Exam 2 Flashcards
1
Q
Prototroph
A
able to synthesize all organic compounds required for growth
2
Q
Auxotroph
A
unable to synthesize all organic compounds, must have some provided
3
Q
Chemotrophs
A
obtain energy from chemical compounds
4
Q
Phototrophs
A
obtain energy from light
5
Q
Lithotrophs
A
use inorganic electron donors (e.g., H2S, H2, Fe2+)
6
Q
Organotrophs
A
use organic electron donors (e.g., sugars,
amino acids, fatty acids).
7
Q
Heterotrophs
A
use organic carbon sources
8
Q
Autotrophs
A
use inorganic CO2 which they fix into organic form
9
Q
What must general growth media provide?
A
- CHNOPS
- Energy source
- H2O
- Trace metals (Fe, Mg, Zn, Cu, a few more)
10
Q
What do bacteria need the essential nutrients for (CHNOPS, micronutrients)
A
-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
11
Q
Where does carbon come from?
A
- Heterotrophs→organic molecules (sugars, amino acids, etc.)
- Autotrophs→CO2
12
Q
Where does H come from?
A
Water
13
Q
Where does N come from?
A
- Many potential sources: Take up amino acids, ammonium (NH4+) or nitrate (NO3-), a few can fix atmospheric nitrogen (N2)
14
Q
Where does O come from?
A
From water, organic compounds, or (rarely) O2
15
Q
Where does P come from?
A
Inorganic PO42- or phosphate containing organic compounds like nucleotides
16
Q
Where does S come from?
A
From H2S, SO42- or cysteine
17
Q
Steps in NH3 assimilation
A
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
18
Q
What is the preferred source of nitrogen in most bacteria?
A
NH3
19
Q
How do other biomolecules get N?
A
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
20
Q
Where does the NH3 come from?
A
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
21
Q
What is nitrogenase?
A
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
22
Q
How does nitrogenase work?
A
- 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.
23
Q
Why is nitrogenase senstive to oxygen?
A
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+).
24
Q
How does Clostridia protect nitrogenase from O2?
A
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.
25
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.
26
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.
27
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.
28
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).
29
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:
30
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.
31
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
32
How is peptidoglycan linked?
The building blocks are assembled into glycan strands
crosslinked via the peptides on the NAM residues
33
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.
34
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)
35
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
36
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
37
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.
38
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.)
39
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.
40
. 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.
41
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.
42
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).
43
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.
44
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.]
45
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.
46
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.
47
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.
48
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.
49
When grown in broth, Vibrio parahaemolyticus looks like a normal rod shaped bacterium about 4 m long with a single polar flagellum and no lateral flagella. This form is called a “swimmer cell.” But on a plate V. parahaemolyticus differentiates into a “swarmer cell.” Swarmer cells are about 20 m and have thousands of lateral flagella.
Studies of swarmer cell differentiation indicate that division is inhibited because FtsZ rings do not form. Propose two plausible explanations for why Z-rings would be lacking,
and indicate how you would test each idea.
i. Not enough FtsZ protein. Test by doing a Western blot to see how much FtsZ is present (compared to swimmer cells).
ii. Nucleoid occlusion blocks FtsZ assembly into rings. Use DAPI staining to see if swarmer cells have continuous staining throughout the cytoplasm without “gaps” between nucleoids where Z-rings can form.
iii. MinCD is overproduced in swarmer cells and blocks division
everywhere. Test with Western blot to see how much MinCD is
present (compared to swimmer cells). Or make a minCD null mutant and see if it no longer becomes filamentous on a plate.
iv. Not enough GTP, which is needed by FtsZ to polymerize into a ring. Test by using TLC or HPLC to measure GTP levels (compared to
swimmer cells).
v. A forest of flagellar basal bodies in the cell cylinder interferes with Z-ring assembly. This is hard to test for technical reasons you could not be expected to know, but ignoring this problem, the logical test would be to knock out the genes for the flagellar basal body and ask whether
such a mutant no longer becomes filamentous on a plate.
