Tuesday Night Kreftacular Flashcards
What do microbes need to grow?
Elements ◦ Think of composition of Proteins & RNA/DNA C, O, H, N, S, P
◦ Add metal ions: K, (Na), (Ca), Mg, (Fe)
◦ Macronutrients: Simply the list of all the above elements
◦ Micronutrients: (Fe), many others
Growth factors (organic compounds) ◦ From none to more than we need ◦ Vitamins (coenzymes) ◦ Amino acids ◦ Purines, pyrimidines, etc.
Macronutrients
◦ In mass: C 48%, H 7%, O 36%, N 9%
◦ Rule of thumb you need to remember C 50% N 10%
Carbon
◦ CO2 as carbon source: autotrophic microbes (autotrophs) phototrophic or chemotrophic ◦ Organic compounds as carbon source: heterotrophs phototrophic or chemotrophic Organic carbon also used as energy source (as electron donor, oxidized with oxygen etc. as electron acceptor) 12
Oxygen
Oxygen in catabolism: electron acceptor in aerobic respiration
Oxygen sources for anabolism: H2O, O2 , organic compounds
◦ Oxygen in air saturated water only ~250 µM ◦ quickly gone, especially when a lot of organic matter is decomposed ◦ gut, sewage treatment, lake/ocean sediments, rice paddies, subsurface
◦ Aerobe only grows in the presence of oxygen and respires oxygen (Homo, Bacillus, Pseudomonas)
◦ Facultative aerobe aerobe that can grow without oxygen, using anaerobic respiration or fermentation (E. coli)
◦ Anaerobe does not grow in the presence of oxygen and does not use oxygen (Clostridium, Methanobacterium (methanogenic archaeon), some fungi and protozoa) 13
Nitrogen
◦ ~10% of biomass: amino acids, bases in DNA/RNA, etc.
◦ N-sources
NH3 (ammonia): all bacteria can use ammonia as N-source, and that is the form of N incorporated into amino groups Organic nitrogen compounds think amino acids
NO3 - (nitrate): needs to be reduced to NH3 before it can be incorporated into biomass, not all bacteria can do that
N2 (di)nitrogen: needs to be reduced to NH3 too, some bacteria and archaea (but never eukaryotes) can do that (Azotobacter and other nitrogen-fixing prokaryotes) ◦ ‘
Further macronutrients
Hydrogen, Phosphorus, Sulfur (SO4 2- and H2S)
Selenium
Metallic Macronutrients
always positively charged: cations
◦ K: Potassium, K+ K required for the activity of many enzymes. K is compatible at high concentrations in the cell, Na+ not, so cells pump Na+ out and K+ in
◦ (Na: Sodium, Na+) Not all organisms need it Many contain some Na but do not use it, less compatible than K Some use it instead of H+ (sodium motive force)
◦ (Ca: Calcium, Ca2+) Stabilizes cell walls, large amount in endospores
◦ Mg: Magnesium, Mg2+ ATP really is MgATP Mg required for stability of nucleic acids/ribosomes, membranes Ani
Other transition metals: Mn, Co, Ni, Cu, Zn, Mo, W ◦ Like vitamins in active sites of enzymes ◦ Only traces required ◦ Often enough in ‘distilled’ water and other media components
Iron
Most need Fe to make cytochromes and FeS proteins for electron transport chains But not all: Lactic acid bacteria (fermentation of sugar does not need respiratory chain), e.g. Lactobacillus acidophilus Solubility low therefore difficult to get
◦ Fe2+ is relatively soluble, but oxidizes (rusts) in presence of oxygen
◦ Fe3+ (rust) very low solubility at neutral pH solubility product of Fe(OH)3 (i.e. fresh rust) = 10-38 ◦ Between pH 0 - 3, Fe3+ is soluble
◦ Many bacteria produce siderophores to scavenge Fe from the environment
◦ These have very high affinity for Fe Usually chelated by catechol or hydroxamate groups
Media for growth
Defined media ◦ you know exactly what molecules are in the medium and how much ◦ essential for physiological studies ◦ can be a long list of components ◦ can contain more than necessary
Minimal media ◦ a defined medium that only contains what’s essential ◦ Lactic acid bacteria need ~40 growth factors
Complex media ◦ usually digested animal or plant matter ◦ beef extract = boiled cow ◦ contain lots of goodies but who knows what
Cell Growth
Definition of Growth: forming 2 cells from 1 ◦ Increase both in Mass and Number ◦ Absolutely essential for survival ◦ Looks simple but is complex ◦ Applications of growth: stop growth of microbes, or exploit it E. coli growth facts ◦ 20 min generation time of cell under optimal conditions ◦ 40 min DNA replication ◦ 20 min from end of replication to cell division They have to start replication 60 min before division, every 20 min, so that several rounds of replication are running simultaneously Bacteria mostly have one chromosome with one origin of replication ◦ Generation time in colon: 12 hours or more!
Growth Equation
Number of cells after n generations (completely generally)
N = N0 · 2^n
Legend: N: Number of cells N0 : Number of cells at the beginning (t0 ) n: number of generations generation time = time / number of generations g = t/n t: time (since t0 )
Specific Growth Rate
Specific Growth Rate = Growth Rate/Biomass
Balanced growth
In balanced growth, all components of the cell increase in proportion
◦ biomass, protein, number of cells, all increase at the same rate ◦ so the composition of the cell does not change
◦ balanced growth characteristic for exponential growth phases Cell number based measures/concepts ◦ Generation time g ◦ Division rate ν = 1/g Biomass based measures/concepts ◦ Doubling time td ◦ Growth rate µ = ln(2)/td
Simple relationship between growth of number and biomass holds only in balanced growth: ◦ generation time g = doubling time td
Measurement of growth
Biomass ◦ Protein assay (50% of biomass is protein, does not change much) ◦ Dry mass (dry washed cells and weigh them) ◦ Optical Density (OD) Particles scatter light (know any examples?) Bacteria are particles OD is a measure of biomass, rather than cell numbers Measure of cell number only so far as numbers correlate with mass (balanced growth) NB: Brock misleading in some places, correct in others
Cell number Total count Count cells in known volume under the microscope (counting chamber) Cells/mL Viable count – CFU (Colony Forming Units) Spread 0.1 mL of (diluted) culture on agar plate and count # of colonies forming CFU/mL
Optical Density (OD): most convenient measure of growth
If OD too high (>0.5), have to dilute sample as response no longer linear OD proportional to biomass per volume
OD not proportional to cell number per volume as Brock figure implies
Cell number correlates with biomass only if the cell size is constant, i.e. in balanced growth, but not in stationary phase
Growth phases in batch culture
Lag Exponential Stationary Death
All trophs together now
SLP – Substrate Level Phosphorylation
Example from Glycolysis
Oxidation of Aldehyde to Acid coupled to phosphorylation of acid
Transfer of Phospho group to ADP Exactly 1 ATP formed Stoichiometric coupling
If not enough ∆G in a reaction, then no ATP can be made, if too much ∆G, surplus is ‘wasted’ No membrane needed No Fe needed
ETP – Electron Transport Phosphorylation
“Brock Fig. 3.20” (13th ed = 14th ed) Paracoccus denitrificans aerobic respiration Some errors/oversights: Succ/Fum +0.033 V (FAD/FADH2 -0.220 V) 4 H+ have to enter Complex I 2 H+ released upon oxidation of NADH + H+ by Complex I Must have lost 10 H+ from inside if 10 H+ appear outside
