Exam 2 Flashcards
Prototroph
able to synthesize all organic compounds required for growth
Auxotroph
unable to synthesize all organic compounds, must have some provided
Chemotrophs
obtain energy from chemical compounds
Phototrophs
obtain energy from light
Lithotrophs
use inorganic electron donors (e.g., H2S, H2, Fe2+)
Organotrophs
use organic electron donors (e.g., sugars,
amino acids, fatty acids).
Heterotrophs
use organic carbon sources
Autotrophs
use inorganic CO2 which they fix into organic form
What must general growth media provide?
- CHNOPS
- Energy source
- H2O
- Trace metals (Fe, Mg, Zn, Cu, a few more)
What do bacteria need the essential nutrients for (CHNOPS, micronutrients)
-Carbon is in everything
-Hydrogen is in everything
-Nitrogen is in proteins, nucleotides, RNA, DNA
-Oxygen is in everything
-Phosphate is in lipids, nucleotides, RNA, DNA
-Sulfur is in proteins and a few coenzymes
-Mg++, K+, Na+ used as counter ions to shield charges or for osmotic balance
-Trace elements are often important catalytic groups in enzymes
Where does carbon come from?
- Heterotrophs→organic molecules (sugars, amino acids, etc.)
- Autotrophs→CO2
Where does H come from?
Water
Where does N come from?
- Many potential sources: Take up amino acids, ammonium (NH4+) or nitrate (NO3-), a few can fix atmospheric nitrogen (N2)
Where does O come from?
From water, organic compounds, or (rarely) O2
Where does P come from?
Inorganic PO42- or phosphate containing organic compounds like nucleotides
Where does S come from?
From H2S, SO42- or cysteine
Steps in NH3 assimilation
Typically NH3 is assimilated directly into Glutamate to make Glutamine
* The enzyme is named Glutamine Synthetase (or GS for short)
* The reaction requires ATP to drive it forward
* The reaction occurs in two steps with a phosphorylated intermediate (this explains how
energy from ATP can be harnessed to drive a reaction)
From Glutamine it is possible to make Glutamate by a transfer reaction
* The enzyme is named Glutamine 2-oxoglutarate aminotransferase (GOGAT)
* The recipient molecule 2-oxoglutarate (also called alpha-ketoglutarate, α-KG) comes
from the TCA cycle
What is the preferred source of nitrogen in most bacteria?
NH3
How do other biomolecules get N?
Glutamate and Glutamine are the major N-donors in biochemistry
About 90% of cellular N comes from the α-amino group of glutamate.
The remaining 10% of cellular N comes from the side chain amino group of glutamine
Where does the NH3 come from?
Almost all N sources are converted to NH3 so they can be assimilated into
glutamate and glutamine
* NH3 is used directly
* Amino acids are deaminated to NH3 plus the corresponding alpha-ketoacid
* Nitrate is reduced to NH3
* N2 gas is “fixed” to NH3
*See lecture 8 slide 12 for diagrams
What is nitrogenase?
Enzyme that allows SOME bacteria to fix their own N.
A very complex enzyme
* Three subunits: NifH, NifD, NifK
* 2 copies of each = 6 polypeptide chains in all
* Has unique cofactor called FeMoCo and iron-sulfur
clusters
* Nitrogenase is extremely sensitive to O2
How does nitrogenase work?
- It is a reduction
- H2 is produced. That’s odd. Nobody understands why this should be.
- Requires a ton of ATP! The ATP is needed to overcome an activation energy barrier.
The N2 molecule has a triple bond and is very stable. The overall reaction is not
“uphill.” There is no phosphorylated intermediate, rather, ATP hydrolysis causes
enzyme conformation changes that drive the electrons into the N2 molecule. Note
that this is fundamentally different than how ATP is used by GS to make glutamine.
Why is nitrogenase senstive to oxygen?
The “weak link” is the metal clusters; those metals react spontaneously with O2,
become oxidized, and the clusters fall apart (for example, Fe2+ becomes Fe3+).
How does Clostridia protect nitrogenase from O2?
Clostridia are obligate anaerobes. Some fix N2. No special adaptations are needed to protect nitrogenase from O2 because if the organism sees O2 it is probably dead anyway.
How does Klebsiella pneumonaie protect nitrogenase from O2?
Klebsiella pneumoniae is a facultative anaerobe. It only expresses the genes for nitrogenase under anaerobic conditions. In other words, the adaptations to protect nitrogenase from O2 are at the level of gene expression, not cellular structures.