50
What are the steps of bacterial cell division in E. coli?
Assembly of FtsZ
Septal ring assembly
Constriction
Cell separation
51
What does fts stand for?
filamentation temperature sensitive
52
What are the two criteria for determine if a gene is a division gene?
* Mutants are filamentous (division defect)
* The protein encoded by that gene localizes to the mid-cell
(a) the mutant has a division defect
(b) the protein localizes to the division site.
53
What does ftsZ do?
* Nearly universal: Bacteria, Archaea, some chloroplasts
* Tubulin homolog
* Assembly of FtsZ ring at mid-cell is the first event in division
* The Z-ring functions as a landing pad to recruit the other proteins and moves by “treadmilling” to guide the PG synthesis enzymes around the septum.
54
What is the septal ring?
Around midline of bacteria. Contains about 30 proteins important for division. Must cut off new bacilli at midline
55
What is treadmilling?
Directional movement of a polymer by addition of
monomers at one end of a filament and removal of
momomers from the opposite end
56
What are ftsW and ftsI
FtsW = PG transglycosylase
FtsI = PG transpeptidase
57
What are AmiA and EncV?
PG hydrolases for septal PG. An envC mutant has a “chaining” phenotype. The mother cell still divides into two
daughter cells, but these remain attached
by a shared PG wall.
58
Overview of cell division
1. Grow
2. Replicate chromosome, segregate
3. Assemble an FtsZ ring
4. Recruit about 30 more proteins
5. Synthesize a division septum through middle of cell
6. Process (carefully degrade) the PG wall
for daughter cells to separate
59
How do Min proteins regulate septum formation?
They prevent FtsZ from accumulating and make sure it accumulates in the middle of the cell when the time is right
60
What is the role of SlmA, a nucleoid occlusion protein?
Prevents FtsZ from forming ring while nucleoid is still in the center of the ring
61
Describe what a Sec dependent signal peptide looks like
Generally short N-term followed by positive charged amino acids hydrophobic stretch of ~20 amino acids
If cleavable by type 1 signal peptidase AxA motif following transmembrane segment
If cleavable by type 2 signal peptidase LxxC motif following transmembrane segment
62
Explain how Tat and Sec secretion systems differ
Tat signal peptides have twin arginines at the N-terminus
Tat secretes folded proteins Sec unfolded
63
Name or outline the steps of fatty acid biosynthesis
Four step repetitive mechanism
1. Condensation
2. Reduction with NADPH
3. Dehydration
4. Second reduction with NADPH
Extension of 2 carbons / cycle
64
What is a lipid?
– Naturally occurring organic molecules
– Hydrophobic
– Diverse chemistry
– Functions
* Cell structure
* Energy source
* Protection, pathogenesis factor
* Enzyme cofactor
65
Key features of fatty acids
* Background:
1. Chains of methylene carbons with carboxylate group
2. Bound to glycerol as acyl esters (“R” in image)
“phosphoglyceride”
* Types:
* Branched
* Saturated
* Unsaturated
* Other modifications
* Many fatty acids are part of polar lipids
* Esterified with glycerophosphate derivates
66
What is biotin
* What is biotin?
1. Vitamin B7 (vitamin H)
2. Enzyme cofactor
* Role
1. Attached to enzymes through amide bond to lysine residue
2. Separate enzyme is required to biotinylate
3. Necessary in carbon dioxide transfer reactions
* Acetyl-CoA carboxylase
Requires biotin to make malonyl-CoA
* Used in biotechnology as a handle
Binds to streptavidin with high affinity
67
Fatty acid biosynthesis overview
* NADPH is reductant (not NADH)
* Pathway requires CO2
* Pathway proceeds via malonyl-CoA
* Acyl carrier is not CoA, instead is ACP
* Aerobic and anaerobic pathways to convert saturated
fatty acids to unsaturated
* Enzymes of synthesis are one polypeptide in eukaryotes.
