Professional Documents
Culture Documents
- Counting bacteria
o Direct microscopic count
Use counting chamber with rids
Count number of bacteria per volume
Problems
Can’t tell between live or dead bacteria
Lack of bacterial motility
o Viable count
Count number of cells capable of forming a colony on solid medium
Problems
Takes time for culture to grow
Can only count living cells
If counting species on environmental sample, medium may be selective
for some species
- Serial dilutions
o Help separate bacteria if they are too densely populated in original sample
o 10-fold serial dilutions before plating
Multiple plate count by dilution factor to obtain original number of bacteria
Ex. if I count bacteria on plate from 10^-6 dilution, I would multiply the
number of CFUs I counted by 10^6
- Turbidity
o OD proportional to number of cells present
o Indirect measurement of bacteria population
o Less light transmitted (more cloudy), the more bacteria there are
o Very fast method
o Data must be graphed
o Problems
Can’t tell if all same bacterial species
Doesn’t distinguish between alive and dead cells
- Culture media
o Rich/complex-not chemically defined, contain complex organic molecules
o Defined/minimal media-chemically defined, contains precise amounts of chemicals
May have organic molecules
Usually has H2O, C, N, P,etc
Total cell yield in culture is determined by limiting nutrient
- Bacterial growth
o Bacteria can go through exponential growth if enough resources are present
o Bacteria double each generation time
o Equation:
N=N0*2n, N=final cell #, N0=initial cell #, n=# of generations
n=t/g, g=generation (doubling) time, t=time elapsed during exponential
growth
o Growth phases
Stationary phase
Environment conditions limit cell growth
o Ex. lack of nutrient, accumulation of toxic waste product
No net increase/decrease in cell number
Energy metabolism can still occur, some biosynthetic processes can
continue
Lag phase
Cells are not in most rapid exponential growth rate
Happens when:
o Stationary phase culture is diluted into fresh medium
o Cells are moved from rich to minimal medium
o Culture is moved to different conditions of temperature or
stress
Bacteria must readjust to new environment and make proteins needed
to survive there
- C and N
o Most animals use NH4+ or NO3- as N source
o Oxygen
Oxic zone- area of tube containing O2
Obligate aerobes-need O2 as terminal electron acceptor
Microaerophiles-live in places with low O2
Aerotolerant anaerobes-neither use O2 nor are harmed by it
Facultative anaerobes-can use O2 or other strategies when no O2 is present
Grow better in O2
Obligate anaerobes-must live in places with no O2
When O2 is present, ROS can be accidentally made by electron-carrying
molecules
ROS can damage cells, so enzymes are made that remove ROS
- Optimal growth temperature
o Minimum temperature-membrane gels, slow transport so growth cant happen
o Optimum-enzymatic rxns run at max rate
o Maximum-enzymes denature, membrane collapses, thermal lysis
o Bacteria do not regulate their internal temperature
Extreme bacteria
Thermophile- >45C
Hyperthermophile- >80C
Psychrophile- <15C
- Adaptations to heat
o Problem-enzymes denature in heat
Solutions
Less glycine residues in protein
Increased ionic bonding between acid/base AAs and their hydrophobic
cores
Chaperones to help refold proteins
Solutes to stabilize proteins
o Problem-membranes are too fluid
Solutions
Increase amount of saturated fatty acid chains in membrane
- Adaptations to cold
o Problem-Proteins have less thermal motion, so they work slower
Solution
proteins are more flexible, so they can move around more even when
the temperature drops
o Problem- membrane fluidity decreases
Solutions
increase amount of unsaturated fatty acids in membrane
increase amount of glycine residues in AAs
o Problem-Ice crystals form and damage cell walls
Solution
Cryoprotectants to help prevent ice crystal formation
- Adaptations to osmolarity
o Hypotonic (hypoosmotic) medium
Cells usually swell
Solution
Have rigid cell wall that can withstand pressure increases
Mechanosensitive channels that can leak solutes when pressure inside
cell increases
- Hypertonic (hyperosmotic) medium
o Cells shrink
Solution
Import or make compatible solutions that increase internal osmolarity
o Ex. sugars
- Central dogma
o Bacterial chromosomes found in cytoplasm
o Transcription and translation can happen simultaneously
o Bacterial genes encoded in operons
Proteins transcribed on same mRNA strand
Translated separately due to having different RBSs
- Control
o Cells control gene expression and enzyme activity