How does Azotobacter vinelandii protect nitrogenase from O2?
Azotobacter vinelandii is unusual in fixing N2 while growing
aerobically.
* Creates a microaerobic environment
➢ Extremely high rate of metabolism consumes the entering
O2, keeping the concentration low.
➢ Produces thick capsule and lives in colonies, slowing the
rate of O2 entry.
* A special protein protects nitrogenase from O2 (it binds to
nitrogenase, holding it inactive).
* Azotobacter is widely used in agriculture as a biofertilizer.
How do rhizobia use nitrogenase?
Various Rhizobia species only fix
nitrogen when in a symbiotic
relationship with leguminous plants.
The Rhizobia are found in specialized
root structures called nodules, which
are red because they contain an O2-
binding protein named leghemoglobin.
The leghemoglobin controls the O2
tension in the nodule, so there is
enough for the bacteria to respire and
make ATP needed to fix N2 but not
enough to kill the nitrogenase enzyme.
The plant supplies the bacteria with
carbon compounds (e.g., malate) and
the bacteria export both NH3 and
alanine to the plant.
How do cyanobacteria solve the problem of O2 that is liberated from the splitting of water during photosynthesis while still using nitrogenase?
Temporal separation
➢ Some Cyanobacteria do photosynthesis during the day and
fix N2 at night.
Spatial separation
➢ Some Cyanobacteria differentiate into “heterocysts”
where nitrogen fixation occurs (but not photosynthesis).
What are heterocysts?
-Only in filamentous Cyanobacteria
-Vegetative cells→ Photosynthesis to generate
energy and fix CO2 but no nitrogenase.
-Heterocysts→Specialized cells where nitrogen
fixation happens. Have nitrogenase but no
photosystem II (the PS that splits water).
-Vegetatvie cells feed carbon compounds to the
heterocysts and get fixed N in return.
Heterocysts differentiation is regulated by
availability of N in environment:
What is peptidoglycan good for
Cell wall that surrounds bacteria
✓ Major functions = Shape, protection against lysis
✓ Useful for identifying/classifying bacteria (Gram stain)
✓ Synthesis of PG illustrates clever solutions to complex
biochemical problems and provides many antibiotic targets
✓ Medical importance (Penicilllin-binding proteins, inflammation)
✓ Peptidoglycan hydrolases are important for growth and cell
division.
What is the structure of peptidoglycan?
A repeating structure made from a building block.
Disaccharide-pentapeptide
* NAG = N-acetylglucosamine
* NAM = N-acetylmuramic acid
➢ Each of these is a glucose derivative
with an amino group
* Pentapeptide extends from the NAM residue
* Note the unusual amino acids
A repeating structure made from a building block
Cartoon modified from Irazoki et
al., Front Microbiol 2019 L-alanine, D-glutamic acid, meso-diaminopimelic acid,
D-alanine, D-alanine
How is peptidoglycan linked?
The building blocks are assembled into glycan strands
crosslinked via the peptides on the NAM residues
What is the benefit of using unusual amino acids in PG?
They are less likely to be broken down by existing enzymes or used for other cellular components.
Major functions of the PG sacculus (PG net surrounding cell)
✓ Confers shape
✓ Protects against lysis
✓ PG fragments activate the innate immune
system (not necessarily a “function” from the bacteria’s
point of view)
What is evidence that PG confers shape?
-One line of evidence that PG confers shape is that
the purified sacculus retains the shape of the
organism it came from
-Another line of evidence is that bacteria lose their
shape when the PG is removed with lysozyme
How can you purify the sacculus?
Boil cells in SDS
➢ Solubilizes the membranes and proteins
Centrifuge at high speed
➢ PG sacculi are intact and large, so end
up in pellet at bottom of tube
Remove and discard supernatant (contains
lipids and proteins)
➢ Leaving behind “pure” PG sacculi
Examine the pellet in electron microscope
How does PG interact with the immune system?
✓ Sometimes bacteria lyse, releasing PG fragments
✓ Bacteria also release some PG during normal growth
because the sacculus is an exoskeleton that has to be
broken down to enable expansion.
Which of these amino acids could plausibly substitute for mDAP in the stem peptide of PG? Explain.
The requirement is a long sidechain with a terminal amino group for crosslinking. Only the top amino acid fits that description. (The amino acids shown are ornithine and aspartate, but naming them was not part of the
question.)