– Dissociated in bacteria
68
Fatty acid biosynthesis review
* The two carbon atoms at the methyl terminus in each
fatty acid molecule derive from acetyl-CoA
* Malonyl-CoA supplies the remaining carbons
* No carbon atoms in fatty acid derive from bicarbonate
* FA are assembled two carbon atoms at a time by the
fatty acid synthase complex
* Odd chain fatty acids require propionyl-CoA start
69
Why is desaturase necessary for cold temps?
Allows for more membrane fluidity
70
What does DesRK do
senses membrane fluidity. be able to diagram
71
steps in beta oxidation of fatty acids
* Activation of terminal carbon
* Four step mechanism
1. Oxidation
2. Hydration
3. Oxidation
4. Cleavage
* Mechanism repeats until acetyl-CoA or propionyl-CoA
is reached
* Acetyl-CoA →TCA
Propionyl-CoA →2-Methylcitrate cycle
72
Properties of phospholipids
1. Permeable only to water, gases, and small hydrophobic
molecules
2. Low ionic conductance
3. Capable of doing “work” through generation of electrochemical
gradient
4. Lipid portion must remain fluid
5. Fluidity due to presence of unsaturated fatty acids
* Fatty acid biosynthesis
1. Making malonyl-CoA, importance of biotin
2. Fatty acid synthase reaction
73
Define translocation
– The transport of proteins through the membrane
74
Define export
– Protein translocated through the inner membrane to the periplasm
– Sec/Tat systems
75
Define secretion
– Protein transported to the extracellular medium, to the cell surface, or
into another cell
76
Define excretion
– Transport of compounds (non-proteins) into the extracellular medium
77
Why do bacteria secrete things?
* Cell envelope proteins
– Cell membranes – hundreds of proteins
– >100 proteins in Gram-negative periplasm
– Cell wall attached proteins of Gram-positives
– Outer envelope and surface layers
* Surface structures
– Flagella (polar, lateral)
– Pili (fimbria)
* Extracellular enzymes
– Hydrolytic enzymes – lipases, proteases, nucleases, saccharidases
* Virulence factors
– Pathogens injecting toxins into hosts
– Secreted virulence factors
78
Describe the structure of an integral protein
A membrane-spanning α-helix is
the most common structural
motif found in cytoplasmic
transmembrane proteins. Positvely charged amino acid anchor. 18-22 hydrophobic aa in between membranes.
79
What are the three signal peptides?
Sec, SRP, TAT, usually 20-30 residues long
80
Describe sec
Basic, pos charged terminal, hydrophobic core, uncahrged C terminal, exported protein
81
How does sec work with lipoproteins
* Lipoproteins have an LxxC box
* The cysteine residue is lipid modified (OM anchor)
* After modification, Type II signal peptidase cuts
* C-terminus bound to cell wall
82
Sec system intiation
* SecA and SecYEG form complex
(“translocase”)
* Translate protein with signal sequence
* Bind chaperone SecB
* Binds to the mature protein domain,
not leader
* Prevent misfolding and aggregation
in cytoplasm
* Thought to recognize the unfolded
topology of proteins
83
Insertion into Sec channel
* SecB targets protein to SecA
* SecB is released
* SecA targets protein to SecYEG
channel
* SecA binds ATP, conf. change
* Leader leaves SecA, “flips”, N-term
stays on cyto side and C-term goes to
periplasm
* Pre-protein begins to enter SecYEG
84
Sec Translocation across
channel
* SecA hydrolyzes ATP to drive protein
through SecYEG channel
Is ADP released?
