerization already

purines (A, G) made from ribose-5-P (21 enzymes)

pyrimidine (T, C, U) made from ribose-5-P and oxaloacetate (24 enzymes)

uses lots of energy, reducing power, and nitrogen

sugars

used in capsules, cell walls, other membranes

also can contribute to energy/ carbon storage (glycogen)

most of these specialized sugars must be manufacture from precursors

fatty acids & lipids

a major requirement for phospholipid membranes and LPS (lipid A)

glycerol for linking fatty acids

fatty acids constructed by adding 2C units to chains

• # of double bonds

• chain length

• branching

• most fluid: short, branched, with more double bonds