When B. subtilis is treated with lysozyme in a buffer containing 400 mM sucrose, the cells become
round but do not lyse. If the same experiment is in buffer containing only 50 mM sucrose, the cells lyse.
How does sucrose protect the cells against lysis?
400 mM sucrose is roughly isotonic with the bacterial cytoplasm, so there is no strong movement of water
into the spheroplasts by osmosis. On the other hand, in 50 mM sucrose water will move across the
membrane by osmosis, causing the spheroplast to swell and burst.
. Where does the energy for the transpeptidation reaction come from?
The peptide bond between D-Ala–D-Ala is the source of the energy; this energy is conserved in the new
peptide bond that is formed. Ultimately, the energy comes from ATP, which is the energy source for
making the D-Ala–D-Ala bond is made in the cytoplasm.
What are PG hydrolases and why are they necessary?
PG hydrolases are enzymes that cleave the PG cell wall. These enzymes open spaces for insertion of
new PG during elongation, process the PG in the division septum so to allow for daughter cell separation,
and make holes for structures that have to go through the PG such as flagella, pili and Type 3 secretion
systems.
Would you expect penicillin to be more effective at killing bacteria in exponential phase or stationary
phase or about the same? Explain.
Exponential phase. Penicillin blocks PG synthesis. In growing bacteria PG hydrolases are active as they open spaces in the sacculus for insertion of new PG strands during elongation and as they degrade
septal PG to allow for daughter cell separation. Ordinarily, this degradation does not lead to lysis because the PG synthesis enzymes are active, but penicillin prevents synthesis (crosslinking). In stationary phase cells there is neither synthesis nor degradation going on, so inactivating transpeptidases
with penicillin has no effect (or at least not much effect).
How do penicillin binding proteins work?
Membrane anchored proteins
Bifunctional:
➢ transglycosylase domain (GT)
➢ transpeptidase domain (TP)
Working together, these domains
add precursors by extending the
glycan chains and then crosslink the
chains by making peptide bonds.
How would you go about isolating cold-sensitive fts mutants?
Mutagenize wild-type E. coli cells, plating on rich medium at 37 degrees. When colonies come up (about 1 day), pick them and test for growth on plates at 22 degrees. Most will grow, but the ones that do not are cold-sensitive mutants. They could have a mutation in any number of important genes, such as those for DNA polymerase, RNA polymerase, components of the ribosome, lipid synthesis, outer membrane assembly, cell division, etc. You need to find the subset of mutants that dies at 22 degrees because of cell division defect. One approach is
to take each cold-sensitive mutant and grow it in broth at 37 degrees to early exponential phase, then shift the tube to 22 degrees and continue growth for several more hours. Finally, examine the culture in the microscope–the mutants
you are interested in will appear as long filaments with regularly-spaced nucleoids (you will need a stain such as DAPI to see the nucleoids). [Mutants with defects in other processes like DNA synthesis or protein synthesis will simply stop growing at 22°C without any striking change in cell size or shape.]
Using this approach you discover a new gene. How will you determine whether the protein it encodes is localized to the division site?
A fusion of the new gene to gfp can be used to determine where the protein goes in the cell. You are hoping to see a fluorescent stripe across the middle of the cell.
What are thought to be the functions of FtsZ and FtsI (a PBP)?
FtsZ (a) recruits other Fts proteins to the division site and (b) guides synthesis of septal peptidoglycan by a process called treadmilling. FtsI is a transpeptidase needed for synthesis (cross-linking) of septal peptidoglycan. FtsI works together
with FtsW, a transglycosylase that polymerizes the NAG-NAM units.
FtsZ is used for division of plant chloroplasts, but not for division of plant cells. What is the evolutionary rationale for this finding?
The chloroplast is derived from a symbiotic relationship with cyanobacteria, so the chloroplast is in many ways very bacterial in its molecular properties. As one example, the chloroplast retains a bacterial division apparatus. The evolutionary origin of the plant cells (the source of the nuclear genome) is not clear, but in any case, the plant cell itself is not nearly so closely related to bacteria as the chloroplast is.
What do you think would happen if you artificially overproduced AmiA and EnvC in an E. coli cell? Do you think a small molecule that strongly activates the AmiA/EnvC complex would be a good antibiotic?
AmiA and EnvC form a PG hydrolase that processes PG in the division septum to enable daughter cells to go their separate ways. Overproducing these proteins is likely to result in over-digestion of the PG, which could cause lysis. Yes, a small molecule that over-activates AmiA/EnvC is likely to kill the bacteria.