* SecA is released from complex
* Translocation continues by membrane
potential
* Process starts again, SecA binds new
protein from SecB chaperone
85
Sec cleavage and release
* Protein translocated across SecYEG
channel
* Signal peptidase removes leader
sequence
* Type I enzyme – secreted proteins
* Type II enzyme – lipo proteins
* Leader peptide degraded by “signal
peptide peptidase”
* Mature protein either released into
periplasm or membrane-bound
86
How does Sec work with membrane proteins
* Protein translocated across SecYEG
channel
* Signal peptidase removes leader
sequence
* Type I enzyme – secreted proteins
* Type II enzyme – lipo proteins
* Leader peptide degraded by “signal
peptide peptidase”
* Mature protein either released into
periplasm or membrane-bound
87
SecDF
* Integral membane proteins
* Required for proper translocation
* Exact role unknown
88
YajC
YajC
* Co-transcribed with secDF
* Integral membrane protein
* Not required for protein
translocation
* Exact role unknown
89
YidC
YidC
* Integral membrane protein
* Required for proper insertion of
membrane proteins
* Essential for viability
* Proper insertion of F1F0-ATPase and other proteins
* Energy not required likely mediated by hydrophobic forces
* Very abundant protein
* ~2500 per cell vs ~500 SecYEG
* Gram negative bacteria have a single YidC (essential)
* Gram Positive bacteria have 2 different YidC (redundant)
90
What is SRP
* Bacterial system similar to the SRP pathway in
eukaryotic cells
* E. coli SRP particle
4.5S RNA (ffs)
48 kDa protein (ffh)
* SRP particle recognize leader peptide
Often more hydrophobic than Sec leader
Non-cleavable
* Nascent polypeptide often recognized co-
translationally
* Most, but not all SRP-dependent proteins, use the
SecYEG translocation channel for membrane
insertion or translocatio
91
SRP mechanism
1. SRP recognizes leader peptide as it
emerges from ribosome
* More hydrophobic sequence
2. SRP brings protein to FtsY membrane
receptor
3. FtsY brings protein to SecYEG,
hydrolyzes GTP, releases protein
4. Translocation of protein through
SecYEG channel
5. YidC protein helps membrane insertion
92
Role of YidC in sec dependent vs independent
* Sec-dependent pathway
* Protein-protein interactions:
* SecYEG
* SecDF-YajC
* Depletion affects protein
insertion
*First proteins are passed from
YidC stabilizes TMs after they leave
the SecYEG translocation channel
* Helps partition substrates into the
lipid environment by contacting TM
segments of substrates
* Sec-independent
* Required for insertion of
phage-coat proteins
* Assembly of membrane protein
complexes?
* Sec-independent
* YidC acts as an insertase
* Required for insertion of
phage-coat proteins
* Depletion of YidC decreases
membrane insertion of proteins
* YidC proteoliposomes are
sufficient to insert some substrates
into membrane
* Pf3 coat and subunit c
proteinAssembly of membrane
protein complexes?
93
What is TAT (twin-arginine translocation)
* Export of folded proteins across CM:
periplasmic proteins (often w. Redox cofactors; i.e.
TAMO-red.)
several IM proteins
proteins later exported via Type II system
Not Essential
* Energy: electrochemical gradient (no
ATP)
* Signal peptide: N-terminal signal
SRRxFLK 'twin-arginine' motif
cleaved off during transport
(Signal peptidase I; LepB)
94
Why does the cell need TAT?
Intracellular Co-factors
* Co-factors that require energy/pre-assembly to function
95
TatA
* Most abundant, 20x more than TatB or TatC
* N-term TM domain, followed by cytoplasmic domain
* Functions in late stage of translocation, likely forms the pore
96
TatB
* N-term TM, followed by cytoplasmic domain
* Forms critical interaction with leader peptide, only when TatC is present
* Mediator between TatA and TatC
97
TatC
* Largest, most conserved Tat component
* Integral membrane protein
* Initial docking site of RR leader peptide, interactions on the cytoplasmic face
98
TatE
* Homologous to TatA
* Not essential absent from many systems
* When overexpressed can complement tatA deletion
99
Tat mechanism
* Substrate binds initially to TatBC and is
independent of TatA
* Once the precursor is TatBC bound to the
substrate associates with TatA
* The active translocon has never been
‘captured’
* Two models of secretion
* The pore model or “trap door”
* The membrane destabilization model
100
DsbA
Thiol redox enzyme
Catalyzes formation of disulfide bonds
101
DsbB
Integral membrane protein
Works with DsbA, oxidation – reduction cycle
Hands off electrons to ETC
102
How do we see disulfide bonds?