- Transcriptional regulation
o RNA polymerase-transcribes DNA into RNA
Contains sigma factor
Binds to specific promoter sequence on gene
o Promoter
Region of DNA where RNA pol binds to
Better match of promoter sequence with consensus sequence for a sigma
factor makes stronger promoter
Poor match yields weaker promoter
o Extra proteins required to help RNA pol bind to promoter
Cell regulates global gene expression patterns by controlling which sigma
factors are present and active
Sigma factor must be degraded after use
o Accessory transcription factors
Activate or repress specific genes
Activator-turns on transcription
ex. CRP for lac operon
Repressor-turns off transcription
Bind to operator sequence of operon, block downstream gene
transcription
Corepressor-helps repressor block transcription
o Bind to repressor
o Sometimes required to block transcription
o Can be end products of biosynthetic pathways
Ex. tryptophan is corepressor of trp operon
Inducer-activate transcription by inactivating repressor
Bind to repressor, inactivating it
Ex. lactose for lac operon
o Catabolite repression
Allows bacteria to use sugars in sequence
Glucose always used first
Diauxic growth curve-lag in growth when glucose is gone
Cells must make new enzymes to metabolize secondary sugar
CRP-regulates catabolite repression
Activator
o Required for transcription of lac, mal, ara operons
Coactivator is cAMP
o cAMP binds to CRP, activating it
o When glucose is high, cAMP is low, and vice versa
- Metabolism
o Two branches-energy conservation (catabolism) and energy consumption (anabolism)
- Metabolic options for energy conservation
o Chemicals-chemotrophy
Chemoorganotrophs-use organic compounds (contain C) for energy
Chemolithotrophs-use inorganic compounds for energy
o Light-phototrophy
Phototrophs-use light for energy
- Redox rxns
o Electron tower
Reduction potential EI0-tendency of oxidized substance to accept electrons
top of tower-lowest EI0, greatest tendency to donate electrons
many compounds can either be electron donors or acceptors, depending on
what other electron carriers are present
o oxidation half rxn
electron donor gives up electrons and becomes oxidized
o reduction half rxn
electron acceptor takes electrons and becomes reduced
o construction of redox rxn: just practice
o Differences in EI0 are expressed as Δ EI0= (EI0 of reduction couple)-( EI0 of oxidation
couple)
If EI0>0, rxn is favorable in direction written
- Relationship between Δ EI0 and ΔGI0
o ΔGI0=-nF Δ EI0
n=total number of electrons, F= 96.5 kJ/vmole-
units are kJ/mole of X oxidized
- How do bacteria store energy they get from redox rxns?
o Proton gradient across cytoplasmic membrane
o High energy compounds that power unfavorable rxns
Ex. ATP, GTP, PEP
o In catabolism, electrons are extracted and transferred to electron carriers
Ex. NAD+, FAD, NADP+
Reduced carriers bring electrons to electron transport chain
- Chemoorgantrophs
o Use organic compound as both electron donor and carbon source
o Aerobic chemoorganotroph- use O2 as electron acceptor
o Some substrates are fully oxidized to CO2 and electrons are used to drive PMF and make
ATP
CO2 excreted as waste product
o Cs in other substrates used to build cellular molecules/structures
- Chemolithotrophs
o Use inorganic compounds to get electrons
o Can use O2 or other molecules as electron acceptors
o Often autotrophs, obtain C for cellular molecules from CO2
Carbon fixation
- Phototrophs
o Get energy from light
o Get electrons from H2O or other compounds
o Get C from CO2 (photoautotrophy) or other organic compounds (photoheterotrophy)
- Fermentation
o Happens when there is no electron acceptor available for respiration
o No ETC
o ATP made via substrate-level phosphorylation
o Energetically inefficient
Respiration preferred if given a choice
o Starting compound is only partially oxidized
Fermentation products- Compounds still containing energy that are excreted
by cell as waste
o NAD+ must be regenerated from NADH
Occurs when oxidized product is reduced to fermentation product
NAD+ is needed for other metabolic rxns in the cell
o Most carbon from substrate is excreted as partially reduced waste product
Only small amount of carbon used in biosynthesis
Cell must use all carbon substrate for energy production
- Glycolysis
o Glucose oxidized to pyruvate
o Pyruvate can be reduced to make fermentation products or fed into the TCA cycle
If respiration is possible, go to TCA cycle
if not, make fermentation products and regenerate NAD+
o Net 2 ATP and 2 NADH made at end of glycolysis
- The Pasteur Effect
o Low net yield of ATP per glucose when fermentation occurs
2 mol ATP/1 mol glucose
o Cell takes in a lot of glucose when going through fermentation since it’s so energetically
inefficient
o When O2 is added and respiration can occur, cell decreases glucose intake and alcohol
production ceases
- Respiration
o Pyruvate completely oxidized to CO2
o Electrons on NADH and FADH2 are moved to an ETC
o TCA cycle also generates intermediates of many other biosynthetic pathways
o Generates much more energy per substrate than fermentation due to complete
oxidation of glucose
- Electron Transport Chain
o Series of electron donor and acceptor molecules that ends in a terminal electron
acceptor
Each electron carrier has reduction potential
o Span cytoplasmic membrane
o Electrons move from lower to higher EI0 as they travel through the complexes
Energy released as electrons are moved
o Energy released is converted into a proton gradient
o Electron carriers:
NAD+/NADH
Carries 2 H+ and 2 electrons
o Involved in other pathways too
FMN
Intermediate carrier in ETC
Carries 2 H+, 2 electrons
Cytochromes
Contain heme cofactors
o Fe in heme group cycles between 2+ and 3+ states as electron is
accepted and donated
Only carry electrons
FeS clusters
Covalently bonded to ETC by cysteine residues
o Fe cycles between 2+ and 3+ states as electron is accepted and
donated
Only carry electrons
Quinone molecules
Diffuse in the plane of the cytoplasmic membrane
Interact with different donor and acceptor complexes
Carry 2 H+ and 2 electrons
When accepting electron from an upstream donor, also takes a H+ from
cytoplasm
o When electron is passed to a downstream acceptor, the H+ is
released into the periplasm, helping to generate PMF
Lecture 8-Chemolithotrophy
- Chemolithotrophs
o Metabolize inorganic compounds
o Have alternative ways of obtaining carbon
Autotrophs-obtain C from CO2
Mixotrophs-carbon source is organic compound, but not used to obtain
electrons
o ATP synthesis is by oxidative phosphorylation, powered by PMF
o NADH must be generated, regardless of the electron donor being used
If reduction potential of the electron donor couple is greater than NAD+/NADH,
reverse electron transport must occur
PMF provides energy for this process
Electrons are pushed uphill onto NAD+
- Hydrogen as an inorganic electron donor
o Some H2 acceptors are aerobes, others are anaerobes
Aerobic H2 acceptors can obtain carbon from CO2 via carbon fixation
Some aerobic H2 oxidizers can also grow as chemoorganotrophs
Would rather use sugars instead of H2
o Sugars repress genes for H2 oxidation and carbon fixation
o Electron flow in aerobic H2 oxidation
Membrane-bound hydrogenase gives electrons to quinone pool
Quinone passes electrons to cytochrome complexes, which give electrons to O2
PMF is generated, contributing to ATP synthesis
Cytoplasmic hydrogenase-uses H2 to reduce NAD+ into NADH
NADH made this way can be used in reduction biosynthetic rxns
- Sulfur as an inorganic electron donor
o Sergei Winogradsky
Observed bacteria that grew in lots of H2S had granules
If deprived of H2S, the granules would disappear, but the bacteria would still
grow for a period of time
These bacteria oxidize H2S to S0
Energy extracted is used for growth
S0 stored in granules
o S0 can be used as an electron source once H2S is depleted
- Iron-oxidizing bacteria (FeOB)
o Fe2+/Fe3+ has different EI0 at different pHs
At pH 7, iron oxidation can be coupled with both O2 and NO3- reduction
Competing rxn: Fe2+ can be abiotically oxidized by O2 to form Fe3+
o FeOB at pH 7 must live in anoxic or microoxic environments
o Metabolism has to compete with abiotic oxidation of Fe2+
At pH2, can be coupled with only O2 reduction
Energetically inefficient
Large amounts of waste products generated
1 electron per Fe atom
o Electron flow at low pH
Fe2+ oxidation is performed by a cytochrome c on the outer membrane
Fe remains extracellular
Rusticyanin moves electrons from cytochrome c into complexes in the ETC
Reverse electron flow occurs-PMF moves electrons to NADH oxidoreductase
that reduces NAD+ to NADH
o Neutral Fe oxidation
Since Fe oxidation only yields 1 electron per atom, lots of abiotic Fe oxidation
occurs
Problem: Oxidized iron forms insoluble crystals
Mariprofundus ferroxydans
o Produces a polysaccharide stalk that extends from its cell body
o Fe3+ minerals precipitate out onto stalk, removing any insoluble
Fe from the cell body
Lecture 8-Phototrophy
- Phototrophs
o Obtain energy from light, but must still obtain electrons from chemical sources
Electrons used to generate PMF or make FADH2, NADH for redox rxns
- Photosynthesis
o O2 is electron acceptor