* Change in mobility on an gel
(Western or immunoblot)
* Mutants or changes in condition
* DTT (Reductant)
* dsbA mutant
* site directed cysteine mutant
Disfulfide bonds will be lower on the western blot than unbound proteins
103
What is hyperosmotic shock
Cells shrink due to water loss
104
What is hypoosmotic shock
Cells lyse from having too much water
105
What is the response of microbes in a hypertonic solution?
Decrease of intracellular water causes proteins, etc. to
precipitate out of solution, stop functioning. Hyperosmotic shock
106
How do cells deal with a hypertonic
environment (low water activity)?
* K+ ions
– Halophiles maintain [K+] at very high
levels, >3M
* Compatible solutes
– Do not harm cells
– Transported in/out to maintain internal
solute concentration
107
What are compatible solutes?
* small neutral molecules accumulated in cytoplasm when
external environment is hypertonic.
* No net charge, not acidic or basic.
108
Order of events for B. subtillus response to hyperosmotic stress
* Order of events
– Import of Potassium ions
– Import Organic osmolytes (proline, betaine etc)
– Synthesize Organic osmolytes
– Export of Sodium Ions
– Export of Potassium ions
109
hich transporters are required for potassium uptake?
Potassium uptake is dependent upon 2
transporters KtrAB and KtrCD
* Mutants in ktrABand ktrCDare
unable to grow at lower KCl
concentrations
* Why does the ktrABktrCDdouble
mutant still grow at high KCl
concentrations?
110
What is required for potassium uptake other than transporters?
NaCl
111
What is an osmoprotectant?
Things like glycine betaine. Higher salt means more glycine betaine. Acts as an osmolyte and thus brings water with it
112
Why are integral membrane proteins positively charged?
Combats negatively charged phospholipid bilayer
113
What is SRP good for?
Really hydrophobic proteins, cotranslational
114
How are mechanosensitive channels opened?
Lipid bilayer Tension or stretch model: Tension in the lipid bilayer triggers conformational changes, thus leading to the opening of the channels. The tension perceived by the protein comes from the lipids. It has been demonstrated
that the tension/stretch profile in the lipid bilayer is originated by membrane curvature and bilayer-protein hydrophobic mismatch.
115
What are mechanosensitive channels used for?
To relieve hypoosmotic stress. Open to release select substances
116
What is LPS
* Only in Gram negative bacteria
* Part of the outer membrane (OM)
* helps protect organism from environment
* Complex polymer of polysaccharide and lipid
* LPS may cause host reactions, symptoms of infectious
disease
* endotoxin (due to toxic lipid A); e.g., Salmonella
117
LPS composition
* Hydrophobic region embedded in membrane = Lipid A
* Core polysaccharide region projecting from surface
Connected to lipid A through “KDO” (3-deoxy-D-manno-octulosonate)
* Outermost polysaccharide region = O-antigen
Repeating part up to 30 units
118
What is lipid A
* Synthesized from UDP-N-acetyl-glucosamine
* Fatty acids anchor lipid A to membrane
* 4 fatty acids attached directly to glucosamine hydroxyls
In E. coli: C14 b-hydroxymyristic acid
* Esterified to hydroxyl group are C12 fatty acids
119
σE Regulon
* Increase expression of
* periplasmic proteases
* Outer membrane biogenesis Lol, Lpt and Bam complexes
* Tuns off expression of OMPs
* Old observation that if you increase expression of one
OMP you decreas expression of the others?
* SigE upregulates synthesis of siRNA
* siRNA target degradation of other OMP transcripts
* ECF sigma factors are important in virulence for a
number of pathogens
* Salmonella, Psuedomonas, Mycobacterium, Vibrio
120
What is bam
BamA is a β-barrel (rolled up to create pores), outer membrane protein found in Gram-negative bacteria and it is the main and vital component of the β-barrel assembly machinery complex in those bacteria.
121
What is lol
lipoprotein transport through outer membrane
122
what is lpt
lipopolysaccharide transport from IM to OM