o Oxygenic photosynthesis- H2O is electron donor
O2 produced as waste product
o Anoxygenic photosynthesis-H2S or another reduced chemical is electron donor
O2 not produced as waste product
o Light and dark rxns
Light rxn-light energy stored as chemical energy and reducing power
phototrophy
Dark rxn-chemical energy and reducing power are used to reduce CO2 into
organic compounds
Carbon fixation
- Phototrophy
o Light harvested with photosynthetic reaction centers (RC)
Chlorophyll pigments are bound to proteins or associated with photosynthetic
membranes via a phytol group
ex. chlorophyll a absorbs red and blue light and reflects green
ex. bacteriochlorophylls absorb light of all different wavelengths
Type 1-Photosystem I (PSI)
First stable electron acceptor is a Fe/S cluster
Fe/S type RC
Type 2- Photosystem II (PSII)
First stable electron acceptor is a quinone molecule
Q-type RC
o PSI or PSII can be used individually to generate PMF and reducing power
o Oxygenic photosynthesis uses both PSI and PSII in the same membrane
- Principles of phototrophic transport
o Light excites chlorophyll, allowing it to donate electrons to an electron acceptor to due
decrease in reduction potential
o Electrons move down as reduction potential increases
o PMF is generated
o Reverse electron flow occurs to make NADH if first stable electron acceptor has higher
reduction potential than NAD+
- Electron flow in anoxygenic photosynthesis
o After being excited by light, chlorophyll P870 donates electron to the next acceptor
o Cyclic electron flow-electron is passed through acceptors, until it returns to the original
chlorophyll
P870Quinone moleculescytochromesP870
During transfer of electrons from quinone to cytochrome bc1, PMF is generated
When electrons are removed from the pathway to generate NADH (reverse
electron flow), external electrons must be provided to reduce P870
Come from electron donors like H2S
o PSI (Fe/S type RC) can also be used, but unknown if cyclic electron flow is utilized
- Electron flow in oxygenic photosynthesis
o Z-scheme
Uses both PSI and PSII
Part 1
LightP680PhQAQBPQ poolcyt b6fplastocyaninP700
When PQ accepts electrons, it also picks up protons from the
dissociation of water
o When it donates electrons to cyt b6f, protons are pumped into
periplasm to generate PMF
Part 2
LightP700chl aFe/S clusterFdFNRPQ pool (cyclic electron
flow)
o Fd can keep electrons for use in other reactions
o In linear electron flow, FNR reduces NADP+ to NADPH for use in
other rxns
o In cyclic electron flow, FNR donates electrons to the PQ pool to
generate more PMF
No reducing power is made though
- Internal membrane systems
o Used to accommodate all of the photosynthetic proteins, since they span such a huge
area
- Light-harvesting pigments
o Chlorosomes
Contain tightly packed crystalline array of bacteriochlorophyll (Bch)
Excitation energy transferred from Bch through intermediate proteins
to rxn centers in the cytoplasmic membrane
o Phycobilisomes (antenna complexes)
In cyanobacteria
Absorb light at different wavelengths
Bacteria can use different phycobilisomes in different environments as a
result
Absorb light energy and transfer it to rxn center
- Dark reaction
o Converts CO2 into carbohydrates
o ATP and reducing power generated by phototrophy reduces CO2
o Calvin cycle or Reverse TCA cycle are used
- Calvin cycle
o Uses RuBisCo and PRK enzymes
o Requires NADPH and ATP
o RuBisCo
Catalyzes conversion of Ribulose bisphosphate to PGA
Carboxylation
Competing rxn: Ribulose bisphosphate+O21 PGA + 1 phosphoglycolate (PG)
PG is toxic to cells
Mechanisms required to favor carboxylation
o PRK
Catalyzes conversion of ribulose phosphate to ribulose bisphosphate
- Concentrating carbon
o Problem
CO2 is natural substrate of Rubisco, but HCO3- is more prevalent
Can’t accumulate CO2 inside lipid-bound compartment
o Solution
Carboxysomes
Cellular compartment that helps concentrate CO2 inside cell
Protein shell-controls what goes in and out of compartment
Inside contains high amount of Rubisco and carbonic anhydrase
Physical, functional and genetic organizations
o Transcribed on operon
o CO2 can’t diffuse out, O2 can’t diffuse in, favoring carboxylation
rxn
Inducible high-affinity transporters maximize uptake of CO2 and HCO3-
o CO2 is reduced to HCO3- after import
o HCO3- cannot pass through cytoplasmic membrane
HCO3- influx into carboxysome powered by Na+ influx
or ATP
HCO3- + H+CO2+ H2O
- Reverse TCA Cycle
o found in green sulfur bacteria and archaea
o ATP and reduced Fd from light rxn are used to drive TCA cycle in reverse
o ATP citrate lyase is diagnostic for this pathway